ACMI conference posters 2019

Access Microbiol. 2019 Apr 8;1(1A):15.

Viral hijacking of the nucleolar DNA-damage response machinery: a novel mechanism to regulate host cell biology

Stephen M Rawlinson ¹, Tianyue Zhao ¹, Ashley M Rozario ¹, Christina L Rootes ², Paul J McMillan ³, Anthony W Purcell ¹, Amanda Woon ¹, Glenn A Marsh ¹, Kim G Lieu ¹, Lin-Fa Wang ⁴, Hans J Netter ⁵, Toby D M Bell ¹, Cameron R Stewart ², Gregory W Moseley ^1,^*

Abstract

Recent landmark studies indicate that the nucleolus plays key roles in stress responses including the DNA-damage response (DDR). The latter involves interactions of components of the DDR machinery including NBS1 with the sub-nucleolar protein Treacle, a key mediator of ribosomal RNA (rRNA) transcription and processing, implicated in Treacher-Collins syndrome. Using comparative proteomics, confocal and single molecule super-resolution imaging, and infection under BSL-4 containment, we have shown for the first time that the nucleolar DDR pathway is targeted by infectious pathogens [1]. We found that the matrix (M) proteins of Hendra virus and Nipah virus, highly lethal viruses of the Henipavirus genus (order Mononegavirales), target Treacle to inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhanced henipavirus production. These data identify a novel mechanism for viral subversion of host cell biology by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intra-nucleolar function for proteins of any mononegavirus [1, 2]. For the presentation I will discuss our new data, which is advancing our understanding both of the mechanisms impacted by the Henipavirus-Treacle interaction, and potential roles of such interactions in infection by other viruses, including highly lethal lyssaviruses [3].

[1] Rawlinson et al. Nature Communications 2018.9 : 3057 (2018)

[2] Rawlinson et al. Cellular Microbiology 2015. 17(8):1108–20

[3] Oksayan et al. Journal of Virology 2015.89(3):1939–43

Access Microbiol. 2019 Apr 8;1(1A):15.

Microbiological profile and risk factors for in-hospital mortality of infective endocarditis intertiary care hospitals of south Vietnam

Hoang Tran ^1,^*

Abstract

Objectives

We aimed to evaluate the microbiological characteristics and risk factors for mortality of infective endocarditis in two tertiary hospitals in Ho Chi Minh City, south Vietnam.

Materials and methods

A retrospective study of 189 patients (120 men, 69 women; mean age 38±18 years) with the diagnosis of probable or definite infective endocarditis (IE) according to the modified Duke Criteria admitted to The Heart Institute or Tam Duc Hospital between January 2005 and December 2014.

Results

IE was related to a native valve in 165 patients (87.3%), and prosthetic valve in 24 (12.7 %). Of the 189 patients in our series, the culture positive rate was 70.4 %. The most common isolated pathogens were Streptococci (75.2%), Staphylococci (9.8%) followed by gram negative organism (4.5%). The sensitivity rate of Streptococci to ampicillin, ceftriaxone or vancomycin was 100 %. The rate of methicillin resistant Staphylococcus aureus was 40 %. There was a decrease in penicillin sensitivity for Streptococci over three eras: 2005 ± 2007 (100 %), 2008 ± 2010 (94 %) and 2010 ± 2014 (84 %). The in-hospital mortality rate was 6.9 %. Logistic regression analysis found prosthetic valve and NYHA grade 3 or 4 heart failure and vegetation size of more than 15 mm as strong predictors of in-hospital mortality.

Conclusion

Streptococcal species were the major pathogen of IE in the recent years with low rates of antimicrobial resistance. Prosthetic valve involvement, moderate or severe heart failure and vegetation size of more than 15 mm were independent predictors for in-hospital mortality in IE.

Access Microbiol. 2019 Apr 8;1(1A):15.

The effect of epinephrine and norepinephrine on the growth and pathogenicity of Campylobacter jejuni

Brendha Truccollo ^1,^2,^*, Paul Whyte ², Declan J Bolton ¹

Abstract

Campylobacter spp. is a leading cause of foodborne illness globally. The pathogen colonises the gastrointestinal tract of the host, where small concentrations of neuroendocrine hormones are also secreted. Epinephrine and norepinephrine are neuroendocrine hormones involved in the stress response that have been shown to promote the expression of virulence factors in pathogens including E. coli, Salmonella spp., and Campylobacter spp. In our study Campylobacter jejuni strains from human infection and broiler farms that were supplemented with epinephrine and norepinephrine showed increased growth characterised by shorter lag phases and higher maximum OD595, and enhanced pathogenicity characterised by increased motility, attachment to and invasion of Caco-2 cells. The data obtained suggests that host stress may promote C. jejuni proliferation and pathogenicity.

Access Microbiol. 2019 Apr 8;1(1A):15.

In-ovo antiviral assay of methanolic leaf extract of Cymbopogon citratus (Lemon grass) on Newcastle disease virus

Joseph Abraham-Oyiguh ^1,^2,^*, Abigail W Zakka ², Joseph Taiwo C Onwuatuegwu ³, Lanre K Sulaiman ⁴, Salamatu Y Muhammad ²

Abstract

This study determined the in-ovo antiviral effect of crude Methanolic leaf extract of Cymbopogon citratus on Newcastle disease virus. Cold extraction was carried out using analytical grade methanol. Phytochemical screening of the crude extract was carried out using standard procedures. Antiviral assay was carried out in nine-day old specific pathogen free embryonated hens’ eggs in three designs made of five eggs per group (Virus with extract, virus only and un-inoculated groups) with concentrations ranging from 12.5 to 100 mg ml⁻¹. Egg toxicity of the extract was determined for concentrations of 12.5, 25, 50, 100, 200, 300 and 400 mg ml⁻¹. Inoculated eggs were incubated at 37 °C and observed daily for 96 h for embryo survival and mortality. Spot haemagglutination was carried out on bacteria-free allantoic fluid from the embryonated eggs to detect the presence of the virus. Phytochemical assay revealed the presence of saponins, flavonoids, steroids, terpenes, phlebotannins and terpenoids. Mild toxicity was observed at concentrations of 100 mg ml⁻¹ and above. There was no haemagglutination of fluid from the eggs inoculated with a combination of Virus and extract at concentrations of 50 and 100 mg ml⁻¹. The current findings demonstrated that leaf extract of Cymbopogon citratus has potential medicinal value as well as antiviral activity against Newcastle disease virus in-vivo. The specific mechanism of action remains to be studied to further elucidate on its potential as a therapeutic product for the treatment of Newcastle Disease.

Keywords: Cymbopogon citratus, Newcastle disease virus, Phytochemical, embryonated hens eggs, Spot haemagglutination, Methanol extract

Access Microbiol. 2019 Apr 8;1(1A):15.

Mathematical modelling to characterise the in vivo dynamics of Salmonella in the naïve and immunised host

Myrto Vlazaki ^1,^*, Olivier Restif ¹

Abstract

Systemic salmonellosis encompasses typhoid and paratyphoid fever, and invasive non-typhoidal salmonellosis, with high mortality and morbidity amongst children and the immunocompromised in low-resource settings. Immunisation efforts remain hampered by the unavailability of safe vaccines with cross-protectivity against causative Salmonella strains. Characterisation of the within-host Salmonella dynamics in the naïve and immunised host can elucidate the mechanisms by which different vaccine types exert their protective effect, and help in vaccine selection and design. Experimental data tracking the changes in bacterial population composition in the different tissues of the host at different timepoints can be coupled with mechanistic mathematical models to estimate the parameters governing the processes of bacterial replication, killing and inter-organ migration. Using a recently described minimisation-divergence estimation approach, we extend a three-compartmental mechanistic model and re-analyse existing datasets to better characterise the bacterial migratory processes between the blood, liver and spleen in the early stages of infection, and the overall Salmonella dynamics in the later phases in the naïve host. We apply the same model to published data from mice immunised with either a live-attenuated or killed whole-cell vaccine to identify their in vivo differential impacts on Salmonella migration, replication and death. Finally, we identify alternative experimental designs to improve the statistical qualities of the mathematical model and allow better inference of parameters governing the unobserved processes of bacterial dynamics.

Access Microbiol. 2019 Apr 8;1(1A):15.

Structural elucidation of viral antagonism of innate immunity: the STAT1 interface

Gregory W Moseley ^1,^*, Alamgir Hossain ², Florence Larrous ³, Jingyu Zhan ², Ashish Sethi ², Youssef Ibrahim ², Maria Aloi ¹, Stephen M Rawlinson ¹, Kim G Lieu ², Yee-Foong Mok ², Michael D W Griffin ², Naoto Ito ⁴, Toyoyuki Ose ⁵, Hervé Bourhy ³, Paul R Gooley ²

Abstract

To evade innate immunity, many viruses express interferon-antagonists that target STATs, critical mediators of immune signalling. Virus-STAT interfaces may provide new therapeutic targets but progress is hindered by a lack of direct structural data, owing to poor tractability of antagonists/full-length STATs for structural/biophysical approaches. By applying cross-saturation transfer NMR, we report the first direct structural analysis of binding of full-length STAT1 to an interferon-antagonist of a human pathogenic virus, the first such study of the virus-host interface. Analysis using mutation of the interface, biophysical characterization, immune signalling/protein-protein interaction assays including PCA, reverse genetics and animal infection demonstrated the significance of this interface in immune signaling suppression, and in disease caused by a pathogenic field-strain lyssavirus. Importantly, NMR/mutagenesis also revealed that the interface comprises multiple surfaces/domains in both the viral and cellular partners, indicating that antagonism involves extensive interactions consistent with a multifaceted inhibitory mechanism, distinct from ‘simple’ mechanisms such as tethering. Furthermore, by elucidating the spatial relationship of interactions critical to immune evasion and replication, the data provide insight into how ostensibly simple viruses can regulate these central functions via a single multifunctional protein. These data provide novel insights into fundamental viral biology, and potential exploitation of these mechanisms as new targets for antivirals and vaccine development. The study also demonstrates the power of biophysical/NMR approaches to elucidate the atomic interface of full-length STATs with regulatory proteins, providing a framework for studies to reveal immune evasion mechanisms of other pathogens in their full complexity.

Access Microbiol. 2019 Apr 8;1(1A):15.

Shining new lights on chytrid cell biology: quantitative live cell imaging of rhizoid development in an early-diverging fungus

Davis Laundon ^1,^2,^*, Glen Wheeler ¹, Thomas Mock ², Michael Cunliffe ^1,³

Abstract

Chytridiomycota (Chytrids) are the most basal lineage within the true fungi, however they have largely remained in the dark in terms of their fundamental cell biology. In aquatic ecosystems, chytrids can dominate ‘dark matter’ surveys and are important saprotrophs of recalcitrant organic carbon. They therefore play an integral biogeochemical role in carbon cycling. Unlike ‘higher’ dikaryan fungi that feed via multicellular hyphae, chytrids are unicellular and develop an anucleate rhizoid system that acts as the trophic interface of the cell. Understanding the functions of the rhizoid has the potential to shed light on the trophic biology of ‘dark matter’ chytrids. We applied 3D and 4D live-cell confocal microscopy to morphometrically quantify rhizoid development in the model saprotrophic chytrid Rhizoclosmatium globosum under different nutrient treatments. Rhizoid branching was highest under carbon-rich conditions, whereas under carbon-starved treatments, rhizoids grew significantly longer and were less branched, in what we interpret to be a ‘search strategy’ for nutrient sources. F-actin and the cell wall were identified throughout the rhizoid system. Chemical inhibition of actin and cell wall glucan synthesis induced the development of hyperbranched paramorphs, suggesting that these components underpin rhizoid branching and organising cell polarity at the rhizoid tip. Previous studies have shown that inhibition of these components induces an identical phenotype in dikaryan fungi. These findings represent an important step in understanding the trophic biology of a biogeochemically important microbe and unveil striking similarities in cell development between early-diverging and ‘higher’ dikaryan fungi.

Access Microbiol. 2019 Apr 8;1(1A):15.

The HamE scaffold positively regulates MpkB phosphorylation to promote development and secondary metabolism in Aspergillus nidulans

Dean Frawley ^1,^*, Betim Karahoda ¹, Ozlem Sarikaya Bayram ¹, Ozgur Bayram ¹

Abstract

Mitogen-activated protein kinase (MAPK) pathways are conserved signalling cascades in eukaryotes which regulate a myriad of processes in fungi from sexual reproduction to stress responses. These pathways rely on recruitment of three kinases on a scaffold protein to facilitate efficient kinase phosphorylation and subsequent downstream signalling to the nucleus. The model filamentous fungus Aspergillus nidulans utilises a MAPK pathway termed the pheromone module to regulate both development and secondary metabolism. This complex consists of the MAP3K (SteC), MAP2K (MkkB), MAPK (MpkB) and adaptor protein SteD. To date, there has been no scaffold protein identified for this MAPK pathway. In this study, we characterised a protein termed HamE, which we propose as a scaffold that regulates kinase phosphorylation and signalling in the pheromone module. Mass spectrometry analysis and BIFC experiments revealed that HamE physically interacts with both MkkB and MpkB and transiently interacts with SteC. Deletion of hamE or any of the pheromone module kinases results in reduced sporulation and complete abolishment of cleistothecia production. Mutants also exhibited reductions in expression of secondary metabolite gene clusters, including the velvet complex and sterigmatocystin genes. HamE acts as a positive regulator of MpkB phosphorylation, allowing for HamE to subsequently regulate development and secondary metabolism.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of Acinetobacter baumannii type VI secretion system effectors and characterisation of a novel effector/immunity pair

Jessica Lewis ^1,^*, Deanna Deveson Lucas ¹, Marina Harper ¹, John Boyce ¹

Abstract

The type VI secretion system (T6SS) is a bacterial nanomachine utilised by many Gram-negative bacteria, including Acinetobacter baumannii, to deliver toxic effectors for microbial warfare. These toxic effectors are often delivered via specific non-covalent interactions with cognate VgrG proteins, which form part of the T6SS tip. In A. baumannii, each vgrG gene is usually located in the same locus as two other genes, one encoding the cognate effector and one encoding an immunity protein that protects against self-intoxication. Bioinformatic searches of ninety seven A. baumannii genomes using a highly conserved domain found within the VgrG proteins, enabled the identification of more than 250 genes encoding putative effectors and, in most cases, the gene encoding the corresponding immunity protein. Phylogenetic analysis revealed that the predicted effectors clustered into 33 distinct groups, some of which contained predicted amidases, chitinases, lipases, nucleases and deaminases. Two effectors, Tse5Ab, containing no toxic domains and Tse6Ab, containing a Tox-GHH nuclease domain characteristic of nucleases, were chosen for functional analysis. The C-terminal region encoding the predicted toxic domain of each effector was cloned and expressed in E. coli. Expression of this region of Tse5Ab did not perturb E. coli growth. In contrast, expression of Tse6Ab was toxic but toxicity could be neutralised by the co-expression of the cognate immunity protein. However, Tse6Ab did not exhibit DNase activity and instead may function as an RNase. Further characterisation of the diverse A. baumannii T6SS effectors may lead to the identification of antibacterial molecules with novel activities.

Access Microbiol. 2019 Apr 8;1(1A):15.

Inter-species transmission of avian influenza virus to dogs: 10 years experience

Daesub Song ^1,^*, Woonsung Na ¹, Minjoo Yeom ¹

Abstract

Influenza viruses have continuously evolved into multiple mutant strains from several regions, resulting in aggravated endemic or epidemic outbreak conditions. In the 2000s, several outbreaks of inter-species transmission were reported, such as, the avian H3N2 influenza virus that crossed the host barrier to dogs. The inter-species transmission gave rise to the H3N2 canine influenza virus (CIV) that spread from East Asia to North America. The newly emerged H3N2 CIV was likely to infect to cats; however, ferrets, which had a SA receptor-binding pattern similar to that of humans, were not suitable natural hosts. In addition to avian-to-dog transmission, the infectivity of pdm H1N1 and seasonal H3N2 viruses in dogs was proven when artificial inoculation of the viruses with active viral shedding in dogs caused pathologic changes in the lungs. Studies on sero-prevalence and artificial infection suggested the possibility of co-infection of and reassortment between the two viruses in dogs; later, H3N1 and variants of M-variant H3N2 reassortants between pandemic H1N1/2009 and prototype H3N2 CIV were isolated. Notably, the H3N2 CIV with the matrix gene of the pdm H1N1 virus showed more efficient transmission in ferrets than the classic H3N2 CIV. These results implied that this primary companion animal, which lives in closer proximity to humans than pigs, might act as a mixing vessel or a source of novel influenza A virus in humans. Our findings emphasized the necessity of intensive monitoring for influenza infection in companion animals for investigating the potential for the emergence of novel human influenza strains.

Access Microbiol. 2019 Apr 8;1(1A):15.

Understanding the killing mechanism of action by virus-infected yeasts

Duygu Dikicioglu ^1,^*

Abstract

Killer yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via viral infection. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts have been considered as beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill species off starter cultures and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action in silico. Our deterministic model explains how killer Saccharomyces cerevisiae cells distress and consequently kill the sensitive members of the species, accounting for the K1, K2 and K28 toxin mode of action at high or low concentrations. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death, and allowed us to gain novel insight on these mechanisms. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately 3 times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.

Access Microbiol. 2019 Apr 8;1(1A):15.

Regulation of exploratory growth and antibiotic production in Streptomyces venezuelae by the two-component system CutRS

Thomas C McLean ^1,^*, Barrie Wilkinson ², Matthew I Hutchings ¹

Abstract

Antibiotic production and cellular development in bacteria are intimately linked to the extracellular environment. One key mechanism by which bacteria recognise and respond to these external cues is through two-component regulatory systems (2CS). Consisting of a membrane-bound sensor kinase and a cognate DNA-binding response regulator, these 2CSs are essential in the response to a myriad of signals including antibiotic attack, microbial interaction and nutrient availability. The soil-associated filamentous actinobacteria Streptomyces spp. are prolific antibiotic producers with a large number of 2CS. In addition to their complex life cycle a new developmental stage has recently been described termed exploration. When exploring, the streptomycetes rapidly expand via non-branching vegetative hyphae when contacted by fungi, a juxtaposition to the canonical lifecycle which ends in sporulation. Fifteen of the fifty-six 2CS in the model organism Streptomyces venezuelae are highly conserved throughout Streptomycetaceae. Having developed and screened a 2CS operon deletion library within S. venezuelae we determined that one of these, cutRS, is involved in co-ordinating exploration and antibiotic production. The cutRS deletion mutant displays unrepressed exploration and overproduction of chloramphenicol. With a greater understanding of how Streptomyces spp. identify and respond to external signals we can imitate and subvert these systems. Using this we aim to activate cryptic biosynthetic gene clusters enabling the discovery of novel antimicrobial products which may prove beneficial in the clinical setting.

Access Microbiol. 2019 Apr 8;1(1A):15.

Study on seroprevalence of IgG antibody of Varicella-Zoster virus in Pregnant mothers, Neonates&childrens of six month age

Selina Yasmin ^1,^*, Mosharof Hossain Chowdhury ¹

Abstract

This was a cross sectional and observational study, conducted in the Department of Microbiology, Sylhet MAG Osmani Medical College, Sylhet The study period was from July 2010 to June 2011 in objective to explore the Varicella-Zoster virus immune status (VZV-IgG) in pregnant mother and neonates up to six months post birth. For this purpose, 60 pregnant women, 60 new born babies and 60 infants aged six months were selected. The mean age of the pregnant woman was 28.8 (SD±4.7) years. The sex of new born babies and six months aged infants was identical [29 (48.3 %) male vs 31 (51.7 %) male; P=0.715]. Seroprevalence rate of IgG antibody of Varicella-Zoster Virus in Pregnant, mothers 81.7%, in new born babies 78.3 % and in infants aged six months were 10.0 %. The seropositivity of VZV IgG level in both pregnant mothers and newborn babies were almost similar but the infant aged six months were significantly lower than that of new born babies (p This showed that a significant proportion of Bangladeshi pregnant mother is susceptible to varicella and infant aged six months is highly susceptible to varicella. Any vaccination strategy must have to take into account these epidemiological variability of the country.

Access Microbiol. 2019 Apr 8;1(1A):15.

Redefining a new genomic blueprint of the human gut microbiota

Alexandre Almeida ^1,^2,^*, Alex Mitchell ¹, Miguel Boland ¹, Samuel Forster ^3,⁴, Gregory Gloor ⁵, Aleksandra Tarkowska ¹, Nicholas Thomson ², Trevor Lawley ², Robert Finn ¹

Abstract

The human gut microbiota composition is linked to both health and disease, but knowledge of individual microbial species is needed to decipher their biological role. Despite extensive culturing and sequencing efforts, the complete bacterial repertoire of the human gut microbiota remains undefined. Here we identify 1952 uncultured candidate bacterial species by reconstructing 92 143 metagenome-assembled genomes from 11 850 human gut microbiomes. These uncultured genomes substantially expand the known species repertoire of the collective human gut microbiota, with a 281 % increase in phylogenetic diversity. Whilst the newly identified species are less prevalent in well-studied populations compared to reference isolate genomes, they improve classification of understudied African and South American samples by over 200 %. These candidate species encode hundreds of novel biosynthetic gene clusters and possess a distinctive functional capacity that might explain their elusive nature. We also highlight newly identified species overrepresented in patients with gastrointestinal diseases, suggesting an underappreciated role in human health and disease. Our work uncovers the uncultured gut bacterial diversity, providing unprecedented resolution for taxonomic and functional characterization of the intestinal microbiota.

Access Microbiol. 2019 Apr 8;1(1A):15.

When the metabolic model says NO: untangling the Gordian knot of TB’s intracellular metabolism

Piyali Basu ¹, Noor Sandhu ¹, Apoorva Bhatt ², Albel Singh ², Ricardo Balhana ¹, Irene Gobe ¹, Nicola Crowhurst ¹, Tom Mendum ¹, Liang Gao ³, Jane Ward ³, Mike Beale ³, Johnjoe McFadden ¹, Dany Beste ^1,^*

Abstract

The causative agent of TB, Mycobacterium tuberculosis (Mtb) is once again the world’s number one infectious killer. M. tuberculosis resides primarily within macrophages and metabolic reprogramming within this intracellular niche is a crucial determinant of virulence. We previously applied the metabolic modelling-based tool 13C-flux spectral analysis (13C-FSA) to show that intracellular M. tuberculosis co-metabolises multiple gluconeogenic and glycolytic carbon substrates by utilizing the reactions of the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate (OAA) or anaplerotic (ANA) node. However, predicting the metabolic mode of operation required for intracellular survival is chellenging using a metabolic network as the ANA node consists of several apparently functionally redundant bidirectional reactions. Here we use multiple techniques including 13C isotopomer profiling, lipid analysis and fluorescent reporter strains to dissect the role of the ANA node. We show that this node has unexpected roles in the life cycle of M. tuberculosis including lipid biosynthesis, protection from known toxic intracellular carbon sources and redox regulation. Inhibiting enzymes at this node with novel therapeutic compounds restricts the growth of M. tuberculosis and limits the ability of this formidable pathogen to survive within the human host cell identifying the ANA node as a potential druggable pathway for controlling TB.

Access Microbiol. 2019 Apr 8;1(1A):15.

The chemical ecology of protective microbiomes in plant roots and leafcutter ants

Sarah Worsley ^1,^*, Matt Hutchings ¹, Colin Murrell ¹

Abstract

Actinobacteria are ubiquitous in soil and well-known for producing antimicrobial compounds. Increasingly, members of this phylum are found to form symbiotic relationships, for example with plants and insects, and are thought to provide protection against host infection. However, it remains poorly understood how Actinobacteria are recruited to microbiomes and whether secondary metabolites are produced in vivo. Acromyrmex echinatior leaf cutter ants transmit Pseudonocardia bacteria between generations and also recruit Streptomyces to their cuticular microbiome. We show that Pseudonocardia species isolated from the ant cuticle inhibit the fungal nest pathogen Escovopsisweberi and dual RNA-sequencing confirmed that Pseudonocardia secondary metabolite gene clusters are expressed in vivo on the ant cuticle. RNA stable isotope probing showed that ants supply cuticular resources to their microbiome which may fuel interference competition and select for antibiotic-producing bacteria. Similar to leaf cutter ants we also show that plant roots recruit growth-promoting and antibiotic-producing Streptomyces bacteria, but appear not to transmit them via their seeds. Root exudates are hypothesized to play a major role in root microbiome recruitment and DNA stable isotope probing coupled with Illumina sequencing showed that these were actively metabolized by many bacterial genera. However, Streptomyces appeared to be outcompeted by more abundant Proteobacteria, despite the fact that isolates could grow on purified exudates in the absence of competition. Streptomyces root exudate preferences are now being evaluated using comparative metabolomics. Defining the factors that influence the competitiveness of protective bacteria when colonizing microbiomes has implications for the development of more consistent biocontrol strategies and prebiotic techniques.

Access Microbiol. 2019 Apr 8;1(1A):15.

Rabies virus: defining antigenic requirements for pan-lyssavirus neutralisation

Rebecca Shipley ^1,^2,^*, David Selden ², Wu Guanghui ², Edward Wright ¹, Anthony R Fooks ^2,^3,⁴, Ashley C Banyard ²

Abstract

The lyssavirus genus is a diverse group of viruses all capable of causing an invariably fatal disease known as rabies, most commonly caused by the prototype species rabies virus (RABV). Alongside RABV the lyssavirus genus currently contains 15 other viruses capable of causing rabies. These viruses are broadly categorised into phylogroups according to the predicted level of vaccine protection, with protection from current vaccines and therapeutics afforded against phylogroup I but not II or III. Current evidence suggests that for a protective neutralising antibody response against RABV a neutralising antibody titre of 0.5 IU ml⁻¹ is sufficient. This arbitrary value has been developed and promoted as a serological cut-off based on the reactivity of defined sera with a standardised dose of RABV. Studies using cross protection assays, have suggested that for protection against more divergent members of the genus, even those in phylogroup I, 10-fold or greater than the 0.5 IU ml⁻¹ antibody titres are required. The continued discovery of novel lyssaviruses globally warrants an in-depth assessment of the protective titre required to protect against all the lyssaviruses to inform occupationally high-risk groups (e.g., scientists, bat workers and speleologists). Based on live virus neutralization assays, a minimum of 7 distinct lyssavirus glycoprotein antigens would have to be included in any pan-lyssavirus vaccine. Certainly, representative immunogens from all lyssavirus species characterized in phylogroups II and III are required to stimulate a pan-lyssavirus response.

Access Microbiol. 2019 Apr 8;1(1A):15.

iPSCs derived from endothelial progenitors model latency and reactivation during human cytomegalovirus infection

Emma Poole ^1,^*, Chris Huang ¹, Jessica Forbester ¹, Miri Shnayder ², Aharon Nachson ², Baraa Kweider ¹, Anna Basaj ¹, Daniel Smith ¹, Sarah Jackson ¹, Fedir Kiskin ¹, Kate Roche ³, Eain Murphy ³, Mark Wills ¹, Gordon Dougan ¹, Noam Stern-Ginossar ², Amer Rana ¹, John Sinclair ¹

Abstract

To date, models of human cytomegalovirus (HCMV) latency and reactivation have depended on the use of primary myeloid cells, which have limited availability, are difficult to culture and are challenging to genetically modify. We now show that induced pluripotent stem cells (iPSCs) derived from circulating late outgrowth endothelial progenitors (EPC) can be differentiated down the myeloid lineage, where HCMV latent carriage and reactivation is known to occur in vivo, and act as a model to allow the interrogation of viral and cellular factors involved in latency and reactivation of this persistent human pathogen. In contrast, monocytes generated from iPSCs derived from de-differentiated fibroblasts failed to support HCMV latent carriage. These iPSCs derived from EPCs may also be suitable for in depth genetic interrogation of other viruses which also infect cells of the myeloid lineage, such as HIV and Zika.

Access Microbiol. 2019 Apr 8;1(1A):15.

Hydrogenotrophic methanogenesis dominates at high pH

Richard Wormald ^1,^*, Paul Humphreys ¹

Abstract

One potential design for a geological disposal facility (GDF) for intermediate level radioactive waste (ILW) involves the use of a cement base grout which will establish a highly alkaline environment for extended time periods [1]. Methane generation by colonising microbes could impact the long-term performance of the facility by influencing gas pressures and potentially leading to the migration of 14C to the biosphere [1]. Sediments acquired from a wide-range of anthropogenic alkaline sites in the UK were used to develop acetoclastic and hydrogenotrophic methanogen enrichment cultures over a broad range of pH values (7.0–12.0). The generation of methane from hydrogen and acetate was assessed to determine the dominant methanogenic pathways. Archaeal community analysis via Illumina MiSeq was employed to describe the populations involved and the acetoclastic inhibitor methyl fluoride was utilised to confirm the lack of acetate-dependent methane generation under alkaline conditions. High pH (pH>9.0) microcosms employing alkaline sediments were dominated by hydrogen-consuming methanogens of the orders Methanobacteriales and Methanomicrobiales, with no acetate consumption detected under these conditions. In contrast, neutral pH microcosms employing control sediments were dominated by acetoclastic methanogens of the order Methanosarcinales and demonstrated high acetate consumption rates. The rate of acetate consumption and proportion of acetoclastic methanogens decreased in a linear fashion as the pH within cultures was increased, however hydrogen consumption rates remained stable up to pH 11.0. The data shown suggests hydrogenotrophic methanogenesis is the dominant methanogenic pathway at high pH which could have important consequences on gas pressures within a GDF.

Access Microbiol. 2019 Apr 8;1(1A):15.

Epidemiology of tick borne pathogens of dogs in Nigerian communities

Ternenge Thaddaeus Apaa ^1,^2,^*, Stephen Dunham ¹, Rachael Tarlinton ¹

Abstract

Tick borne diseases (TBDs) have significant impact on the health and welfare of domestic animals and humans. There is extremely little data on the prevalence of tick species, TBDs or their impact in Nigeria. Nigeria’s scenario is further worsened by lack of basic diagnostic facilities and treatment, compared with the average person’s income. The multiple climate zones and animal husbandry practices in Nigeria also make it difficult to extrapolate studies from one zone to the other five geopolitical zones. TBDs reported in Nigerian dogs include Anaplasma species. (A. platys A. omatnenne), Babesia species (B. rossi, B. canis and B. gibsoni), Theileria species. (T. equi, T. sable), Ehrlichia sp. (E. canis, E. ruminantum), Hepatozoon species (H. canis) and Candidatus Neoehrlichia mikurensis. The prevalence of zoonotic pathogens of dogs such as Borrelia sp., in humans in West Africa also indicates that these are likely to present in dogs in Nigeria. This main study aim is to identify ticks taken from dogs in Nigeria using morphological and molecular methods, determine which host species the ticks have fed on, and identify pathogens that they are carrying. It also aims to compare molecular and point of care diagnostics for TBDs in blood from Nigerian dogs. The overall objective is to provide robust data on which tick species and TBDs are present in Nigerian dogs and the potential zoonotic or epizootic risk.

Access Microbiol. 2019 Apr 8;1(1A):15.

The MtrAB-LpqB two component system links development with secondary metabolite production in Streptomyces species and can be manipulated to switch on silent secondary metabolite clusters

Nicolle F Som ^1,^*, Daniel Heine ², Neil A Holmes ¹, John T Munnoch ³, Felicity Knowles ⁴, Govind Chandra ⁵, Ryan F Seipke ⁶, Paul A Hoskisson ⁷, Barrie Wilkinson ⁵, Matthew I Hutchings ¹

Abstract

Streptomyces species are important producers of bioactive compounds such as antibiotics, antitumor and immunosuppressant drugs. Around two-thirds of all known natural antibiotics are produced by these bacteria and antibiotic production is linked to sporulation. The discovery of new bioactive compounds has declined since the 1960s but genome sequencing has revealed the potential to identify many more bioactive compounds. They are many secondary metabolite gene clusters which are inactive (silent) under laboratory conditions. We characterize the highly conserved actinobacterial two component system MtrAB which coordinates sporulation with secondary metabolite production in the two model organisms Streptomyces venezuelae and S. coelicolor. Deletion of the histidine kinase gene mtrB resulted in increased production of the antibiotic chloramphenicol in S. venezuelae and actinorhodin and undecylprodigiosin in S. coelicolor. Chloramphenicol is not usually produced under laboratory condition which suggests that deleting mtrB can activate silent antibiotic clusters. Additionally, we introduced point mutations at the D56 phosphorylation site of MtrA by CRISPR-Cas9 to abolish phosphorylation. This mutant shows the same phenotype as the ΔmtrA strain and chloramphenicol production is increased. Chromatin immunoprecipitation and sequencing (ChIP-seq) was used to identify MtrA targets and revealed that MtrA likely controls secondary metabolite production by binding to the promoter regions of cluster situated regulators in both model organisms. Additionally, MtrA binds upstream of genes involved in DNA replication and cell division. To our knowledge this is the first evidence of the connection of development and secondary metabolite production by a two component system.

Access Microbiol. 2019 Apr 8;1(1A):15.

Marine fungal dark matter in the global ocean

Nathan Chrismas ^1,^*, Michael Cunliffe ¹

Abstract

Marine fungi are a major part of ‘microbial dark matter’, with most organisms known from sequence data and currently not in culture. Interest in marine fungi has substantially increased over the past decade, and studies using culture independent methods have indicated that fungal diversity in the oceans may be greater than previously estimates based on cultivation alone. There remains much to learn about the true diversity of marine fungi in the global oceans and the ecological roles that they could play. The Tara Oceans expedition has allowed for significant advancements in our understanding of the global diversity of planktonic microorganisms. Interrogation of the Tara Oceans 18S rRNA gene dataset for fungal sequences shows that fungi are found throughout the global oceans, appearing in all marine regions covered by the Tara Oceans expedition. In the survey, Ascomycota and Basidiomycota were common, while some locations had increased abundances of Chytridiomycota. Differences in community composition were observed between oceanic regions and, although clear signals were not apparent due to the nature of the sampling, there was some indication of community variation between upwelling, coastal, shelf and gyral provinces. Different size sampling fractions appeared to capture different portions of the pelagic fungal community. These findings highlight a number of ecological questions: How important are oceanic currents in determining fungal biogeography? What is the relationship between marine fungi and biogeochemical processes? What is actually there? By targeting these questions directly, we will be able to bring the dark matter of marine fungi into the light.

Access Microbiol. 2019 Apr 8;1(1A):15.

The Sodalis system and flux balance analysis as a tool for investigating insect-microbe interactions and the evolution of symbioses

Rebecca Hall ^1,^*, Lindsey Flanagan ¹, Michael Bottery ¹, Vicki Springthorpe ¹, Stephen Thorpe ¹, Alistair Darby ², Jamie Wood ¹, Gavin Thomas ¹

Abstract

The development of new microbial growth and analytical techniques is becoming increasingly relevant in relation to ‘unculturable’ organisms. This may involve the modification of existing methods or the development of new, custom procedures. One important application of this is in the symbiotic bacteria of insects. Symbionts, due to adaptations to their host, are often difficult to culture in vitro. With the growing interest in the use of modified microbiomes to control vector-borne diseases, improved culture techniques that further the understanding of an insect’s microbiome are becoming increasingly important. The tsetse fly, genus Glossina, is the insect vector for Trypanosoma brucei. This parasite is responsible for human African trypanosomiasis (HAT), endemic in sub-Saharan Africa, as well as the wasting disease nagana in cattle. The tsetse’s secondary symbiont, Sodalis glossinidius, provides a unique potential target for reducing the spread of T. brucei. Here, we describe the use of metabolic modelling to design an entirely defined growth medium for S. glossinidius. This medium was used to verify predictions about carbon and nitrogen usage in the symbiont, including amino acid and vitamin auxotrophies. Furthermore, we discuss the use of multiobjective evolutionary algorithms combined with flux balance analysis to investigate computationally the evolution of symbioses, with S. glossinidius and its free-living relative S. praecaptivus as an exemplar. This work not only improves our understanding of the metabolic interactions within the tsetse microbiome, but serves also as a template for future investigations into symbiont evolution.

Access Microbiol. 2019 Apr 8;1(1A):15.

Understanding aurodox: A Type III secretion system inhibitor from Streptomyces goldiniensis

Rebecca Elizabeth McHugh ^1,^2,^*

Abstract

Aurodox, a specialised metabolite from the soil bacterium Streptomyces goldiniensis, has been shown to inhibit the Enteropathogenic Escherichia coli (EPEC) Type III Secretion System (T3SS). To further assess the utility of this molecule as an anti-virulence compound, a better understanding of its mechanism of action is required. We used whole transcriptome analysis, cell infection and GFP-reporter assays to show that Aurodox transcriptionally downregulates the expression of the Locus of Enterocyte Effacement (LEE) pathogenicity island-which encodes for the T3SS, acting via its master regulator, Ler. We have also observed similar effects across other enteric pathogens carrying a homologous T3SS such as Enterohemorrhagic Escherichia coli (EHEC). These properties suggest Aurodox may have potential for the treatment of E. coli infections of the gut. Despite the recent interest in the compound, the biosynthesis of Aurodox by Streptomyces goldiniensis is still poorly understood. To gain insight in to this, we have sequenced the whole genome of S. goldiniensis and identified a putative Aurodox biosynthetic gene cluster (BGC) which shares a high level of functional homology with the BGC encoding Kirromycin, a non-methylated Aurodox derivative. In-depth analysis of the BGC supports a model where a unique polyketide synthase pathway involving a combination of both Cis and Trans-Acyltransferases synthesise the Aurodox polyketide backbone, followed by decoration and finally the addition of a methyl group. Future work will include the heterologous expression this BGC to confirm its role in Aurodox biosynthesis, with the ultimate aim to produce novel Aurodox derivatives.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating the role of nitric oxide in plant root colonisation by Streptomyces spp.

Jake Newitt ^1,^*, John Munnoch ², Sarah Worsley ¹, Matt Hutchings ¹

Abstract

Streptomyces is a genus of soil dwelling actinomycetes that play an important role in plant health through association with plant roots. They provide an array of benefits to the plant host such as infectious disease prevention and plant growth promotion. We investigate the role of nitric oxide (NO), a ubiquitous signalling molecule used by plants and bacteria alike, in root colonisation by Streptomyces coelicolor. Plating studies were conducted for Arabidopsis thaliana and Triticum aestivum. Relative colonisation was determined by comparing selective recovery of marked mutant strains, alongside a marked control. The effect of increased endogenous NO was interrogated with a deletion mutant of nsrR-hmpA – genes responsible for NO detoxification in Streptomyces. Strains were also engineered to express recombinant NO synthase genes, to investigate the impact of NO production by the bacteria. We show that S. coelicolor ΔnsrR-hmpA is significantly more competent at colonising T. aestivum rhizosphere compared to the control (P<0.005). Endosphere colonisation is sporadic for both mutant and control, this observation is supported by fCLSM imaging. Preliminary data indicates that the Streptomyces strains engineered to express NO synthase at high levels, colonise poorly. This suggests that NO is a dynamic and finely tuned signalling molecule. We are excited to present promising new evidence to support an as yet undescribed link between NO and plant root colonisation by Streptomyces coelicolor. Understanding the mechanisms that underpin this process is the first step in exploiting these interactions for agricultural technology.

Access Microbiol. 2019 Apr 8;1(1A):15.

Surfactants from the sea: rhamnolipid production by marine bacteria

Matthew Twigg ^1,^*, Lakshmi Tripathi ¹, Katerina Zompra ², Karina Salek ³, Victor Irorere ¹, Tony Gutierrez ³, Georgios Spyroulias ², Roger Marchant ¹, Ibrahim Banat ¹

Abstract

Biosurfactants produced from microbial sources are increasingly viewed by industry as more sustainable and less toxic alternatives to their chemically derived counterparts. One major class of biosurfactant that has the potential for commercial exploitation are the rhamnolipids. Rhamnolipids are composed of one or two rhamnose monosaccharides covalently bonded to fatty acid chains of varying molecular weights. The major microbial producer of rhamnolipid is Pseudomonas aeruginosa, however as this is a known human pathogen many industries are reluctant to utilise rhamnolipids synthesised by this bacterium. In order to avoid this problem a consortium of both academic and industrial partners have been screening marine bacteria for their ability to synthesis biosurfactants in a project called MARISURF. Here we report our findings of rhamnolipid production by two marine bacterial strains. Rhamnolipid production by these strains was first identified via the phenotypic screening of surface tension reduction. Rhamnolipid synthesis was then confirmed and characterised via HPLC-MS and NMR. Both 16S rDNA and subsequent genomic sequencing revealed these strains to be Marinobacter sp. and Pseudomonas mendocina, both species where rhamnolipid production was previously un-reported. Finally, both strains were assessed for potential pathogenicity using the Galleria mellonella model. Importantly for commercial exploitation, neither strain was shown to be harmful to G. mellonella over a 72 h infection period. Confirmed identification of rhamnolipid production in bacterial strains isolated from the marine environment highlights global oceans as an untapped resource in the ocean for the discovery of novel sources of biosurfactants.

Access Microbiol. 2019 Apr 8;1(1A):15.

Discovering novel antimicrobials from Streptomyces formicae, a symbiont of fungus farming plant ants, using CRISPR/Cas9 genome editing

Rebecca Devine ^1,^*, Zhiwei Qin ², Barrie Wilkinson ², Matt Hutchings ¹

Abstract

Finding new antimicrobial compounds is vital to combat the growing threat of resistance. Most currently used antibiotics originate from actinomycetes discovered more than half a century ago. We recently reported the new species Streptomyces formicae, isolated from the African fungus-farming plant-ant, Tetraponera penzigi. S. formicae produces a novel family of polyketide antibiotics, the formicamycins, that have potent activity against resistant pathogens including MRSA and vancomycin-resistant Enterococci (VRE). Using CRISPR/Cas9, we have identified and characterised the genes responsible for formicamycin biosynthesis in the native producer. In addition, we used cappable RNA- and ChIP-sequencing to determine the transcriptional organisation of the pathway. We exploited this information to generate multiple mutants of S. formicae that overproduce formicamycins and their biosynthetic intermediates, some of which also have bioactivity. Furthermore, the potential for novel chemistry from S. formicae is not limited to the formicamycin pathway; antiSMASH analysis shows this talented strain contains at least 45 secondary metabolite biosynthetic gene clusters (BGCs). Under standard laboratory conditions, wild-type S. formicae also exhibits antifungal activity against the drug resistant Lamentospora prolificans, and when the formicamycin BGC is deleted, the strain produces even more potent antibacterial activity against MRSA. To identify the biosynthetic pathways for these metabolites, entire BGCs up to 208 kbp were deleted using CRISPR. Overall, this work demonstrates that searching under-explored environments for new species combined with genome editing is a promising route towards finding novel anti-invectives.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of novel host factors influencing human cytomegalovirus replication using a two-step siRNA screen

Chen-Hsuin Lee ^1,^*, Samantha Griffiths ², Paul Digard ¹, Nhan Pham ², Manfred Auer ², Juergen Haas ², Finn Grey ¹

Abstract

As an obligate intracellular parasite, human cytomegalovirus (HCMV) completely relies on host machinery to replicate. Understanding which host factors are required for virus replication contributes to our understanding of virus biology and cell biology, identification of potential targets for antiviral therapy. High-throughput small interfering RNA (siRNA) screens are a powerful approach to identify novel host-virus interactions. Conventional screens often use reporter genes as a proxy for virus replication, rather than measuring production of infectious virus. We developed a two-step siRNA screen that independently measured primary replication and virus production. Screening with a library targeting almost 7000 genes, we identified 37 genes involved in early stages of HCMV replication and 15 genes specifically involved in later aspects, such as late gene expression, assembly and egress. These include factors in ubiquitin-dependent protein degradation pathway, and components of the mediator complex. Furthermore, we showed that the induction of SIN3A, a transcriptional regulator that forms a repressor complex with histone deacetylase 1 and 2, is essential for late gene expression and virus production. This study demonstrates a powerful two-step high throughput approach which identifies key host factors underpinning HCMV replication and informs our understanding of how the virus interacts with its host.

Access Microbiol. 2019 Apr 8;1(1A):15.

In-depth profiling of calcite precipitation by environmental bacteria reveals fundamental mechanistic differences with relevance to application

Bianca Reeksting ^1,^*, Kevin Paine ¹, Susanne Gebhard ¹

Abstract

Microbially-induced calcite precipitation (MICP) is ubiquitous in nature and has become an area of interest for environmental, geotechnical, and civil engineering applications. These include bioremediation, soil engineering, and self-healing of cementitious materials. To date, ureolytic bacteria have been favoured due to their ability to rapidly increase the pH of the environment through the hydrolysis of urea and thereby induce precipitation of calcite. However, the requirement for urea can contribute to nitrogen-loading in the environment and prove to be incompatible in certain applications, such as in self-healing concrete where it delays setting. Non-ureolytic bacteria are thought to be less efficient at MICP as they lack the ability to hydrolyze urea and thus to induce rapid increases in pH. Profiling of environmental bacteria has revealed the fundamentally different mechanisms that ureolytic and non-ureolytic bacteria utilize to precipitate calcite. These affect the timing of MICP and morphology of the crystals, but not necessarily the overall quantity of calcite precipitated. Furthermore, we show that MICP facilitated by non-ureolytic bacteria results in precipitates that contain significant organic components. These precipitates appear to have increased volume and cohesiveness, which may prove advantageous in application. Our findings offer important new insights into the use of MICP for geotechnical and environmental engineering and will enable us to create a toolbox of microbial precipitators tailored for specific applications.

Access Microbiol. 2019 Apr 8;1(1A):15.

Illuminating the molecular choreography of multi-segmented RNA genomes

Alex Borodavka ^1,^2,^3,^*, Sebastian Strauss ³, Aaztli Coria ⁴, Eric Dykeman ⁵, Ralf Jungmann ³, Don Lamb ²

Abstract

Genome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently we have identified sequence-specific inter-segment interactions between rotavirus (+)ssRNA genome segment precursors. We have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. These protein-RNA interactions result in the stabilisation of extended intermolecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation and replication. Using this approach, we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which are likely to be involved in genome segment assortment process. Having established the role of NSP2 in promoting inter-segment RNA-RNA contacts, we have developed multiplexed imaging tools for directvisualization of the RNA assortment process in rotavirus-infected cells by employing single-molecule RNA FISH. To unravel the mechanisms, by which NSP2 controls the formation of inter-molecular RNA helices, we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for theformation of the RNA assortment complex. Our findings open up unique avenues for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.

Access Microbiol. 2019 Apr 8;1(1A):15.

The effect of antibiotic and nutrient limitation to antibiotic resistant bacteria in single-cell level

Li Liu ^1,^*, Jan Kreft ², Daniele Vigolo ²

Abstract

Heterogeneity in bacterial populations can manifest in various ways, such as resistant cells, which can be observed in harsh environments after the use of antibiotics. Many studies have looked at the evolution of resistance and the effect of inhibitory and sub-inhibitory concentrations of antibiotics by batch culture measurements without considering the heterogeneity of bacterial populations. But antibiotic susceptibility and fitness costs of resistance mutations or plasmids are affected by the growth rate and physiology of individual cells. Single-cell analysis in microfluidic systems has opened up new possibilities enabling us to investigate the various putative mechanisms behind the persistence phenomenon required direct observation under the microscope. In this study, we use a gradient mixer and a novel micro-chemostat, to create concentration gradients of growth substrates and/or antibiotics to study the effect of nutrient and antibiotic concentration on individual cells growing under constant and defined conditions in cell-sized channels. The single-cell elongation, morphology and growth rate of ribosome-targeting antibiotics resistant E. coli was tracked by combining the microfluidics, microscope phase contrast imaging and fluorescent tag in high throughput mode. A mechanistic cellular model was used to describe the reaction between antibiotics and ribosome and the resulting effects on bacterial growth; then we linked the intracellular chemical-reaction kinetics processes to the population level and predicted the behaviour of population responses. Our approach has enabled the investigation of single-cell individuality and predictions of population dynamics under different environment.

Access Microbiol. 2019 Apr 8;1(1A):15.

Optimisation of an in vitro cell system using pseudoviruses to investigate HBV entry mechanisms

Chun Goddard ^1,^*, William Irving ¹, Alexander Tarr ¹

Abstract

Aim

To explore the roles of large (L), middle (M) and small (S) surface antigens in an in vitro model of hepatitis B virus (HBV) entry, and thereby to achieve an optimal in vitro cell system using pseudoviruses to investigate HBV entry mechanisms.

Methods

RNA encoding sodium taurocholate co-transporting polypeptide (NTCP) was extracted from human hepatocytes and cloned into the pHIV-EGFP expression vector. The resulting pHIV-NTCP-EGFP construct was delivered into Huh7 hepatoma cells with the aid of pCMVR87.4 (packaging vector) and pCMV.VSV.G (glycoprotein), subsequently, a cell line over-expressing NTCP was generated. Meanwhile, by silencing start codons at L, M or S, seven constructs were obtained, i.e. L + M -S-, L-M + S -, l -M-S+, L + M + S-, L-M + S +, L + M -S+, l -M-S-. A matrix with various amounts of the seven constructs was used to generate HBV pseudoparticles (HBVpp) using a Luciferase-based HIV (pNL4.3.luc.R-E-) pseudotype entry model system. The infectivity of the HBVpp was tested in NTCP naïve and NTCP over-expressing Huh7 hepatoma cells.

Results

The relative amounts of L, M and S were critical in determining the efficiency of entry of HBVpp into NTCP + ve Huh7 cells.

Conclusions

Creation of NTCP-over expressing cells together with optimisation of conditions to maximise HBVpp entry provides an important tool to investigate the entry step in the HBV life cycle, and may allow identification of non-NTCP-dependent viral entry pathways.

Access Microbiol. 2019 Apr 8;1(1A):15.

HIV-1 Vpr accessory protein interacts with REAF and mitigates its associated anti-viral activity

Joseph Gibbons ¹, Kelly Marno ¹, Rebecca Pike ¹, Jason Lee ¹, Christopher Jones ¹, William Ogunkolade ¹, Claire Pardieu ¹, Gary Warnes ¹, Paul Rowley ², Richard Sloan ¹, Aine McKnight ^1,^*

Abstract

The accessory protein Vpr of Human Immunodeficiency Virus type 1 (HIV-1) enhances replication of the virus in macrophages. Virus particle packaged Vpr is released in target cells shortly after entry, suggesting it is required early in infection. Why it is required for infection of macrophages and not cycling T-cells and why it induces G2/M arrest in cycling cells are unknown. Here we observe, by co-immunoprecipitation assay, an interaction between Vpr and endogenous REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a protein shown previously to potently restrict HIV infection. After HIV-1 infects macrophages, within 30 min of viral entry, Vpr induces the degradation of REAF. Subsequently, as replication continues, REAF expression is upregulated – a response which is curtailed by Vpr. REAF is more highly expressed in differentiated macrophages than in cycling T-cells. Expression in cycling cells is cell-cycle dependent and knockdown induces cell-cycle perturbation. Therefore, our results support the long held hypothesis that Vpr induces the degradation of a factor involved in the cell cycle that impedes HIV infection in macrophages.

Access Microbiol. 2019 Apr 8;1(1A):15.

Signal-integration through PhoPQ enables Salmonella to adapt to heterogeneous tissue microenvironments

Francesca Romana Cianfanelli ^1,^*, Dirk Bumann ¹

Abstract

Enteric fever is major health issue in developing countries and it is becoming progressively untreatable due to increase of antimicrobial resistance. The causative agent, Salmonella enterica, replicates in host phagocytes in various organs and regulates expression of hundreds of genes in response to host signals. PhoPQ is one of the key regulators and essential for virulence in humans and in a mouse typhoid fever model. The sensor kinase PhoQ responds to diverse stimuli (Mg2+ limitation, low pH, cationic antimicrobial peptides, high osmolarity, and, indirectly, to reducing conditions). However, which signals are predominant in vivo remains unclear. To address this issue, we determined the activity of the PhoPQ regulon using a chromosomally encoded fusion of the PhoP-dependent phoNp promoter to gfp-ova and we quantified single-cell fluorescence levels of Salmonella in spleen of infected mice using flow cytometry. The results show extensive heterogeneity in PhoP-activities in the Salmonella population. Comparison of mutants with sensing defects suggests that, in vivo, PhoQ responds to a combination of antimicrobial peptides, acidic pH and low Mg2+, but not to reducing conditions. Negative feedbacks have also only a minor impact. Single-cell analysis of phoNp and asrp promoters suggests differential environmental pH as a major driver of heterogeneous PhoP activities. A combination of immunohistochemistry and proteomics of Salmonella from infected samples was used to validate this hypothesis. Together, our data show how Salmonella uses the PhoPQ system to integrate various host signals in order to tune expression of virulence factors to the diverse tissue microenvironments that this pathogen inhabits.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of viral transcripts in RNA-seq datasets from bees, ants, wasps and mites

Katherine Brown ^1,^*, Ingrida Olendraite ¹, Andrew Firth ¹

Abstract

Many honey bee colonies suffer large losses due to colony collapse disorder. This phenomenon, which has dramatically increased in frequency since 2006, has led to widespread efforts in sequencing honey bee pathogens, including RNA viruses such as deformed wing virus. However, honey bees coexist with a number of other arthropods, whose viruses are less thoroughly characterised. Many viruses currently classified as honey bee pathogens may therefore have a much wider host range. In particular, ants, which like bees are members of the Hymenoptera order, often coexist with bees and the two groups have previously been shown to exchange viruses. Parasitism by Varroamites, known to act as effective vectors for a number of RNA viruses, is also almost ubiquitous amongst honey bees, but little is known about viruses endemic to mites. We have previously demonstrated that it is possible to detect and characterise viral RNA in publicly available RNA-seq datasets. There are over 3000 such datasets for diverse Hymenoptera and mite species. We have developed a computational pipeline to identify viral transcripts in these datasets. This pipeline performs quality control, removes low complexity reads and reads generated from host RNA and various known contaminants, assembles the remaining reads into transcripts and detects the presence of regions with homology to known RNA viruses. Viral fragments identified with this pipeline will be examined phylogenetically to identify novel pathogens, clarify host range and specificity, and characterise transmission patterns.

Access Microbiol. 2019 Apr 8;1(1A):15.

Of mice or men? Developing an ex vivo model of Staphylococcus aureus infection in the cystic fibrosis lung

Esther Sweeney ^1,^*, Freya Harrison ¹, Katerina Guzman ², M Angeles Tormo-Mas ²

Abstract

Staphylococcus aureus is one of the dominant organisms isolated from the airways of cystic fibrosis (CF) patients, particularly early in life, and is usually regarded as pathogenic. However, there remains significant gaps in our understanding of the role of S. aureus in the progression of pulmonary infection and lung disease in CF. Mouse models of S. aureus lung infection, even in CF animals, frequently demonstrate pneumonia and abscesses of the lung, a phenomenon very rarely observed in people with CF. Furthermore, live host models are associated with high costs and are limited in duration and sample size for ethical reasons. Most in vitro models fail to consider the influence of host tissue interaction or spatial structure on the development and persistence of infection. We have previously described an ex vivo pig lung model (EVPL) of cystic fibrosis for Pseudomonas aeruginosa lung infection. Here we show the progression of this model to support the growth of Staphylococcus aureus. Our data suggests that, in our model, S. aureus cells may preferentially aggregate in artificial sputum rather than adhere to lung tissue. In the context of historical case reports, this result potentially reflects the clinical situation in cystic fibrosis more accurately than mouse models and could have substantial clinical significance.

Access Microbiol. 2019 Apr 8;1(1A):15.

Inflammation associated ethanolamine facilitates infection by Crohn’s disease-linked adherent-invasive Escherichia coli

Michael Ormsby ^1,^*, Michael Logan ², Síle Johnson ¹, Anne McIntosh ¹, Richard Hanson ³, Umar Ijaz ², Richard Russell ³, Konstantinos Gerasimidis ⁴, Donal Wall ¹

Abstract

The predominance of specific bacteria within the Crohn’s disease intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. Here we show that the intestinal short chain fatty acid propionic acid stimulates increased ethanolamine degradation by one such Crohn’s disease associated pathogen, adherent-invasive Escherichia coli (AIEC). This degradation occurs within bacterial microcompartments that are subsequently excreted in outer membrane vesicles. Additionally ethanolamine, added extracellularly at concentrations comparable to those in the human intestine, is accessible to intracellular AIEC and stimulates significant increases in growth within macrophages. Finally, expression of the operon for ethanolamine degradation (eut) is increased in children with active Crohn’s disease compared to healthy controls. After clinical remission was seen with exclusive enteral nutrition treatment, Crohn’s disease patient’s exhibit significantly reduced eutexpression. Our data indicates a role for ethanolamine metabolism in facilitating AIEC colonization of the Crohn’s disease intestine and warrants further study of its potential use as an indicator of inflammatory status in Crohn’s disease.

Access Microbiol. 2019 Apr 8;1(1A):15.

Co-selection of antibiotic resistance caused by a legacy of PTE pollution in Gram-negative bacteria

Rebecca Tonner ^1,^*, Tanya Peshkur ¹, Kiri Rodgers ², Ian MacLellan ², Roderick Williams ², Andrew Hursthouse ², Fiona Henriquez ², Charles Knapp ¹

Abstract

Antimicrobial resistant bacteria can become harboured in sediments of post-industrial estuaries. Subsequently, their resistance traits could be enriched by pollutants deposited in the sediments. Recent evidence strongly suggests this may pose hazards that not only affects the health care sector, but could also impact tourism and the aquaculture industries. The River Clyde, UK was chosen for this study due to its extensive industrial history, and three sites were chosen to sample from representing different levels and types of industrial activities—two highly polluted and one relatively ‘pristine’ site. We extracted and analysed for metal pollutants (or ‘potentially toxic elements’, PTE), and other geochemical characteristics for all sediment cores. Gram-negative, enteric bacteria were isolated from all sediment cores from the three sites. Their susceptibilities to antibiotics and metals were assayed—determining minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The results indicate that co-selection of PTEs and antibiotic resistance does occur, and this impacts bacteria that are potential human pathogens. Higher concentrations of metals in the environment correlated to antibiotic resistance and higher MICs to metals than among bacteria found in less polluted sites. To continue to protect human health, the interactions between environmental and human health must be fully understood. This study provides critical information behind the specific causes of antibiotic resistance due to a legacy of pollution.

Access Microbiol. 2019 Apr 8;1(1A):15.

Use of nanosensor technology to investigate biofilm formation and resulting malodour in washing machines

Birte Blunk ^1,^2,^*, Mark Perkins ^1,², Dean Walsh ^1,², Veeren Chauhan ², Miguel Camara ¹, Paul Williams ¹, Jonathan Aylott ², Kim Hardie ¹

Abstract

Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infection for animals and plants. Our investigation is focussed on a current and growing concern: the distribution and formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures around 30 to 40 °C degrees rather than at higher temperatures above 60 °C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosainto the environment. P. aeruginosais one of the predominant bacteria found in washing machines and is highly resistant to many antibiotics. Little is known about environmental microniches present in biofilms. In this work, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilm formation. Here, we use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to variation in pH. Confocal laser scanning microscopy revealed that the nanosensors can penetrate biofilms within minutes and interact with the biofilm structure. Different washing detergents were tested resulting in altered biofilm formation and killing abilities. Using time lapse imaging, pH changes were tracked in real time at a microcolony and single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms, underpinning the development of more effective antimicrobials to limit the emergence of AMR.

Access Microbiol. 2019 Apr 8;1(1A):15.

Assembly of a portal-like structure in feline calicivirus following receptor engagement

Michaela Conley ^1,^*, Marion McElwee ¹, Ian Goodfellow ², David Bhella ¹

Abstract

The mechanism by which non-enveloped RNA viruses, such as the caliciviruses, escape the endosome, is poorly understood. The Caliciviridae are a family of viruses which include many important human and animal pathogens, most notably norovirus which causes winter vomiting disease. We used cryoEM and asymmetric three-dimensional reconstruction to investigate structural changes in the capsid of feline calicivirus (FCV) that occur upon virus binding to its cellular receptor; feline junctional adhesion molecule-A (fJAM-A). We discovered that following receptor engagement substantial conformational changes in the FCV capsid lead to the assembly of a portal-like structure at a unique three-fold symmetry axis. Atomic models of the major capsid protein, VP1, in the presence and absence of fJAM-A were calculated, revealing the conformational changes induced by the interaction. In the course of this analysis we discovered a large portal-like structure which assembles at a unique three-fold axis. The portal-like complex comprises 12 copies of the minor capsid protein VP2. We calculated an atomic model of VP2 and revealed structural changes in VP1 that lead to the formation of a pore in the capsid shell at the portal vertex. VP2 is encoded by all caliciviruses although despite being critical for the production of infectious virus, its function and structure were, until now, undetermined. We hypothesise that the VP2 portal-like complex is the method by which the virus escapes the endosome during virus entry, allowing delivery of the viral genome into the cytoplasm for replication to then ensue.

Access Microbiol. 2019 Apr 8;1(1A):15.

BactiVac, a network to support the study, development and implementation of bacterial vaccines

Adam Cunningham ¹, Johanna Dean ¹, Susan Pope ¹, Evelina Balandyte ^1,^*, Calman A MacLennan ^2,³

Abstract

Infections account for >20 % of all deaths worldwide, and are particularly problematic in low-middle income countries (LMICs), with bacterial infections killing approximately 5 million people annually. The crisis of antimicrobial resistance means our options for controlling infections are narrowing. Vaccines are a cost-effective approach to prevent infectious disease. However, there are many bacterial infections against which we lack any licensed vaccine. The BactiVac Network (birmingham.ac.uk/bactivac), was launched in August 2017 and is led by Profs Cal MacLennan and Adam Cunningham. BactiVac is a global bacterial vaccinology network established to accelerate the development of vaccines against bacterial infections, particularly those relevant to LMICs. BactiVac brings together academic, industrial and other partners involved in vaccine research against human and animal bacterial infections from the UK and LMICs. The Network fosters partnership and provides catalyst pump-priming project and training funding to encourage cross-collaboration across our membership. Full details are available on our website. BactiVac has >600 members across >60 countries with 39 % based in LMICs and 10 % in industry. Membership is free – join us at bit.ly/apply BactiVac Benefits of membership include: Access to catalyst funding – third round for pump-priming projects closes 5 May 2019, open call for training awards Invitation to subsidised Annual Network Meetings–next meeting will be held in March 2020 Access to Members’ Directory – to develop collaborations and access distinct expertise. We encourage you to become a member, be involved in this initiative, and to be part of its growing success now and in the future.

Access Microbiol. 2019 Apr 8;1(1A):15.

Bactericidal fully-human monoclonal antibodies can be cloned from patients convalescing from invasive meningococcal disease

Fadil Bidmos ^1,^*, Simon Nadel ¹, Gavin Screaton ², Simon Kroll ¹, Paul Langford ¹

Abstract

Reverse vaccinology 2.0 (RV 2.0), in which the cloning and recombinant expression of antigen-specific antibodies is followed by determination of their functional activity, is a valuable approach that can unravel novel vaccine antigens, or reinforce the vaccine candidacy of already known antigens. Invasive meningococcal disease (IMD) remains a serious source of concern even with the availability of vaccines. Incomplete strain coverage is a limitation of current vaccines, hence efforts to identify candidate antigens that will compose supplementary or replacement vaccines are necessitated. In this proof-of-principle study, we sought to assess the applicability of RV 2.0 to anti-meningococcal vaccine antigen discovery. Antibody-secreting cells (ASCs) obtained from a patient convalescing from serogroup B (MenB) IMD, were isolated and sorted singly using FACS. The specificity and functionality of each antibody produced by individual ASCs were assessed in ELISA and bactericidal assays, respectively. Eight cross-reactive anti-meningococcal antibodies were successfully cloned; three of these mediated complement-dependent killing of antigenically-heterologous MenB strains. Western blot data shows binding of these three bactericidal antibodies to a ∼35 kDa antigen. None of the three bactericidal antibodies were reactive with a target in current vaccine formulations strongly suggesting that the ∼35 kDa antigen does not compose available vaccines. Unequivocal determination of the identity of the ∼35 kDa antigen is ongoing. Given the need for antigens that would compose improved or novel anti-meningococcal vaccines, this study shows that the RV 2.0 approach has the potential to be a powerful tool in the identification of functionally-immunogenic anti-meningococcal antigens.

Access Microbiol. 2019 Apr 8;1(1A):15.

The application of CRISPR/Cas9 system in the generation of viral vectored avian influenza vaccines

Pengxiang Chang ^1,^*, Jean-Remy Sadeyen ¹, Joshua Sealy ¹, Sushant Bhat ¹, Munir Iqbal ¹

Abstract

Avian influenza is highly contagious poultry disease and the mortality can reach 100 %, leading to huge economic burden and threat to food security. Vaccination is the most effective strategy for prevention and control of influenza. Recombinant vector vaccines are effective promising vaccines capable of immunizing against multiple pathogens. Traditional methods for constructing recombinant vaccines involve homologous recombination or bacterial artificial chromosomes but these methods can be time-consuming and labour-intensive. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a recently developed gene editing technology which has proven beneficial to gene modification and offers an alternative for constructing recombinant vaccines. There are mainly two methods used for gene insertion; error-prone non-homologous end joining (NHEJ) and the high-fidelity homology-directed repair (HDR) pathway. Owing to its high fidelity, most studies focus on using HDR for vaccine development but NHEJ offers some attractive advantages through its high efficiency. In our study, both HDR and NHEJ dependent CRISPR/Cas9 systems were explored for the rapid generation of recombinant influenza vaccines using duck enteritis virus and herpesvirus of turkeys as vectors.

Access Microbiol. 2019 Apr 8;1(1A):15.

A novel enterovirus protein modulates infection in gut epithelial cells

Valeria Lulla ^1,^*, Adam Dinan ¹, Myra Hosmillo ¹, Yasmin Chaudhry ¹, Lee Sherry ², Nerea Irigoyen ¹, Komal Nayak ¹, Nicola Stonehouse ², Matthias Zilbauer ¹, Ian Goodfellow ¹, Andrew Firth ¹

Abstract

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all the enteroviral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF). We report that many enterovirus genomes also harbor an upstream ORF (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed expression of uORF protein (UP) in infected cells. Using ribosome profiling (a technique for global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, UP-knockout echoviruses are attenuated compared to wild-type virus at late stages of infection where membrane-associated UP facilitates virus release. Thus we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells – the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy, and have important implications for understanding enterovirus pathogenesis.

Access Microbiol. 2019 Apr 8;1(1A):15.

Species-specific restriction of Bluetongue virus replication correlates to host resilience

Alexandra Hardy ^1,^*, Meredith Stewart ¹, Andrew Shaw ¹, Mariana Varela ¹, Sam Wilson ¹, Richard Randall ², Massimo Palmarini ¹

Abstract

Bluetongue is a vector-borne disease of ruminants caused by bluetongue virus (BTV). BTV can infect essentially all domestic and wild ruminants but the clinical outcome of infection differs substantially between host species. Clinical disease induced by BTV, including haemorrhagic fever in severe cases, is normally evident only in sheep. Conversely, cattle are more resilient to BTV infection, as they develop high levels of viremia and can be reservoirs of infection, but rarely show clinical signs. Here, we concentrated on BTV-host cell interactions using primary cells as an experimental system. First, we determined that BTV reaches higher titres in ovine cells, compared to bovine cells although it induces comparable levels of antiviral cytokines in both cell types. Importantly, these differences are abolished by inhibiting the Jak/Stat pathway. In addition, pre-treatment with interferon (IFN) severely hampers BTV replication in bovine, but not in ovine, primary cells. These data suggest that bovine, unlike ovine, IFN-stimulated genes (ISGs) are effective in controlling BTV replication. Using a high-throughput flow cytometry approach, we screened an expression library of over 300 bovine ISGs to identify genes with antiviral properties against BTV. We have identified ∼10 bovine ISGs that negatively impact BTV replication (by at least 50%). Currently, we are assessing the sheep orthologues to the bovine ISGs of interest in order to investigate host-species differences. Our study provides novel insights on how bovine cells restrict BTV replication and could provide an intellectual framework to understand the host determinants involved in disease severity.

Access Microbiol. 2019 Apr 8;1(1A):15.

Understanding the population dynamics of organisms exposed to the predatory activity of myxobacteria

Natashia Sydney ^1,^*, David Whitworth ¹

Abstract

Predatory myxobacteria have an antimicrobial nature that dominates their interactions with neighbouring organisms. They are abundantly found in soil, water, dung of herbivores, and have the potential to significantly affect the microbiome of an environment. In this project, we hypothesize that potential prey organisms evolve in response to the selective pressure exerted by predatory microbes. Using a variety of nutrient media, we isolated bacteria from soil samples to test their susceptibility and resistance to the laboratory strain Myxococcus xanthus DK1622 and its predatory secreted outer membrane vesicles (OMVs). Soil (with and without heat treatment) was spread onto plates which had been pre-inoculated with myxobacteria, OMVs, or no pre-treatment. Plates with myxobacteria lawns or OMVs exhibited reduced diversity of isolates compared to control plates. The yield and diversity of isolates obtained also depended on the media used. Heat pre-treated soil gave rise to distinct morphologies and fewer slime producers. Co-existence and competition were exhibited by soil isolates, which were identified using 16S rRNA gene sequencing and phylogenetic analysis. Purified isolates were also characterised for their resistance and/or susceptibility to predatory attack by a variety of myxobacteria. The bacterial isolates obtained varied when exposed to seven different myxobacteria predators. Our data suggest that the addition of myxobacteria to isolation plates biases isolation towards relatively predation-resistant prey organisms. Our next goal is to isolate myxobacterial predators from the same soil samples (on different prey isolates) and test predator-prey interactions quantitative using pure strains. The genetic basis of differential predatory activity and prey susceptibility can then be investigated.

Access Microbiol. 2019 Apr 8;1(1A):15.

Infectious bronchitis virus modulates cellular stress granule signalling

Matthew Brownsword ^1,^2,^*, Nicole Doyle ¹, Michele Brocard ², Nicolas Locker ², Helena Maier ¹

Abstract

Infectious bronchitis virus (IBV), a gammacoronavirus, causes the economically important poultry disease, infectious bronchitis, resulting in reduced weight gain and egg quality. As observed for many viruses, during replication, IBV shuts off translation of host proteins, preventing synthesis of important products of the innate immunity, which are pivotal in fighting viral infection. This work investigates the role of stress granules in IBV translational control. Stress granules are membranes-less aggregations of stalled translation initiation complexes comprising translation initiation factors, 40S ribosome and RNA binding proteins. These structures serve as sites of storage and sequestration of translational machinery and cellular mRNA while simultaneously enabling intracellular signalling and antiviral responses. It is shown here by immunofluorescence that IBV induces stress granules in only a proportion of infected cells. These stress granules occur late in the virus life cycle and appear canonical, containing multiple stress granule markers and showing mRNA exchange with ribosomes. In addition, stress granule markers are not diverted to sites of virus replication, as seen during replication of some other viruses. Interestingly, IBV infection results in resistance to chemicals that induce stress granules via eukaryotic initiation factor 2α (eIF2α). Consistent with this, eIF2α is not phosphorylated at any time during IBV infection. This also indicates a non-canonical signalling pathway for IBV-induced stress granules. Significantly, stress granule formation is uncoupled from translational arrest as visualised using ribopuromycylation. Therefore, IBV replication both induces and inhibits cellular stress granule signalling in a process that is uncoupled from shut off of host translation.

Access Microbiol. 2019 Apr 8;1(1A):15.

Replication of the Chikungunya virus genome requires cellular chloride channels

Marietta Müller ^1,^*, Natalie Jones ¹, Eleanor Todd ¹, Henna Khalid ¹, Andres Merits ², Jamel Mankouri ¹, Andrew Tuplin ¹

Abstract

Chikungunya virus (CHIKV) causes fever and debilitating joint pain, with frequent long-term health implications and cumulating fatalities worldwide. There are no specific antivirals and vaccines, therefore understanding CHIKV replication is essential to establish treatments and preventative measures. Cellular ion channels are druggable targets and are known to facilitate replication of RNA viruses. To determine if the activities of cellular chloride channels (Cl--channels) are required during CHIKV replication, we applied broad-ranging inhibitors and siRNA to mammalian and invertebrate cells. The Cl--channel inhibitors DIDS, 9-ACA and NPPB significantly reduced the titre of released CHIKV progeny at 12 h post-infection in a dose-dependent manner suggesting that Cl--channels are pro-viral factors. Analysis of viral protein expression and time-of-inhibitor-addition studies indicated that CHIKV requires Cl--channels at post-entry and pre-egress stages. Replication of a sub-genomic replicon was restricted and genome copy numbers reduced by Cl--channel inhibition, implying that Cl--channels are involved in genome replication. siRNA knock-down identified the chloride intracellular channels (CLIC) 1 and 4 to be required for the CHIKV infectious cycle with CLIC1 interacting with the viral protein nsP3. We hypothesise that the channels play a role in formation or maintenance of the membranous, viral replication-complexes and that this important role is conservt amongst the mammalian and invertebrate hosts. These findings advance our understanding of CHIKV replication in the two host environments and help to identify drugs/druggable targets for treatment and prevention of CHIKV disease.

Access Microbiol. 2019 Apr 8;1(1A):15.

Host-derived markers of Lyme disease: their discovery and diagnostic potential

Greg Joyner ^1,^*, Nick Beeching ^1,², Julian Hiscox ¹, Amanda Semper ³

Abstract

Lyme disease (LD) is a multisystem infection caused by tick-borne spirochaetes of the Borrelia burgdorferii sensu lato group. UK and US laboratory diagnosis of LD involves the two-tier serological approach. The negative predictive value of the test has been challenged, particularly in early stage LD. There is considerable interest, therefore, in the development of improved diagnostic tests. The main aim of the project is to identify new markers that could form the basis for improved tests. A mass spectrometry biomarker discovery study was undertaken on LD positive and negative residual diagnostic samples from UK LD testing by Public Health England and a cohort of patient samples from collaboration with a research group in the Czech Republic. A ‘related-disease control group’ including serum samples from syphilis, leptospirosis and chronic fatigue syndrome was also included. Several proteins were found at a significantly higher or lower in abundance in the ld-positive patients compared with ld-negative. Of particular interest was Lipocalin-2 (LCN2), a protein involved in immunity. LCN2 has previously been found in increased abundance in mice exposed to B. burgdorferi. Further analysis of LD samples using Illumina RNA sequencing revealed further markers of interest. Transcriptomic analysis including Ingenuity Pathway Analysis (IPA) gave insights into the host response to LD infection. Proteins of interest from proteomic and transcriptomic analysis were taken forward for further analysis by WB or ELISA in a larger sample set.

Access Microbiol. 2019 Apr 8;1(1A):15.

Genome-led discovery of novel microbial natural products

Alicia Russell ^1,^*, Rodney Lacret ¹, Andrew Truman ¹

Abstract

Microorganisms have been increasingly exploited for their remarkable ability to produce diverse natural products, which display bioactivities ranging from antimicrobial and anticancer to signalling and developmental. Genome sequencing has revealed that bacteria harbour many more biosynthetic gene clusters (BGCs) for natural products than are currently characterised. Whilst several genome mining tools have been developed to aid discovery, some classes of BGCs remain difficult to identify. One such class is ribosomally-synthesised and post-translationally modified peptides (RiPPs). RiPP BGCs are small with few regions of homology, and the short precursor peptides are rarely annotated in genomes. After developing a targeted genome mining approach for RiPPs, we identified a novel family of BGCs spanning over 200 actinobacterial genomes. The presence of diverse biosynthetic enzymes and sequence variation of the precursor peptides suggest that these BGCs may produce several structurally diverse molecules. We have successfully TAR cloned one such BGC from Streptomycesand expressed it heterologously, and metabolomic analysis led to identification of the pathway product. Gene deletion experiments have also confirmed the involvement of individual biosynthetic enzymes. The compound has been purified and the structure elucidated by NMR. Further work will focus on exploring RiPP BGCs from other species and investigating the biological role of these molecules. The discovery of such a diverse and highly conserved group of BGCs highlights that we are still scratching the surface of the huge biosynthetic capacity of microorganisms, and the use of more sophisticated genome mining tools could help unveil many more important molecules in the future.

Access Microbiol. 2019 Apr 8;1(1A):15.

Dimerisation of the influenza virus RNA polymerase during viral genome replication

Alexander Walker ^1,^*, Haitian Fan ¹, Loic Carrique ², Jeremy Keown ², David Bauer ¹, Jonathan Grimes ², Ervin Fodor ¹

Abstract

Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA, that carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins. Replication of negative-sense viral RNA (vRNA) is a two-step process, progressing via a positive-sense complementary RNA (cRNA) intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP dimerisation may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography, SAXS and cryo-EM, we identify the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We use bimolecular fluorescence complementation (BiFC) to show that influenza RdRP forms dimers in mammalian cells through the interface identified in solution. Using a combination of cell-based and in vitro assays, we show that influenza RdRP dimerisation via the PA-C terminal domain is necessary for copying cRNA back into vRNA during viral genome replication. In addition, we show that a nanobody (a small-domain antibody) that interferes with dimerisation attenuates influenza A virus growth in cell culture. These data provide insight into the mechanism of influenza viral genome replication, and identify a potential novel drug target against influenza A virus.

Access Microbiol. 2019 Apr 8;1(1A):15.

Characterising Birnaviridae replication and reassortment in vitro: virus factories derived from distinct input viruses form in the cytoplasm of co-infected cells and coalesce over time

Elle Campbell ¹, Joanna Wells ¹, Alice Gray ¹, Andrew Broadbent ^1,^*

Abstract

The Birnaviridae family is comprised of non-enveloped viruses with a double-stranded RNA genome that is divided into two segments, A and B. Birnaviruses are responsible for major economic losses to the poultry and aquaculture industries, and reassortment complicates their epidemiology and control. However, little is known about the nature of theirreplication in cells, or the molecular mechanism underpinning reassortment. In order to address this, we rescued two recombinant infectious bursal disease (IBD) viruses, with either a GFP11 or Tetracysteine (TC) tag at the 3’ end of segment B (IBDV-GFP11 and IBDV-TC, respectively). DF-1 cells were either transfected with GFP1-10 prior to IBDV-GFP11 infection, or stained with ReAsH following IBDV-TC infection, which led to the apprearance of green or red foci in the cytoplasm, respectively. Foci co-localised with VP3 and dsRNA, suggesting these were virus factories (VFs). The average number of VFs significantly decreased from 60 to 5 per cell between 10 and 24 h post infection (P<0.01), while the average area significantly increased from 1.24 µm² to 45.01 µm² (P<0.01), suggesting VFs coalesce in the cytoplasm over time. Red, green and yellow foci were observed in the cytoplasm of co-infected cells, suggesting that VFs are initially derived from distinct input viruses prior to coalescence. Live cell imaging revealed that larger VFs were more static while smaller VFs were more mobile, and fusion events were observed. We speculate that VF coalescence is required for birnavirus reassortment, and current work is aimed at determining the cellular factors that drive coalescence.

Access Microbiol. 2019 Apr 8;1(1A):15.

Encapsidation of viral RNA in Picornavirales: studies on cowpea mosaic virus demonstrate dependence on viral replication

Hadrien Peyret ^1,^*, Inga Kruse ¹, Pooja Saxena ¹, George Lomonossoff ¹

Abstract

To elucidate linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of the plant-infecting member of the order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (eVLPs) can be generated by transiently co-expressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24K protease, in the absence of the replication machinery (Saunders et al., 2009). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We show here that the remarkable specificity of packaging observed in CPMV is due to a functional linking between the two processes of viral replication and encapsidation. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effect of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation, and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication, which opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

Access Microbiol. 2019 Apr 8;1(1A):15.

clusterTools: functional element identification for the in silico prioritization of biosynthetic gene clusters

Emmanuel Lorenzo de los Santos ^1,^*, Gregory Challis ^1,²

Abstract

The large amount of data from inexpensive sequencing means that the number of putative biosynthetic gene clusters (BGCs) far exceeds our ability to experimentally characterize them. This necessitates the need for development of further tools to analyze putative BGCs to flag those of interest for further characterization. clusterTools implements a framework to aid in the in silico characterization of BGCs by identifying regions of the DNA, containing homologous proteins, or coding sequences containing specific functional domain compositions using user-built HMM rules, in close proximity, reporting results in an easy to visualize manner. clusterTools complements existing software for BGC analysis in two ways. First, by running clusterTools on databases of genomic sequences in an exploratory mode, the user can identify and download regions of interest in the DNA for further processing and annotation in programs such as antiSMASH. Second, if clusterTools is run on databases constructed from putative gene clusters generated by antiSMASH, one can rapidly identify clusters on interest from the group that warrant further analysis and experimental characterization. We demonstrate the use of clusterTools as part of our workflow to identify BGCs of specific classes of natural products that would be difficult to identify with existing methods, particularly clusters containing assembly line domains as components, including those involved in bacterial polyketide alkaloid biosynthesis. clusterTools can also be used to identify novel BGCs by incorporating regulatory and antibiotic resistance elements. Standalone versions of clusterTools are available for Macintosh, Windows, and Linux.

Access Microbiol. 2019 Apr 8;1(1A):15.

A high throughput siRNA screen to identify host membrane trafficking proteins that restrict pneumovirus egress

Stacey Human ^1,^*, Dalan Bailey ¹

Abstract

The pneumoviruses human and bovine respiratory syncytial virus (RSV) are significant global respiratory viral pathogens responsible for causing lower respiratory tract infections in humans and cattle, respectively. The formation of progeny virions at the cell surface requires the coordinated assembly of glycoproteins trafficked to the apical surface of cells via the host secretary pathway, other viral protein complexes assembled in cytoplasm inclusion bodies and lastly viral ribonucleoproteins. Although some host proteins and pathways have been implicated in pneumovirus budding and assembly (e.g. HSP90, Rab-11 and apical recycling endosomes), the full spectrum of virus-host interactions has not been fully elucidated. Using a siRNA library targeting membrane trafficking and the Incucyte^® Live Cell Imaging System, we have developed and optimised a high-throughput siRNA protocol for characterising RSV egress. The siRNA library targets human proteins which are known, or predicted, to be involved in membrane trafficking or remodelling, while the Incucyte^® System allows near real-time imaging over an extended time-course, generating high quality data that allows the monitoring and quantification of multiple parameters such as cell viability, viral replication and syncytia formation. Using a recombinant human RSV expressing a GFP reporter we have identified a number of proteins involved in pneumovirus trafficking in infected cells. Our techniques provide a robust and sensitive mechanism for genetic screening and the identification of pneumovirus-protein interactions.

Access Microbiol. 2019 Apr 8;1(1A):15.

A pilot study to determine the relationship between bacterial populations in the cloaca and the caecum of broiler chickens

Nadia Andreani ^1,^*, Matthew Goddard ¹

Abstract

The study of animal digestive tracts reveals important information on the host’s health status. For livestock, being able to predict the effect of different treatments on the gut microbiome has important implications for increased sustainability, enhanced animal welfare and increased food safety. However, gut contents can be investigated only after the slaughter of the animal, but cloaca/rectal samples may be collected from live animals and reduce the number of animals killed for experimental purposes. The aim of this study is comparing the microbial communities of caecum and cloaca associated with eight poultry broiler flocks from two English farms. 16S amplicon libraries were run on a MiSeq with a 250 bp PE read metric. The data were evaluated with in qiime1 and qiime2. Comparisons of bacterial communities of cecum and cloaca revealed they are significantly different in terms of the number and types of bacterial species, as well as their abundance (P-value Indicator species analysis of cecum samples showed the class Bacilli were enriched, while Clostridia had greater prevalence in cloaca. Finally, no pathogenic bacterial species of poultry were identified in the analysed animals. Despite the fact sampling cloaca content could be a method to reduce cost and suffering for research purposes, this study reveals the limit of the use of cloacal microbiomes to provide a window into poultry alimentary canal microbiomes.

Access Microbiol. 2019 Apr 8;1(1A):15.

Prevalence of microbial parasites in captive animals across wildlife parks

Emma Betts ^1,^*, Eleni Gentekaki ², Angus Carpenter ³, Adrian Harland ⁴, Anastasios Tsaousis ¹

Abstract

Microbial eukaryotes (parasites/protists) are widely distributed and are common inhabitants of the gastro-intestinal (GI) tract of humans and animals. Some species, including Giardia, Entamoeba and Cryptosporidium are associated with symptomatic gastro-intestinal illness. However, others, for example Blastocystis, have questionable pathogenicity as they can be found in symptomatic and asymptomatic individuals. The aim of this study is to investigate selected protists which present health concerns to humans or animals. To date, approximately 180 faecal samples from 33 mammalian species, four bird species and one reptile across two wildlife parks in the Southeast England have been collected. A combination of cell culturing techniques, microscopy and molecular biology have been carried out to positively identify different protists including Blastocystis, Cryptosporidium, Eimeria, Entamoeba, Giardia and Isospora. Preliminary data show over fifty percent of the animals are sequence positive for at least one species, with approximately thirty percent exhibiting co-habitation with two or more different species. This study provides one of the first thorough investigations into distribution and prevalence of GI tract protists in wildlife parks in the UK. As a result, it has enhanced our awareness regarding what may constitute a normal eukaryotic component of the gut microbiome, in addition to aiding conservation efforts by examining the impact captivity has on an animal’s microbiome and potential implications this may have on their release.

Access Microbiol. 2019 Apr 8;1(1A):15.

Transformation of the dinoflagellate chloroplast

Christopher Howe ^1,^*, Ellen Nisbet ¹, Adrian Barbrook ¹, Isabel Nimmo ¹

Abstract

Dinoflagellate algae are ecologically and environmentally important, as symbionts of corals and many other aquatic organisms, and the causative agents of red tides. However, attempts over the last twenty years to establish genetic manipulation systems for dinoflagellates have met with little success. We have exploited the unusual chloroplast genome of dinoflagellates to establish a system for transformation of this organelle. The chloroplast genome of peridinin-containing (the ancestral state) dinoflagellates is highly reduced and composed of a number of small, plasmid-like molecules, referred to as ‘minicircles’. We have constructed shuttle vectors that are fusions of minicircles and Escherichia coli plasmids and carry selectable markers. We used biolistic transformation to introduce these into the model dinoflagellate Amphidinium carterae. We found that the plasmids confer the expected phenotype on the dinoflagellate cells, and we can detect the plasmid DNA and associated transcripts following selection, indicating successful transformation. This opens up the possibility of studying many aspects of dinoflagellate chloroplast biology, including the maintenance and expression of the minicircles, and the role of the chloroplast in phenomena such as coral bleaching.

Access Microbiol. 2019 Apr 8;1(1A):15.

Mutasynthesis of novel prodiginines derived from the antibiotic prodigiosin by exploiting substrate specificity of H2MAP oxidases PigB and HapB

Maxime Couturier ^1,^*, Hiral Bhalara ¹, George PC Salmond ¹, Finian J Leeper ¹

Abstract

The natural product, prodigiosin, caught researchers’ interest more than a century ago because of its bright red color. Today, interest in this tripyrrolic secondary metabolite remains strong due to its biological effects, including potent antibiotic activity against various Gram-positive bacteria. Further exploration of the potential of this class of molecules requires libraries of analogues. Yet, the total synthesis of prodigiosin-like compounds (prodiginines) proves challenging. This can be overcome by highjacking the bacterial biosynthetic machinery via mutasynthesis. Although a number of different bacteria produce prodigiosin, its biosynthetic pathway is well conserved. The final precursors, MAP and MBC, are always produced independently, before a terminal condensation reaction. Our work focuses on the last step of the formation of MAP: the oxidation of H2MAP. We were able to isolate and characterize HapB, the enzyme catalyzing this reaction in Hahella chejuensis. In addition, we showed that some modifications of alkyl substituents on the C2 and C3 positions of H2MAP did not alter HapB activity significantly. We then fed these analogues of H2MAP into Serratia ΔpigD, a mutant of Serratia ATCC sp.39006 which does not produce any endogenous H2MAP. As expected from the in vitro testing, chain elongation past two carbons on the C2 position could not be accepted whereas all substrates with a modification on the C3 position restored pigmentation, leading to the formation of eight novel prodiginines.

Access Microbiol. 2019 Apr 8;1(1A):15.

Food or a free ride? The ability of a marine microbial community to degrade plastics

Robyn Wright ^1,^*, Matthew Gibson ¹, Joseph Christie-Oleza ¹

Abstract

Recalcitrant polymers are widely distributed in the environment. This includes natural polymers, such as chitin, but also synthetic polymers, which are becoming increasingly abundant, and for which biodegradation is uncertain. Distribution of labour in microbial communities commonly evolves in nature, particularly for arduous processes, suggesting that a community may be better at degrading recalcitrant compounds than individual microorganisms. Previous work, carried out by us, showed that a microbial community could be selected for the efficient degradation of chitin, but if the community was left for too long then the function decayed as the active members were replaced by cheaters and grazers. Here we aimed to determine whether (i) a marine microbial community may be capable of degrading the common packaging plastic poly(ethylene terephthalate) (PET), (ii) whether we see the same pattern of community succession as we do with chitin and (iii) if this community may be better able to degrade PET than an individual organism. We incubated several different types of PET with the natural microbial community found colonising coastal marine debris, and characterised the microbial community succession across the incubation period. We show an enrichment of taxa that have previously been shown to be capable of the degradation of recalcitrant compounds, and we show that this community is capable of growing faster than an individual organism. We are currently determining the correlation between microbial community structure and the concentration of breakdown products from PET, as well as the mechanisms that are used for this degradation.

Access Microbiol. 2019 Apr 8;1(1A):15.

Analysis of bacterial competition using imaging mass spectrometry

Laia Castaño ^1,^*, Ian Gilmore ², Blair Johnston ¹, Michael Chrubasik ¹, Katherine Duncan ¹

Abstract

The bacterial order actinomycetales are responsible for the production of 65–70 % of microbially produced specialised metabolites with diverse biological activities, with some actinomycetale strains containing over 30 Biosynthetic Gene Clusters encoding for these metabolites. However, only approximately 10 % of these genes are typically transcribed in a mono-culture setting. Furthermore, it has been observed that microbial interactions may induce these cryptic gene clusters providing an ecological advantage to the producer strains. To understand the chemical exchange between strains isolated from the marine environment, microbial interactions were assessed using 49 actinomycetale strains, two Pseudomonas and one Bacillus strain. In total, 72 tri-cultures (three strains) were analysed resulting in 29 strains that showed an altered phenotypes as a result of the interaction. These were then evaluated in a one-to-one culture (two strains) followed by bioactivity screening. Using this data, nine tri-cultures and 27 one-to-one cultures were evaluated using tandem Mass Spectometry, enabling chemically interesting interactions to be prioritized for Time of Flight Secondary Ionisation Mass Spectometry (ToF-SIMS) analysis. ToF-SIMS enables the spatial distribution of parent ions within a sample, in this case, two bacterial strains interacting in a Petri dish. The results that will be presented demonstrate that microbial interactions induce the production of metabolites and ToF-SIMS represents an exciting strategy to study bacterial chemical ecology.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of sites of Infectious Bronchitis Virus RNA synthesis

Nicole Doyle ^1,^*, Selma Rayon ¹, Jennifer Simpson ¹, Pippa Hawes ¹, Helena J Maier ¹

Abstract

Infectious bronchitis virus (IBV), an avian gammacoronavirus, is an important pathogen causing significant animal welfare problems and economic losses to the global poultry industry. Positive-strand RNA viruses, including coronaviruses, induce cellular membrane rearrangements during replication forming replication organelles, which are thought to support efficient viral RNA synthesis. IBV replication has been shown to induce the formation of double membrane vesicles (DMVs), zippered ER and tethered vesicles, known as spherules. Although these are proposed to be the site of viral RNA synthesis, this is as yet unconfirmed and is therefore the focus of these studies. Historically, dsRNA has been used as a marker for sites of coronavirus RNA synthesis, however IBV-associated dsRNA and nsp12 (the viral RNA-dependent RNA polymerase) do not colocalise in infected cells. We have determined the cellular location of the viral genome using Fluorescence In Situ Hybridisation (FISH). By comparing the immunofluorescence labelling of cells permeabilised with different detergents, we have demonstrated that dsRNA can be found within membrane-protected compartments, while nsp12 is not, indicating that the virus could be isolating the dsRNA in DMVs or spherules, affording protection from the host immune response. By incorporating uridine analogues over the course of infection with IBV, we have visualised sites of nascent viral RNA synthesis using super-resolution microscopy. This has shown that dsRNA appears to colocalise more strongly with nascent RNA than nsp12. Using these methods as well as looking at the ultrastructural level we are able to begin to discover the location of sites of IBV RNA synthesis.

Access Microbiol. 2019 Apr 8;1(1A):15.

Evolutionary genomics of the host-restricted pathogen Staphylococcus aureus subsp. anaerobius reveals extensive genome decay and signatures of adaptation

Gonzalo Yebra ^1,^*, Maan M Neamah ², Bryan A Wee ¹, Emily J Richardson ¹, Ricardo de la Fuente ³, J Ross Fitzgerald ¹, José R Penadés ²

Abstract

Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.

Access Microbiol. 2019 Apr 8;1(1A):15.

Understanding how bacterial products from the microbiota enter the host, determining where they aggregate, and their influence over immune cells at these sites

Christine Jordan ^1,^*, Tom Clarke ¹

Abstract

The microbiota is crucial for gut homeostasis by aiding in nutrient uptake, and protecting against pathogens. Recent evidence suggests the benefits provided by the microbiota are not restricted to the intestine but also extend to systemic sites. Systemic benefits are hypothesized to be mediated by bacterial products, derived from the microbiota, such as peptidoglycan and lipopolysaccharide, entering the bloodstream and acting as novel signalling molecules at distal sites. However the precise way in which these microbial products enter the bloodstream remains largely unclear. Our data suggest bacterial products can cross the intestinal epithelium, and that routes across may vary between different bacterial products. Using in vitro and in vivo models, we find that host processing of cell wall molecules, by host antimicrobial lysozyme, promotes their translocation across the epithelium. Once they have traversed the intestinal barrier our preliminary data provide support that the liver plays a role in clearing bacterial products from the blood, as here we see a reservoir of peptidoglycan. This increased dissemination of cell wall molecules additionally enhances resistance to pulmonary infection. Therefore lysozyme treatment enhances bacterial product migration and increases host protection against systemic pathogens. Our work provides mechanistic insight into how the gut microbiota exerts systemic effects. Furthermore it provides a basis on which to launch further investigations, including examining the influence these aggregated cell wall proteins have over innate immune cells at these sites. Giving us greater insight into how the host controls microbial signalling and the benefits provided to our innate immune system.

Access Microbiol. 2019 Apr 8;1(1A):15.

Decaying Ascophyllum nodosum as a source of algal cell wall degrading enzymes with potential utility in enzyme-assisted extraction technologies

Maureen Ihua ^1,^*, Halimah Mohammed ¹, Freddy Guihéneuf ², Lekha Margassery ¹, Stephen Jackson ¹, David Clarke ¹, Alan Dobson ¹

Abstract

Seaweeds are of huge interest in the food, pharmaceutical and agricultural industries due to their high nutritional content and the prevalence of useful bioactive compounds. Current extraction methods of macroalgal-derived metabolites are however problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular as they allow the development of more efficient and eco-friendly processes. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25°C and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina Miseq sequencing and culture-dependent approaches, including the novel iChip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay with a substantial decrease in the relative abundances of certain phyla including Planctomycetes and Verrucomicrobia observed during the decay period. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species from the phylum Bacteroidetes together with a number of Vibrio species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculous and were shown to be equally efficient as commercial enzymes in their extraction efficiencies.

Access Microbiol. 2019 Apr 8;1(1A):15.

Development of streptomyces to utilise sustainable feedstock in fermentations

Anna Birke ^1,^*, Steve Kendrew ², Benjamin Huckle ², Iain Hunter ¹, Paul Hoskisson ¹

Abstract

The genus Streptomyces is comprised of soil-dwelling Gram-positive Actinobacteria that are widely used for the industrial production of antibiotics. S. clavuligerus is used for the industrial production of clavulanic acid, which is a potent b-lactamase inhibitor, and is, therefore, able to restore the sensitivity of b-lactamase-producing bacteria penicillins and cephalosporins. In fermentations, the carbon sources available for utilisation by the producing organism have profoundly impact central carbon and specialised metabolic pathways. We have a long-term goal of using carbon sources from food waste to produce clavulanic acid with a view to developing more sustainable fermentations. To achieve this, the carbon utilisation profile of S. clavuligerus has to be diversified. Wildtype S. clavuligerus is a natural glucose auxotroph and has adapted to utilise glycerol most efficiently. It has been shown that the lack of glucose utilisation by S. clavuligerus is due to the insufficient expression of genes whose products are required for glucose uptake (glcP) and phosphorylation (glk). To enable glucose utilisation by S. clavuligerus strains, we have constructed strains for heterologous expression of either glcP or glk from different Streptomyces species. Further, the range of utilisable carbon sources for growth and clavulanic acid production has been investigated. Growth on solid media has revealed interplay between carbon and nitrogen metabolism, with extracellular protease production being regulated in a carbon source-dependent manner. Therefore, the role of protease secretion and its relationship with clavulanic acid production has been examined, revealing a complex role between carbon catabolite repression, protease production and clavulanic acid biosynthesis.

Access Microbiol. 2019 Apr 8;1(1A):15.

Mechanistic analysis of the minimalistic twin-arginine translocation system found in Bacillus subtilis

Jon Cherry ^1,^*, Tracy Palmer ¹

Abstract

The twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.

Access Microbiol. 2019 Apr 8;1(1A):15.

Generation of recombinant avian coronaviruses indicates the S gene is a factor in pathogenicity

Phoebe Stevenson-Leggett ^1,^*, Sarah Keep ¹, Michael Oade ¹, Paul Britton ¹, Erica Bickerton ¹

Abstract

The avian coronavirus infectious bronchitis virus (IBV) is the most economically important disease of chickens in the UK, causing significant losses as a result of poor weight gain and reduced egg quality in infected birds. IBV expresses a large spike (S) glycoprotein on the surface of the virion which is responsible for attachment to host cells and is the main antigenic target for neutralising antibodies during infection. Previous work has also demonstrated that the S protein determines cell tropism in vitro. In order to investigate the involvement of the S gene in IBV pathogenesis and explore the potential for vaccine propagation in cell culture, recombinant viruses were generated using vaccinia virus based reverse genetics. Two isolates of the pathogenic M41 strain were mutated to include the S gene from a non-pathogenic lab strain with extended tropism (Beau-R) or a heterologous pathogenic field strain with restricted tropism (4/91), resulting in two recombinant IBVs termed M41K-BeauR(S) and M41K-4/91(S), respectively. These viruses were characterised in vitro and in vivo to determine the involvement of the S gene in IBV replication and pathogenicity. M41K-BeauR(S) was attenuated in vivo but exhibited the extended host tropism of the S donor strain. M41K-4/91(S) remained pathogenic and also adopted the restricted in vitro tropism of 4/91. This indicates that the S gene not only determines the cellular tropism of the virus but also plays a key role during in vivo infections, and that replacing the ectodomain of IBV S can significantly alter the pathogenicity of the resulting virus.

Access Microbiol. 2019 Apr 8;1(1A):15.

Attenuation of E. coli O157:H7 virulence by a combination of natural plant extracts and organic acids before and after refrigerated storage

Alexandros Stratakos ^1,^*, Mark Linton ¹, Patrick Ward ², Filip Sima ¹, Carmel Kelly ¹, Laurette Pinkerton ¹, Ozan Gundogdu ³, Nicolae Corcionivoschi ¹

Abstract

Anti-virulence strategies are an alternative approach to combat zoonotic bacterial pathogens. Although control measures are in place against E. coli O157, it still remains a global health concern with cattle being the most important reservoir. Different feed constituents have varying effects on the virulence of E. coli O157 present in the gut which then are transferred to meat processing surfaces and meat during slaughter. This study explored the anti-virulence properties of a mixture of natural plant extracts and organic acids (citrus, grape and oregano extracts, lactic and citric acid) before and after refrigerated storage as means of reducing the risk from E. coli O157 by reducing its virulence. Assessment of the effects of sub-inhibitory concentrations (0.1 and 0.5 %, v/v) of the antimicrobial mixture before refrigerated storage showed that the pathogen’s motility and adhesion onto HCT-8 cells was significantly reduced (P<0.05) in a dose-dependent manner. Shiga-toxin 2 production was also significantly reduced. Real-time PCR analysis revealed that the mixture of natural antimicrobials repressed expression of adhesion (eae) and shiga-toxin 2 (stx2) genes, which was consistent with the observed reduction in adhesion and toxin production. The same virulence factors were investigated after simulated storage of E. coli O157 in meat simulation medium. Results revealed that after exposure to the antimicrobial mixture, the virulence was significantly lower compared to non-treated control after refrigerated storage. The present work shows the potential of the antimicrobial mixture in reducing virulence and thus risk from E. coli O157 by applying it as a feed additive.

Access Microbiol. 2019 Apr 8;1(1A):15.

Studies of cell surface and soluble HLA class I expression in pancreatic beta cells exposed to interferons or Poly I:C

Pouria Akhbari ^1,^*, Noel Morgan ¹, Sarah Richardson ¹

Abstract

HLA class I (HLA-I) molecules play a crucial role in cell-mediated immunity by presenting peptide antigens to cytotoxic CD8+T cells. A pathological hallmark of Type 1 diabetes (T1D) is the hyperexpression of HLA-I in pancreatic islets that contain residual insulin producing beta cells. The expression of HLA-I can be induced following exposure to interferons (IFN). Previous studies have implicated enteroviruses (EV) as major players in triggering an autoimmune response against beta cells and it seems likely that the hyperexpression of HLA-I contributes to the recognition and targeting of beta cells by CD8+T cells. Whilst HLA-I molecules are expressed on the surface of nucleated cells, there is increasing evidence that HLA-I can also be found in a soluble form in the plasma, including in patients with viral infections. Intriguingly, soluble HLA-I (sHLA-I) levels are significantly elevated in the serum of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and T1D, yet the function of sHLA-I is still unclear. In this project, we are investigating the impact of interferons and poly I:C, a viral dsRNA mimetic, on the expression of surface and soluble HLA-I in the human pancreatic beta cell line EndoC-βH1, the human pancreatic ductal cell line PANC1 and in HeLa cells. We show that surface HLA-I is upregulated in response to these stimuli in each of the cell lines, with greatest magnitude in EndoC-βH1. We also show for the first time that release of sHLA-I is significantly increased in response to IFNγ in EndoC-βH1 cell line.

Access Microbiol. 2019 Apr 8;1(1A):15.

The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus replication

Stephen Polyak ^1,^*, Morgan Herod ², Oluwapelumi Adeyemi ², Joseph Ward ², Toby Tuthill ³, Stephen Berryman ³, Mark Harris ², Nicola Stonehouse ²

Abstract

Arbidol (arb, umifenovir) is used clinically in several countries as an anti-influenza virus drug. We have previously shown that arb inhibits many viruses including hepatitis C virus, Ebola and Zika, and that the primary mode of action appears to be via inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. We have also shown that arb is a good inhibitor of (non-enveloped) poliovirus types 1 and 3. Here, we evaluate the antiviral potential of arb against another picornavirus, foot-and-mouth disease virus (FMDV), an important veterinary pathogen. Sub-cytotoxic concentrations of arb inhibited the replication of FMDV replicon RNA. arb inhibition of FMDV RNA replication was not a result of generalised inhibition of uptake of cargo (e.g. plasmid DNA or RNA), nor did arb inhibit FMDV replication when added at 4 h post-transfection of FMDV replicon RNA. FMDV replication was blocked by the replication inhibitor guanidium hydrochloride (GuHCl). Upon GuHCl removal, FMDV replication was restored, and arb suppressed this recovery of virus replication. For other picornaviruses, recovery of virus replication upon GuHCl removal has been shown to require translation. However, arb did not suppress cap- or internal ribosome entry site (IRES)-dependent translation. arb also inhibited infectious equine rhinitis associated virus (ERAV), a close relative of FMDV. Testing of arb against infectious FMDV is in progress. Collectively, the data demonstrate that arb inhibits certain picornaviruses by a mechanism that appears to be independent of effects on virus entry but involves inhibition of genome replication.

Access Microbiol. 2019 Apr 8;1(1A):15.

The effect of Soil pH and phosphorus interactions on nitrous oxide emissions and microbial communities involved in nitrogen cycling

Meritxell Grau Butinyac ^1,^2,^*, Vincent O’Flaherty ², Karl Richards ¹, David Wall ¹, Fiona Brennan ¹

Abstract

Excess application of Nitrogen (N) to agricultural soils can lead to environmental pollution. As nitrous oxide (N₂O) is a potent greenhouse gas, it is of critical importance to reduce its emission for climate change mitigation. Availability of nitrogen can facilitate denitrification, an anaerobic respiratory pathway carried out by microbial communities in which N₂O is an intermediate product. An understanding of soil, climatic and edaphic factors influencing microbial communities and their activity is key to reducing N₂O emissions. Soil pH strongly impacts microbial community structure, with a direct effect on NosZ, the pH-sensitive enzyme catalysing N₂O reduction. We thus expect that microbial communities in acidic soils have a reduced capacity to mitigate N₂O emissions. It is likely that other management factors, like phosphorus application, interact with pH; causing changes to chemical nutrient availability and direct effects on microbial composition. The complexity linking N₂O emissions and microbial activity (impacted by soil pH), and the interacting role phosphorus availability plays in this relationship, is not yet understood. In this study, the capacity of microbial communities to denitrify, as well as the functional microbial community impacted by soil pH, were analysed by potential denitrification assays, measuring N₂O fluxes and N₂O/(N₂O+N₂) ratios; and qPCR analysis of denitrification genes. This was investigated across two soil types with a pH gradient and range of phosphorus application rates. Understanding the link between the microbial communities and N₂O production can be applied in agricultural management to reduce emissions from fields.

Access Microbiol. 2019 Apr 8;1(1A):15.

The microbial diversity of a sulfur-rich and saline cold pool in the Canadian high Arctic

Michael Christopher Macey ^1,^*, Ben Stephens ¹, Mark Fox-Powell ², Susanne P Schwenzer ¹, Victoria K Pearson ¹, Claire R Cousins ², Karen Olsson-Francis ¹

Abstract

Currently, the surface of Mars cannot sustain liquid water, but there is evidence suggesting that water was present in the Noachian. Although water might exist in the subsurface of Mars, it could not sustain in the present day unless it was highly saline. Thus, saline springs in polar desert environments are analogues with which to investigate martian conditions. An example of this is Axel Heiberg Island, located in a region of continuous permafrost in the Canadian High Arctic, which hosts sulfidic and highly saline springs. In this study, cultivation-dependant and independent techniques were used to investigate the microbial diversity of a sediment sample collected from a saline cold (3–8 °C) pool at Colour Peak Springs on Axel Heiberg. Both DNA and RNA were extracted from the samples, and the microbial community was characterised using the 16S rRNA gene from the extracted nucleic acids. The metabolic profile was characterised by screening DNA and cDNA for functional genes relating to the cycling of carbon (coxL, xoxF, cbbL), nitrogen (nifH, nosZ, nod) and sulfur (dsrB, soxB). The community profiles were used to inform enrichment strategies, allowing for the isolation and characterisation of several halophilic isolates including strains of Marinobacter, Halomonas, Halanaerobium and Loktanella. Through this work we have been able to develop an in-depth characterisation of the metabolic and phylogenetic diversity that is present and viable within this analogue site. This allows us to start building an understanding of the underlying mechanisms and strategies that enable organisms to persist in these environments.

Access Microbiol. 2019 Apr 8;1(1A):15.

Development of in vitro functional antibody assays to identify and assess vaccine targets against E. coli bacteraemia

Kin Chan ^1,^*, Richard Hesp ¹, Steve Thomas ¹, Steve Taylor ¹, Stephanie Leung ¹, Elizabeth Penn ¹

Abstract

Extra-intestinal pathogenic E. coli (ExPEC) is one of the leading causes of bacteraemia and urinary tract infections (UTI) worldwide. At present, there is no licensed vaccine available and with the increasing incidence of multidrug resistance (MDR) the demand for developing alternatives to antibiotics is paramount. We have developed in vitro functional antibody assays to fast track screening of patient convalescent sera from ExPEC infections to identify potential vaccine antigens. A functional immunological assay will also facilitate the assessment of vaccine candidates against the many disease-causing serotypes circulating in the population. Outer membrane vesicles (OMVs) purified from a mutant (ΔlpxM) MDR O25 (ST131) strain were used to immunise mice to raise antiserum against the bacterial strain. Immunised mice were protected in a mouse model of E. coli bacteraemia. The antiserum from protected animals was used to develop an antibody-mediated bacterial killing assay and a complement deposition assay (CDA). The antiserum mediated killing with human IgG and IgM-depleted plasma as the complement source, and also showed deposition of C3b and C5b-9 onto the bacterial surface determined by flow cytometry. This may be a useful screening tool for mining for new antigens and in assessing the antisera raised against the candidates for cross serotype protection.

Access Microbiol. 2019 Apr 8;1(1A):15.

Bacterial coping mechanisms for aging: using an individual-based model to study aging in biofilms

Robyn Wright ^1,^2,^*, Robert Clegg ², Jan-Ulrich Kreft ²

Abstract

Aging can be defined as an accumulation of damage, or a loss of function, with increasing age. For bacteria, it has generally been assumed that the mechanism used to cope with aging is the asymmetric segregation of damage at division, so that all of the damage is inherited by one cell and the other is therefore rejuvenated. Another, often neglected, mechanism is to repair the damage; our previous computational modelling work has found that an optimized, fixed rate of repair is fitter than damage segregation in well-mixed environments such as chemostats. The predominant mode of growth for bacteria is in biofilms, however, and here we investigate aging in biofilms using the individual-based model iDynoMiCS. In addition to the previously used damage segregation and fixed repair strategies, we introduced adaptive repair: sensing the current damage levels within the cell and responding to this by investing in damage repair machinery. We found that the optimal method for dealing with cellular damage varies with the environment being investigated. The investment of additional resources into adaptive repair is only beneficial when competition is sufficiently strong, in the chemostat and in biofilms, and the speed at which the fittest strategy becomes apparent depends upon the initial density of cells. When the bacterial cells are dense initially, and thus the competition between strategies is stronger, the adaptive repair strategy emerges as the winner much more rapidly.

Access Microbiol. 2019 Apr 8;1(1A):15.

Chain termination of structurally disparate non-ribosomal peptides by a trans-acting β-lactamase

Asif Fazal ^1,^*, Divya Thankachan ¹, Daniel Francis ¹, Lijiang Song ², Michael Webb ¹, Ryan Seipke ¹

Abstract

Non-ribosomal peptide synthetases (NRPSs) are responsible for the natural in vivo production of a large number of therapeutically relevant compounds. The non-ribosomal peptides (NRPs) formed by their action are the product of the long modular assembly lines, whilst the terminal step in the biosynthesis is the hydrolytic release, and frequently, macrocyclisation of the aminoacyl-S-thioester by an embedded thioesterase (TE). The surugamide biosynthetic pathway is composed of two NRPS assembly lines. One produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded TE, which led us to question how the peptides are released from the assembly line (and cyclised in the case of surugamide A). We characterised an alpha/beta hydrolase and β-lactamase in vivo and established that the former is a type II TE for surugamide A, but not surugamide F, and that the latter is a trans-acting release factor for both compounds. In vitro substrate utilisation assays unambiguously established that the β-lactamase can produce mature surugamides A and F from N-acetylcysteamine (SNAC) thioester mimics of the cognate terminal biosynthetic intermediates. Using bioinformatics, we estimate that ∼12 % of filamentous Actinobacteria harbor an NRPS system lacking an embedded TE and instead use a trans-acting β-lactamase release strategy. This expands the paradigmatic understanding of how non-ribosomal peptides are released from the terminal NRPS module and adds a new dimension to the synthetic biology toolkit, potentially useful in the search for novel antibiotics.

Access Microbiol. 2019 Apr 8;1(1A):15.

Proteome-wide analysis of CD8+ T cell responses to EBV lytic infection

Calum Forrest ^1,^2,^*, Andrew Hislop ², Alan Rickinson ², Jianmin Zuo ²

Abstract

Epstein-Barr virus (EBV) is one of 9 Human herpesviruses that can develop lifelong persistence. These viruses provide an antigenically complex challenge that induce strong CD8+T cell immunity during primary infection and continue to shape this immunity through recurrent lytic reactivation. Here is described the first lytic proteome-wide analysis of CD8+T cell responses to EBV and the first to compare primary vs memory CD8+T cell responses to any human herpesvirus. Primary CD8+T cells were mitogenically expanded directly from the blood of infectious mononucleosis (IM) patients. Comparatively, memory CD8+T cells required pre-enrichment using autologous dendritic cells loaded with a lytically-infected EBV cell lysate and FACS selection based upon the activation marker 4-1BB. Enriched cells were then expanded in vitro as for IM cells. Preparations from 7 IM patients and 7 healthy carriers were screened against each of the 70 EBV lytic cycle proteins in combination with the donors’ HLA-I alleles. Multiple reactivities were identified across the full lytic cycle with 146 responses identified amongst the 7 IM patients and 96 amongst the 7 healthy carriers. However the distribution of responses varied between the 2 cohorts with primary responses targeting IE and a small group of E proteins whereas memory responses targeted all phases but with some prominent responses against L proteins. This infers that responses in primary infection therefore appear to be shaped by presentation on the infected cell surface prior to the activity of viral evasins. However long-term carriage appears to re-shape the virus-specific response.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of novel antimicrobial-producing bacteria from an ancient water source by Oxford Nanopore Whole Genome Sequencing and Natural Product Chemistry

Tim Walker ^1,^*, Paul Stapleton ¹, Simon Gibbons ¹

Abstract

Microbially-derived antimicrobial compounds are a rich source of clinical antibiotic leads. However, discovery rates have declined over the past 40 years due, in part, to high rediscovery rates of known compounds from traditional soil-based screening approaches. In this study, an ancient hot-spring water source was tested for the presence of antimicrobial-producing bacteria using culture techniques which led to isolation of two organisms capable of inhibiting the growth of multiple bacterial species. Oxford Nanopore whole genome sequencing was used to identify these two isolates as being in one of two genera; Streptomyces and Paenibacillus. Bioinformatic analysis revealed both isolates to have multiple novel secondary metabolite gene clusters. Investigations of the Streptomyces sp. by natural product chemistry techniques showed the organism to produce multiple antimicrobial compounds, these were effective methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. This study underlines the value of investigating non-traditional habitats in the search for novel antibiotic-producing organisms.

Access Microbiol. 2019 Apr 8;1(1A):15.

Understanding metabolic processes shaping adaptation of E. coli to the gut

Miguel Pedro ^1,^*, João Batista-Barroso ², Catarina Pinto ³, Joana Dias ⁴, Isabel Gordo ¹, Karina Xavier ¹

Abstract

Many microbes colonise the gut establishing interactions with their host and their nutritional environment. Studying genetics and metabolism brought about the drive and potential to engineer communities to promote health and improve industrial processes. However, structuring artificial communities in predictable ways is underdeveloped. We studied Escherichia coli’s genetic targets and physiological mechanisms during gut colonisation and adaptation and how metabolic environment/microbiota complexity shape these processes. We introduced a tractable E. coli K-12 in mice Germ-free or with polymicrobial communities. Whole Genome Sequencing identified potential adaptive targets. Here, we established phenotypic assays as well characterising effects of key mutations and metabolomics was performed with 1H-NMR of intestinal contents. Genes for sugar alcohol metabolism (gat) was the only target common to both mouse models, evidencing specificity. Facing complex microbiota E. coli targeted use of sugar alcohols (srlR, kdgR) and anaerobic respiration (dcuB, focA) [1] whereas alone, we observed instead mutations pointing to increased ability for amino acid use (lrp, dtpB, alaA). Mutations selected correlated dinamically with metabolomics: our results fit the model whereby other microbiota members scavenge oxygen and breakdown complex sugars, limiting E. coli to anaerobically respire simple by-product carbon sources. In the opposing scenario (functional absence) improved amino acid use are favoured colonisation factors. Through experimental evolution we gained insight on shaping E. coli’s metabolic traits through genetic engineering to colonise specific host environments. This work also highlights the versatility of E. coli as potential biotic sensor. [1] Barroso-Batista, J. et al. The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps, 2014.

Access Microbiol. 2019 Apr 8;1(1A):15.

Coupling of subunit availability to activation of PMF-driven flagellar type III secretion

Owain Bryant ^1,^*, Betty Chung ¹, Gillian Fraser ¹

Abstract

Bacterial flagella are assembled from thousands of protein subunits that are unfolded and exported via a specialized type III secretion system. Subunit export is fuelled by the proton motive force (PMF) facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. What is unclear is how FliJ activation of FlhA is regulated to prevent constitutive proton influx when there are no subunits available. FliJ-mediated export gate activation could be regulated by other proteins that bind FliJ. We have shown that FliJ recruits unladen export chaperones, transferring them to their cognate subunits to create a local cycle of chaperone-subunit binding. To investigate whether chaperones also regulate FliJ activation of FlhA, we sought to isolate chaperone variants that were defective in FliJ binding but retained their ability to bind subunits and other export components. Disruption of chaperone-FliJ binding attenuated motility and cognate subunit export. To test whether chaperones blocked the FlhA-FliJ interaction, we developed in vitro and in vivo competition assays. Our data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.

Access Microbiol. 2019 Apr 8;1(1A):15.

Genome epidemiology of Mycobacterium bovis infection in contemporaneous, sympatric badger and cattle populations in Northern Ireland

Adrian Allen ^1,^*, Paul McAdam ¹, Eleanor Presho ¹, Carolyn Hughes ¹, David Rutherford ¹, Colin Bell ¹, Lucy Watson ¹, Grainne Mullan ¹, Rowland Kao ², Joseph Crispell ³, Assel Akhmetova ⁴, Fraser Menzies ⁵, Carl McCormick ⁵, Paddy McGuckian ⁵, Raymond Kirke ⁵, Robin Skuce ^1,⁶

Abstract

Introduction

Bovine tuberculosis (bTB) is an epidemiologically complex disease affecting both cattle and badgers in the UK and Ireland. Traditional molecular typing schemes have been used to characterise the spatial structure of the pathogen and relationship of M. bovis derived from sympatric animals. However, these methods lack the resolution to describe transmission dynamics at the farm level or to inform on the extent to which the hosts contribute. Whole genome sequencing can improve resolution of molecular epidemiology investigations in this epi-system.

Methods

We collected 598 M. bovis isolates from contemporaneous badgers (n=119) and cattle (n =479), located in a 100 km2 area of Northern Ireland. Cultures were DNA extracted and genome sequenced. Bioinformatic analysis was undertaken using the reddog pipeline and maximum likelihood phylogenetic analyses were conducted using RAxML, with the major endemic clade in the region subjected to phylodynamic analysis using Bayesian Evolutionary Analysis Sampling Trees (BEAST) software.

Results and Discussion

All 598 isolates produced reads of good quality, aligning to >90 % of the M. bovis reference genome with coverage of at least x10. A total of 1598 SNPs were detected. Phylogenetic analysis indicated the presence of nine major lineages circulating in the region. Eight exhibited long branch-lengths suggesting they were not endemic in the area. One lineage was endemic, comprising isolates from 60 badgers and 363 cattle. From the substitution rate of 0.36 SNPs per annum, this lineage arose in the study area in the mid-1980s. Data were consistent with ongoing transmission within and between both hosts.

Access Microbiol. 2019 Apr 8;1(1A):15.

An uncharacterised protein mediates motility, biofilm formation, and host colonisation in adherent invasive Escherichia coli

Robert Cogger-Ward ^1,^*, Adam Collins ¹, Jacob Dehinsilu ¹, Alan Huett ¹

Abstract

Adherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli patho type with a putativea etiological role in Crohn’s Disease (CD). AIEC pathogenes is ischaracterised by adhesion to, invasion of, and replication within intestinal epithelial cells and macrophages, and biofilm formation. Using a heterologous expression screen, we identified a gene in the AIEC LF82 genome encoding a protein which self-assembles into filaments in HeLa cells, which we hypothesised was a novel pilin or biofilm matrix component, designated bcmA (biofilm coupled to motility in AIEC). Using a crystal violet-based assay, we found LF82ΔbcmA have defective biofilm formation, which can be fully complemented by episomal bcmA expression. Microscopic analysis of LF82 biofilms demonstrated LF82ΔbcmA form patchy, sparse biofilms, and revealed an intracellular localisation for GFP-tagged bcmA, suggesting the protein is not a surface-exposed adhesin or biofilm matrix component. We therefore assessed the role of bcmA in flagellar-mediated motility and found that – despite displaying wild-type flagellar morphologies – LF82ΔbcmA have profound swimming and swarming defects. Work in a Caenorhabdit is elegans infection model suggests bcmA is not required for full virulence; however, preliminary evidence suggests LF82ΔbcmA have defective C. elegans gut colonisation. Taken together, our data demonstrates roles for bcmA in AIEC host colonisation via an undefined role in motility. bcmA is highly conserved among pathogenic gammaproteobacteria, including Salmonella Typhi, Shigella boydii and Klebsiella pneumoniae. This suggests bcmA may not only be significant for AIEC pathogenesis, but may also represent an important virulence determinant in several major gammaproteobacterial pathogens.

Access Microbiol. 2019 Apr 8;1(1A):15.

Genomic analysis of Burkholderia ambifaria identifies key specialised metabolites for biopesticidal applications

Alex Mullins ^1,^*, James Murray ¹, Matthew Bull ¹, Matthew Jenner ², Cerith Jones ^1,³, Gordon Webster ¹, Angharad Green ⁴, Daniel Neill ⁴, Thomas Connor ¹, Julian Parkhill ⁵, Gregory Challis ^2,⁶, Eshwar Mahenthiralingam ¹

Abstract

Background

Burkholderia bacteria are renowned for the biosynthesis of an extensive repertoire of specialised metabolites encoded by biosynthetic gene clusters (BGCs). Burkholderia ambifaria is a historical biopesticide and produces multiple antimicrobials with activity against nematodes, fungi and bacteria. We evaluated the distribution of antimicrobial activity and BGCs across the species which, despite their biopesticidal potency, is poorly understood.

Results

Genomic analysis of 64-B. ambifaria whole-genomes revealed considerable diversity. Pathway prediction identified 1272 BGCs across 64 strains that were de-replicated to 38 distinct BGCs. Known BGCs accounted for 13 of the BGCs; 7 of which corresponded to known antimicrobials, and included enacyloxins and bactobolins. The antimicrobial activity of the strains was assessed in a classical overlay assay against animal and plant pathogens. The combined genome mining and in vitro activity screens identified a potent anti-Gram-positive and anti-Oomycete compound, cepacin. A damping-off disease model with the pathogen Pythium ultimum and legume Pisum sativum (garden peas) highlighted cepacin as a key specialised metabolite in crop protection. B. ambifaria strain persistence in a mouse inhalation infection model was reduced by removal of the third replicon, whilst retaining the biopesticidal properties of the B. ambifaria strain.

Conclusion

This work highlights the diversity and prevalence of specialised metabolites in B. ambifaria, and the importance of the potent antimicrobial cepacin in protection against damping-off disease in agriculture. Construction of a B. ambifaria strain that retained biopesticidal properties yet was attenuated for persistence within a murine infection model provides a potential route towards developing safe biopesticidal agent.

Access Microbiol. 2019 Apr 8;1(1A):15.

Development of low bio-containment assays to characterise the antibody responses in pigs to Nipah virus vaccine candidates

Nazia Thakur ^1,^*, Nagendra Nath Barman ², Li-Yen Chang ³, Keith Chappell ⁴, Sarah Gilbert ⁵, Teresa Lambe ⁵, Glenn Marsh ⁶, Rebecca McLean ¹, Mercedes Mourino ⁷, Miriam Pedrera ¹, Rudiger Raue ⁹, Elma Tchilian ¹, Daniel Watterson ⁴, Paul Young ⁴, Simon Graham ¹, Dalan Bailey ¹

Abstract

Nipah virus (NiV) is a zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. Since 1998, NiV outbreaks have occurred in Malaysia, Bangladesh and India. NiV poses a significant pandemic threat due to its broad host range and the widespread distribution of its natural host, the Pteropus fruit bat. Despite this, there are currently no licenced therapeutics or vaccines for use in either humans or livestock. Aiming to develop a safe and inexpensive vaccine to protect livestock in future outbreaks and to prevent onward transmission to humans, we are evaluating the immunogenicity and efficacy of three candidate vaccines in pigs. These vaccines are based on the NiV G or F surface glycoproteins, proteins which are essential for mediating virus-cell or cell-cell entry. The candidate vaccines include: (i) a recombinant soluble NiV G protein subunit, (ii) a recombinant molecular clamp stabilised NiV F protein subunit and (iii) a replication deficient, adenoviral vectored NiV G protein. We have developed low biocontainment assays to characterise the antibody responses to NiV and to aid identification of immune correlates of protection through quantification of both antigen-specific and neutralising antibody responses to our vaccine candidates. This includes anti-NiV F/G indirect ELISAs, a microneutralisation test using pseudotyped particles, and a microfusion inhibition test using a quantifiable cell-cell fusion assay. Using these assays we have demonstrated that all three of our novel vaccines are immunogenic in pigs and capable of generating a robust antibody response, with evidence for neutralising antibodies.

Access Microbiol. 2019 Apr 8;1(1A):15.

Development of serologic tools for diagnosis and surveillance of Chikungunya

Flávia Fonseca Bagno ^1,^2,^*, Maria Marta Figueiredo ², Lara Carvalho Godoi ², Flávio Guimarães da Fonseca ^2,¹

Abstract

Chikungunya (CHIKV), an arbovirus that belongs to the Alphavirus genus of the Togaviridae family, causes a disease characterized by acute onset of fever accompanied by arthralgia. CHIKV also has been associated with cases of meningoencephalitis (primarily in neonates), Guillain–Barré syndrome and hemorrhagic disease. The clinical similarities, cross-reactivity and cocirculation of arboviruses in Brazil have complicated their differentiation, highlighting the need for new diagnostic tools. A serologic test can be useful for acute detections as well as for surveillance and epidemiological studies. Point of care tests currently available to detect CHIKV infections have been associated with low accuracy. The aim of this work was to design a synthetic gene and generate a recombinant protein to be used as antigens in diagnostic assays. Computational methods were used to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKV) was synthetized and the protein was purified by affinity chromatography. Antigenicity of the protein was initially confirmed by western-blot using sera from CHIKV infected mice. Additionally, the seroreactivity of r-CHIKV protein was evaluated using a panel of sera samples from human patients, CHIKV seropositive or not, by indirect IgG ELISA. The r-CHIKV protein showed sensitivity of 95 % and specificity of 96 %. No cross-reactivity was found against sera of Zika and Dengue positive patients. These results indicate that this proteins maybe useful antigen to detect CHIKV infections in ELISA assays. Our next step includes the development of a rapid test kit.

Access Microbiol. 2019 Apr 8;1(1A):15.

HSV-1 genome decompaction plays an important role in viral DNA sensing by PML-NB intrinsic antiviral regulators

Victor Iliev ^1,^*, Anne Orr ¹, Chris Boutell ¹

Abstract

Constituent proteins that reside within promyelocytic leukaemia nuclear bodies (PML-NBs) are known to play an important role in cellular restriction of herpesvirus gene expression as a component of the intrinsic antiviral immune response to DNA virus infection. However, the precise mechanism(s) of PML-NB genome detection, entrapment, and silencing remain unknown. We have recently reported a sensitive method of viral genome detection that enables the visualisation of infecting EdC (5-Ethynyl-2’-deoxycytidine) labelled HSV-1 genomes (HSV-1EdC). We report that ultraviolet light (UV) irradiation of HSV-1EdC virions inhibits the nuclear recognition of viral DNA (vDNA) by core PML-NB constituent proteins (PML, Sp100, and Daxx). This impairment of recruitment was independent of UV source, but dependent on dose and time course of UV irradiation that correlated with a loss in viral genome decompaction upon nuclear entry. Moreover, UV treatment promoted premature uncoating of viral genomes within the cytoplasm of infected cells in a dose-dependent manner. Our data highlights the importance of genome decompaction in PML-NB sensing of infecting viral genomes. Additionally, it uncovers a previously undescribed mechanism of action for the induction of innate immune defences during herpesvirus infection that have historically utilised UV inactivation to promote the activation of cellular pattern recognition receptors (PPRs) and induction of interferon stimulated gene (ISG) expression.

Access Microbiol. 2019 Apr 8;1(1A):15.

The macrophage intracellular niche and its role in cryptococcosis

Katherine Pline ^1,^*, Simon Johnston ¹, James Bradford ¹

Abstract

Cryptococcus neoformans is an opportunistic fungal infection that causes cryptococcal meningitis in immunocompromised individuals. Macrophages play a critical role in determining the outcome of infection, and can either phagocytose and kill the cryptococcal cells, or disseminate infection. While it is known that macrophages impact the progression of cryptococcal disease, it is not known how the macrophage intracellular niche contributes to complex infection outcomes. Clinical and experimental studies have identified potential genetic differences, in both host and pathogen, but statistical robustness has been difficult to achieve due the large variability in the outcome of infections. Therefore, in attempt to quantitatively explain this variability, we have used a zebrafish model of cryptococcal infection where we can directly relate the initial level of fungal infection with final infection outcome. We find that at low levels of initial fungal burden the outcome of infection is stochastic, while over high ranges of initial infection the outcome is linearly related to the initial burden, but with a further stochastic component that contributes to increased variability. Using these experimental data and data from clinical trials we have generated a computational simulation of in vivo cryptococcal infections which allows us to consider different infection variables and how they alter the progression of cryptococcosis. By combining such computational simulations with our experimental models we demonstrate that the macrophage intracellular niche determines the unknown stochastic component, independent of fungal burden. Using this knowledge, we can better identify the molecular, population genetic, and clinical parameters associated with the outcome of cryptococcosis.

Access Microbiol. 2019 Apr 8;1(1A):15.

The role of RNA-RNA interactions in the assembly and reassortment of influenza A viruses

Michael Knight ^1,^*, Bernadeta Dadonaite ¹, Brad Gilbertson ², Sanja Trifkovic ², Steven Rockman ², Alain Laederach ³, Lorena Brown ², Ervin Fodor ¹, David LV Bauer ¹

Abstract

Influenza A virus has a genome consisting of 8 segments of negative sense RNA. When two influenza A virus strains infect the same cell, there is potential for the progeny to package segments from both strains. This process is termed reassortment and can lead to rapid genetic shifts that have previously generated strains of influenza responsible for pandemic events. Recent evidence suggests that assembly of the eight influenza genomic segments for packaging into a virion is mediated by RNA-RNA interactions between the segments. These interactions are likely to contribute to the varying compatibilities for reassortment observed between segments from different strains of influenza. We have captured complete RNA-RNA interaction maps for several influenza A viruses using a high-throughput sequencing approach and identify extensive, redundant, networks of RNA-RNA interactions between the genomic viral RNA segments. We extended this analysis to H1N1 and H3N2 reassortants, and found that by manipulating these interactions, we can drive preferential co-segregation of segments during reassortment. This work provides the first direct evidence that RNA-RNA interactions between the influenza virus genomic segments are a key factor in driving reassortment between viral strains.

Access Microbiol. 2019 Apr 8;1(1A):15.

In silico identification of two novel antimicrobial peptides with antibacterial activity against multi-drug resistant Staphylococcus aureus

Linda Oyama ^1,^*, Hamza Olleik ², Ana Carolina Nery Teixeira ³, Matheus M Guidini ³, James A Pickup ¹, Alan R Cookson ⁴, Hannah Vallin ⁴, Toby Wilkinson ⁵, Denise Bazzolli ³, Jennifer Richards ⁶, Mandy Wootton ⁶, Ralf Mikut ⁷, Kai Hilpert ⁸, Marc Maresca ², Josette Perrier ⁹, Matthias Hess ¹⁰, Hilario C Mantovani ¹¹, Narcis Fernandez-Fuentes ⁴, Christopher J Creevey ¹, Sharon A Huws ¹

Abstract

Herein we report the identification and characterisation of two linear antimicrobial peptides (AMPs), HG2 and HG4, with activity against a wide range of multi-drug resistant (MDR) bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains, a highly problematic group of Gram-positive bacteria in the hospital and community environment. To identify the novel AMPs presented here, we employed the classifier model design, a feature extraction method using molecular descriptors for amino acids for the analysis, visualization, and interpretation of AMP activitiesfrom a rumen metagenomic dataset. This allowed for the in silicodiscrimination of active and inactive peptides in order to define a small number of promising novel lead AMP test candidates for chemical synthesis and experimental evaluation. In vitrodata suggest that the chosen AMPs are fast acting, show strong biofilm inhibition and dispersal activity and are efficacious in an in vivomodel of MRSA USA 300 infection, whilst showing little toxicity to human erythrocytes and human primary cell lines ex vivo. Observations from biophysical AMP-lipid-interactions and electron microscopy suggest that the newly identified peptides interact with the cell membrane and may be involved in the inhibition of other cellular processes. Amphiphilic conformations associated with membrane disruption are also observed in 3D molecular modelling of the peptides. HG2 and HG4 both preferentially bind to MRSA total lipids rather than with human cell lipids indicating that HG4 may form superior templates for safer therapeutic candidates for MDR bacterial infections.

Access Microbiol. 2019 Apr 8;1(1A):15.

In vivo and ex vivo models of infectious bursal disease virus (IBDV) in inbred chicken lines differing in their resistance to the disease

Amin Asfor ^1,^*, Kate Dulwich ², Andrew Broadbent ²

Abstract

Infectious bursal disease virus (IBDV) targets B cells in the bursa of Fabricius (BF), causing immunosuppression in chickens and mortality. Susceptibility differs between inbred chickens, with 0 % mortality in ‘resistant’ lines and up to 80 % mortality in ‘susceptible’ lines. However, the mechanism of disease resistance is not understood. In order to address this, chickens (n=18) from three ‘resistant’ lines (15, C and O) and one ‘susceptible’ line (W) were infected with the very virulent IBDV strain, UK661. Clinical scores were recorded and tissues harvested at necropsy on day one, two and three post-infection for RNA extraction and virus titration, compared to non-infected controls. Interestingly, within a given line, we observed a range of symptoms, with some individuals experiencing more severe disease than others, despite no difference in viral replication. Line 15 was the least susceptible to disease based on the average clinical scores (3.2 (15), 5.7 (C), 4.8 (O) and 4.7 (W)) and the percentage of birds with a clinical score of 2 or above (17 % (15), 100 % (C), 83 % (O) and 83 % (W)). The average peak virus replication was also significantly lower in line 15 birds (6.3 log10 fold change) compared to lines C or O (7.0 and 6.8 log10 fold change) (P<0.01). RNA-sequence analysis will be performed using BF samples to understand the biological pathways that confer IBDV resistance. Moreover, primary bursal cells harvested from resistant and susceptible lines were infected with IBDV ex vivo and ongoing work aims to quantify differentially expressed genes in these cells.

Access Microbiol. 2019 Apr 8;1(1A):15.

Global mapping of protein subcellular location in apicomplexans: the parasite as we’ve never seen it before

Konstantin Barylyuk ¹, Ludek Koreny ¹, Huiling Ke ¹, Simon Butterworth ¹, Imen Lassadi ¹, Tobias Mourier ², Lisa Breckels ¹, Laurent Gatto ¹, Arnab Pain ², Kathryn Lilley ¹, Ross Waller ^1,^*

Abstract

Apicomplexans are human and animal protozoan pathogens responsible for diseases including malaria, cryptosporidiosis and toxoplasmosis. As obligate intracellular parasites they are highly organised cells with numerous novel and specialised sub-compartments that form the basis of their invasion biology, host defence evasion, and novel metabolic traits. However, our understanding of these cells is highly constrained by our limited knowledge of the locations and functions of most of the cell’s proteome. Even in the best-studied apicomplexans (Plasmodium spp. and Toxoplasma gondii) only a small fraction of proteins’ locations have been experimentally determined, with most assignments based on predictions from orthologues in distant relatives. Moreover, many parasite proteins are annotated as ‘hypotheticals’, for example 4113 of 8121 Toxoplasma proteins, and many are unique to parasites stymying even predictions of location or function by comparative biology. To address this deficit in our basic understanding of the compositional organisation of the apicomplexan cell, we have used a spatial proteomics method called hyper LOPIT to simultaneously capture the steady-state subcellular association of thousands of proteins in the apicomplexan Toxoplasma. These protein atlases reveal: extensive protein association networks throughout the cell providing testable hypotheses of their function; conservation and novelty of compartment proteomes between apicomplexans; differential selective pressures across the different cell compartments; and clear instances of protein relocation from one organelle to a different one during apicomplexan speciation. This new, global view of the organisation of the apicomplexan cell proteome provides a much more complete framework for understanding the mechanisms of function and biology of these cells.

Access Microbiol. 2019 Apr 8;1(1A):15.

Human Cytomegalovirus pUL83 targets core histones to inhibit interferon synthesis and promote viral spread

Anasua Deb ^1,^*, Christina Paulus ¹, Michael Nevels ¹

Abstract

Tegument protein pUL83 is the most abundant component of human cytomegalovirus (hCMV) particles. The viral protein is predicted to be composed of three domains: a pyrin association domain (PAD), a carboxy-terminal domain (CTD), and an intrinsically disordered linker domain (amino acids 388–479) located between the PAD and CTD. Although pUL83 has been shown to antagonize interferon (IFN) responses, it has not been fully elucidated how the viral protein may contribute to hCMV replication. In this study we demonstrate that pUL83 associates broadly with viral and host chromatin including condensed chromosomes during mitosis. We further show that the linker domain in pUL83 is both required and sufficient for host chromatin targeting, and that this interaction depends on two evolutionary conserved arginine residues (R453 and R455) in the viral protein. Our data indicate that the pUL83 linker domain specifically associates with human core histones (but not linker histones). Furthermore, pUL83 inhibits IFN-beta and IFN-lambda gene induction, but not expression of other cytokine genes, via a mechanism that largely depends on the linker domain including R453/455. Although earlier studies suggested that pUL83 is dispensable for productive hCMV infection in fibroblasts, we find that the viral protein is necessary for efficient plaque formation in these cells, specifically in the presence of IFN. Finally, the pUL83 linker domain including R453/455 contributes significantly to the plaque size in hCMV-infected fibroblasts. Overall, we propose that pUL83 promotes spread of hCMV by selectively inhibiting induction of IFN gene expression via a novel chromatin-based molecular mechanism involving core histones.

Access Microbiol. 2019 Apr 8;1(1A):15.

Mapping the pH sensors critical for host cell entry by a complex Nonenveloped Virus

Weining Wu ^1,^*, Polly Roy ¹

Abstract

Bluetongue virus (BTV), in family Reoviridae, is an insect-borne, double capsid virus causing haemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. The recently solved high-resolution structures reveal unique features of BTV VP2 and VP5. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.

Access Microbiol. 2019 Apr 8;1(1A):15.

The great escape: dissecting the interactions between Mycobacterium bovis and the soil amoeba Dictyostelium discoideum

Rachel Butler ^1,^*, Alex Smith ¹, Tom Mendum ¹, Aneesh Chandran ¹, Huihai Wu ¹, Louise Lefrancois ², Thierry Soldati ², Graham Stewart ¹

Abstract

Mycobacterium bovis, a member of the M. tuberculosis complex, causes bovine tuberculosis, one of the most important veterinary health problems in the UK. In the absence of improved control the projected economic burden in the UK over the next decade is predicted to be £1bn. Although transmission of M. bovis occurs primarily between infected mammalian hosts, M. bovis has been demonstrated to persist in soil, suggesting an environmental infectious route. M. bovis is likely predated on by environmental amoebae such as Dictyostelium discoideum and as such may have evolved mechanisms to modulate the interaction with amoebae. In this study we have investigated M. bovis interactions in vitro with D. discoideum. We demonstrate that virulent M. bovis evades destruction by D. discoideum. Using a genome-wide M. bovis transposon mutant library, we selected for mutants that failed to escape D. discoideum after 48 h of infection. Mutants of genes encoding the MCE4 transport system, genes involved in sulpholipid synthesis/transport, and genes encoding PPE and PE-PGRS protein families remained associated with D. discoideum. Most strikingly, mutations in 11 genes of the major mycobacterial virulence locus ESX-1, which encodes a T7 secretion system implicated in bacterial transit through host cell membranes, were significantly enriched in D. discoideum. Our data demonstrate that known virulence factors involved in host-pathogen interactions in mammalian hosts also play a role in D. discoideum-M. bovis interactions. Our data further suggest that M. bovis has evolved to actively transit bacteriverous D. discoideum rather that to use it as a replicative niche.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigation of the Candida-host interaction using dual RNA-seq

Elise Iracane ^1,^*, Hrant Hovhannisyan ², João Miguel Oliveira Pacheco ¹, Marina Pekmezovic ³, Bernhard Hube ^3,^4,⁵, Toni Gabaldón ^2,^6,⁷, Geraldine Butler ¹

Abstract

Candida species are commensal yeasts but are also responsible of life-threatening infection in at-risk populations, like new-born or immunocompromised patients. Candida albicans is the most common causative species, and the most studied. Moreover, non albicans Candida species as Candida glabrata, Candida parapsilosis and Candida tropicalis cause a large proportion of infections. In our study we investigated the interaction between four Candida species and human vaginal epithelial cells A431 by using a dual RNA-seq method. Our aim is to identify the different transcriptomic response of each yeast, and of the host, during the infection of human cells. Gene Ontology analysis of up-regulated genes in the yeasts during infection implicated the ergosterol (ERG) pathway in C. parapsilosis only. We therefore investigated the role of the ERG pathway in the three Candida species in which it is currently possible to generate gene knockouts. The transcriptional factor Upc2 is the main regulator of ERG gene expression in C. albicans and C. parapsilosis. C. glabrata has two UPC2 orthologs, called CgUPC2A and CgUPC2B. We found that deleting CgUPC2A or CgUPC2B or both together does not reduce the damage inflicted by C. glabrata on host cells. However, deleting UPC2in C. albicans greatly reduces damage. Deleting UPC2 in C. parapsilosis appears to reduce damage of host cells; however further investigation is required. Our results show the that the role of ergosterol pathway in the host pathogen interaction differs between Candida species.

Access Microbiol. 2019 Apr 8;1(1A):15.

Modulation of arbovirus infection by mosquito saliva

Daniella Lefteri ^1,^*, Emilie Pondeville ², Steven Bryden ², Marieke Pingen ², Clive McKimmie ¹

Abstract

Arboviruses constitute a major public health problem, in particular mosquito-borne arboviruses that continue to emerge and re-emerge. Arbovirus infection of mammals is enhanced by the presence of a mosquito bite at the inoculation site, in comparison to virus experimentally administered by needle inoculation in the absence of a bite. Inflammatory responses to bites appear to be a key factor in this enhancement. However, the experimental inoculation of mosquito saliva with virus inoculum by needle, in the absence of bite trauma, also has the ability to enhance viral infections. In this study, we have studied the mechanistic basis for these observations. We have studied whether saliva from different mosquito species can enhance virus infection. Interestingly, while saliva from Aedes genus enhanced virus infection, An.gambiae saliva did not. Instead, An.gambiaesalivaactively inhibited infection compared to inoculation with virus alone. This could partly explain why An.gambiae mosquitos are unsuitable vectors for transmitting most arboviruses. By comparing the effects that saliva of these different mosquitoes have at the bite site we have further specified which inflammatory responses at the inoculation site modulate arbovirus infection in the skin. Using an in vivo mouse model we have shown that An.gambiae causes significantly less oedema but a higher up-regulation of key inflammatory genes in the skin than A. aegypti. As such, we are providing important insights into how mosquito saliva modulates infection. A better understanding of this will aid the development of anti-viral treatments by targeting factors within the mosquito bite that are common to many distinct infections.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating the mechanisms of BK polyomavirus egress and virus-host interactions

Sophia Ho ^1,^*, Laura Caller ¹, Rupert Beale ¹, Colin Crump ¹

Abstract

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that can establish a lifelong, silently persistent infection in the kidney and is estimated to infect 70–90 % of the world’s population. In immunocompromised individuals, particularly bone marrow and kidney transplant patients, increases in BKPyV replication can result in significant pathological conditions. In the case of kidney transplant patients, this can result in nephropathy, which in severe cases can result in the deterioration of allograft function and loss of the transplanted organ. There are currently no antiviral treatments with strong evidence of clinical efficacy against BKPyV. Though little is known about BKPyV egress from infected cells, we have evidence showing that BKPyV can be released in a non-lytic manner by an unconventional cellular secretory pathway that bypasses the Golgi apparatus. Here, we investigate the mechanisms behind BKPyV non-lytic egress through studying the effects of knocking out candidate host proteins involved in unconventional secretory pathways on BKPyV release, examining the effects of BKPyV infection on host cell protein secretion, and ascertaining which host proteins are essential for the BKPyV life cycle via a whole-genome CRISPR screen. These experiments are uncovering novel virus-host interactions that, when targeted, could lead to antiviral effects.

Access Microbiol. 2019 Apr 8;1(1A):15.

Metagenomic analysis of open-air and indoor spent fuel storage ponds at Sellafield, UK

Sharon Ruiz Lopez ^1,^*, Lynn Foster ², Hokyung Song ³, Nick Cole ², Jonathan Adams ⁴, Jonathan Lloyd ¹

Abstract

Nuclear power is an important energy source that can compensate for carbon emissions from fossil fuel power plants. However, processing of radioactive waste from nuclear plants is a significant challenge. The current treatment prior to final geological disposal involves wet storage of spent fuel in designated ponds, and microbial colonisation of these ponds can complicate plant operation. To help identify the key microbes that colonise hydraulically interlinked spent fuel storage ponds at Sellafield, UK, a series of samples were collected and analysed using next generation (Illumina) sequencing. Samples were taken from the facility’s indoor Fuel Handling Plant (FHP) pond (feeding head tank, main and subponds), and also from the open-air First Generation Magnox Storage Pond (FGMSP). 16S rRNA gene sequencing revealed that the FHP is colonized mainly by Bacteria (99%), affiliated with species of Curvibacter, Rhodoferax, Sphingomonas and Roseococcus, in addition to the hydrogen-oxidising bacterium Hydrogenophaga. In contrast the open-air FGSMP pond contained species of Hydrogenophaga, Nevskia, and Roseococcus, and also photosynthetic cyanobacteria (Pseudanabaena). Biological function was also assessed by metagenomic sequencing and analyses. The most abundant genes were associated with carbohydrate and protein metabolism, cell wall and capsule synthesis, stress responses and respiration. Genes involved in respiration were also more abundant in the indoor pond microbiome, including genes underpinning hydrogen metabolism, whilst photosynthesis genes were more abundant in the open-air ponds. These datasets give valuable insight into the microbial communities inhabiting nuclear storage facilities, the metabolic processes that underpin their colonisation and can help inform appropriate control strategies.

Access Microbiol. 2019 Apr 8;1(1A):15.

Type I interferon activity promotes a cellular environment that supports the establishment of latency by human cytomegalovirus

Paula Olaizola-Rebe ¹, Chinedu Onyerindu ¹, Kevin Kiesworo ¹, Manivel Lodha ¹, Thomas Stamminger ², Nicholas Peters ¹, Matthew Murray ^1,^*, Matthew Reeves ¹

Abstract

Type I interferons (IFN) are potent inducers of an anti-viral state in response to infection and have been demonstrated to inhibit cytomegalovirus (CMV) replication both in vitro and in vivo. CMV, like all herpes viruses, has the capacity to establish lifelong infections of host through the establishment of latency. As the very early stages of viral entry can trigger IFN responses we investigated the impact of IFN on the establishment of latent human CMV (HCMV) in myeloid progenitor cells. Here we show that priming of myeloid THP1 cells with type I IFN prior to infection skews infection towards a more efficient establishment of latency. This is evidenced by detection of reduced lytic gene expression, increased latent gene expression, and increased levels of reactivation following differentiation. Blockade of IFN signalling with neutralising antibodies antagonised the latent phenotype suggesting that endogenous IFN production upon infection contributed to the effect observed. Intriguingly, whilst both IFNα2 and IFNβ can drive latent infection individually, their effects were dose-dependent and demonstrated a biphasic impact on the establishment of latency, with the highest doses of IFN preventing both lytic and latent infection. These data demonstrate that the HCMV derives an unexpected benefit from IFN production. They support a hypothesis that, although anti-viral in nature, concentration-specific effects of IFN may be evident in the cells which can modulate different outcomes post infection in persistent viruses such as HCMV. Future work is identifying the IFN concentration-specific effects responsible for a cellular environment that favours the establishment of latency.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating the impact of Herpes simplex virus type 1 latency-associated non-coding RNAs on apoptosis in human neuronal cells

Amy Jacobs ^1,^2,^*, Stacey Efstathiou ³, Michael Nicoll ¹

Abstract

Herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons, allowing it to persist for the lifetime of the host. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 2.0 kb major LAT intron and several microRNAs. These non-coding RNAs (ncRNAs) have been reported to influence latency, possibly through limiting apoptosis of infected cells. As these studies have used animal models or non-neuronal cell culture, we have developed a differentiated human neuroblastoma (SH-SY5Y cells) model to examine their effect in human neuronal cells. We have infected these neuronal cultures with replication-defective HSV-1, which establishes a quiescent infection and strongly expresses the latency ncRNAs. We show that quiescent HSV-1 infection reproducibly protects differentiated SH-SY5Y from etoposide-induced apoptosis. We are also further defining the contribution of different LAT ncRNAs using recombinant lentiviruses to drive expression of the LAT intron or microRNAs. Furthermore, we are also currently exploring the mechanisms of this anti-apoptosis effect, and broader virus-neuron interactions by characterising whether the human neuronal transcriptome is altered by LAT RNA expression. Improving our understanding of the molecular interactions underpinning HSV-1 latency in neurons could help develop novel therapies to target HSV-1 latency.

Access Microbiol. 2019 Apr 8;1(1A):15.

Enhancing the unexplored chemical diversity of Streptomyces sp. to produce new antibiotics active against multidrug resistant Acinetobacter baumannii

Joshua Burns ^1,^2,^*, Samantha Law ², Christine Edwards ¹, Linda Lawton ¹

Abstract

Streptomyces species are a major source of antibiotics but are often grown under restrictive conditions that limit biosynthetic gene expression. As a result, the vast majority of secondary metabolites within a single species remain unexpressed in the lab along with the huge variety in chemical structures and bioactivities. The overarching aim of this project was to identify conditions that produce novel antibiotics, specifically against Gram-negative pathogens. The culture collection NCIMB contains hundreds of Streptomyces isolated from around the world. Strains were selected from the collection guided by preliminary bioactivity studies and available literature. These were then grown in an extensive variety of conditions designed to stimulate production of a wide variety of secondary metabolites, detected by UPLC-MS and analysed using the freely available metabolomic tools MZmine, MetaboAnalyst, and GNPS. Conditions included various carbon and nitrogen sources, temperatures, stresses, epigenetic inhibitors, and other microbes. Compounds active against the Gram negative multidrug resistant pathogen Acinetobacter baumannii were detected in the scaleup culture supernatant fractions. Metabolite identification through GNPS did not detect any previously discovered compounds active against A. baumannii, indicating a potentially novel antibiotic against one of the WHO’s priority pathogens.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating the role of the bacterial mechanosensitive channel YnaI in Salmonella pathogenesis

Mimi Asogwa ^1,^*, Samantha Miller ¹, Stefania Spano ¹, Mark Stevens ²

Abstract

Mechanosensitive channels are required for bacteria cells to survive hypoosmotic shock (transition from high to low osmolarity environment). YnaI is one of the mechanosensitive channels found amongst many bacterial species including Salmonella Typhimurium. Previous studies have suggested thatS. Typhimurium YnaI may be implicated in host colonization during infection of farmed animals. Disruption of ynaI impaired intestinal colonization in pigs, cattle and chicken. To investigate S. Typhimurium YnaI structure and function, the S.Typhimurium ynaI was cloned into a plasmid, followed by physiological characterization of the S. Typhimurium YnaI constructs expressed in an E. coli channel-less mutant strain. To further understand the role of YnaI in S. Typhimurium pathogenesis, S. Typhimurium ynaI was deleted and the ability of mutant to survive and replicate in host cells was investigated. The S. Typhimurium YnaI channel has unique characteristics when expressed in E. coli: S. Typhimurium YnaI channel conferred almost complete protection against 0.3 M NaCl hypoosmotic shock when overexpressed, but interestingly, high level expression of S. Typhimurium YnaI inhibited growth in two different complex media and in minimal media. Deletion of ynaI from S. Typhimurium led to increased internalization in macrophages and epithelial cells. Data derived from this study reveals novel characteristics of S. Typhimurium YnaI which may provide insights into other functions of the S. Typhimurium YnaI channel.

Access Microbiol. 2019 Apr 8;1(1A):15.

Characterization the role of key DNA sensors in herpes simplex virus replication

Hanan Moshrif ^1,^2,^*, Peter O’Hare ¹

Abstract

DNA sensors including cGAS, STING and IFI16 are key components of the innate immune response to infection. However, the precise mechanisms of action, in particular the relative importance of direct suppression of replication versus paracrine signalling of an antiviral state to susceptible cells remains unclear. We examined the kinetics of herpes simplex virus infection and spread in a relevant cell type, human keratinocytes, lacking one or other of these DNA sensors using time-lapse microscopy. We also examine transcriptional induction of interferon from the native locus, at single cell and single molecule level using highly sensitive RNA FISH. Our results reveal distinct aspects of the roles of these factors and reveal outcomes not appreciated by other methods. Cells lacking either of these factors showed increased susceptibility to initial infection (prior to any downstream paracrine signalling) but with quite different outcomes. Lack of cGAS resulted in increased cellular migration and cell density at the infection focus. On the other hand, cells lacking STING showed lower cell density and significantly increased cytopathic effect likely curtailing virus yield. Initial results demonstrate that we can analyse interferon transcription at single cell level with exquisite sensitivity down to a few transcripts per cell and reveal profound spatial heterogeneity in responses to induction by PAMP ligands. Altogether, our results reveal new insight into the spatial landscape of the initiation and spread of HSV and key cellular responses which likely integrate pathways including innate immunity, apoptosis and cell migration.

Access Microbiol. 2019 Apr 8;1(1A):15.

Global gene expression profiling of a virulent Klebsiella pneumoniae strain during pulmonary infection

Timothy Kidd ^1,^2,^*, Amy Dumigan ², Brian Forde ¹, Minh-Duy Phan ¹, Rocio Canals ³, Jay Hinton ³, Mark Schembri ¹, Jose Bengoechea ²

Abstract

Background

Klebsiella pneumoniae (Kpn) is an important respiratory pathogen associated with significant mortality, fierce inflammatory responses and high rates of antimicrobial resistance. The increasing incidence of multidrug resistant Kpn has significantly narrowed the therapeutic options available; as such, there is an urgent need to better understand Kpn pathophysiology to identify novel therapeutic targets. Here we performed an in vivo transcriptomic analysis of Kpn isolated from a mammalian host with pulmonary infection.

Methods

C57BL/6 mice were intranasally inoculated with the virulent Kp52.145 strain (serotype O1:K2); with lungs extracted, homogenised and pooled (n=3; in duplicate) at 32 h post-infection for bacterial RNA purification and RNA-Seq (Illumina). Differential gene expression (analysed using Degust [Voom/Limma]; FDR cut-off =0.01, abs log-FC=2) was assessed in comparison with mid-log phase growth in Lennox broth.

Results

Overall, we identified >900 differentially expressed genes (DEGs), comprising ∼17 % of the combined chromosomal and plasmid coding sequence repertoire. 52 % of the DEGs were upregulated during infection, including several siderophore-independent iron-, manganese- and zinc-uptake systems (e.g. hmuRSTUV, sitABCD, mntH and znuACB). We also observed a marked in vivo oxidative stress signal, with several Kpn oxidative stress response genes (e.g. oxyR, katE, katG) upregulated during infection. In contrast, expression of mgrB, a negative-regulator of the PhoPQ two-component system associated with resistance to host antimicrobial peptides was downregulated.

Conclusion

This study provides a novel insight into Kpn gene expression during pulmonary infection. Overall, our data suggest that adaptation to metal starvation, oxidative stress and innate immune defenses are critical for the success of Kpn lung infection.

Access Microbiol. 2019 Apr 8;1(1A):15.

Genomic evolution of Klebsiella pneumoniae clones: the good, the bad and the ugly

Kelly Wyres ^1,^*, Ryan Wick ¹, Louise Judd ¹, Roni Froumine ¹, Alex Tokolyi ¹, Claire Gorrie ¹, Margaret Lam ¹, Sebastián Duchêne ¹, Adam Jenney ², Kathryn Holt ^1,³

Abstract

Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Access Microbiol. 2019 Apr 8;1(1A):15.

Why does a heterotrophic marine protist produce carotenoids? Genetic approaches to investigate the ecophysiology of the thraustochytrid Aurantiochytrium limacinum

Mariana Rius ¹, Joshua Rest ¹, Jackie Collier ^1,^*

Abstract

Thraustochytrids are abundant and ubiquitous osmoheterotrophic marine protists (labyrinthulomycetes, stramenopiles) thought to function ecologically as fungus-like decomposers. Some thraustochytrids have the ability to synthesize carotenoids, including carotenes (e.g. beta-carotene) and xanthophylls (e.g. astaxanthin), which is uncommon among heterotrophic eukaryotes. Carotenogenic thraustochytrids appear to have acquired carotenoid biosynthetic enzymes by horizontal gene transfer from bacteria. Heterotrophic production of carotenoids is typically associated with protection against oxidative stress, and in thraustochytrids may be particularly associated with protecting large amounts of essential omega-3 polyunsaturated fatty acids stored in lipid droplets. To gain better understanding of carotenoid function in thraustochytrids, and thus new insight into the ecophysiology of these organisms, we have produced mutants of the thraustochytrid Aurantiochytrium limacinum in which the trifunctional gene Aurli_150841, encoding the first three carotenogenesis-specific reactions (phytoene synthase, phytoene desaturase, lycopene cyclase), has been interrupted by double homologous recombination with a construct containing a zeocin resistance (BleoR, shble) expression cassette. As predicted, the Aurli_150841 knockout mutants lack the carotenoid pigmentation found in the wild-type. Complementation with the wild-type Aurli_150841 to confirm that this phenotype is due to the knockout is in progress. Differences between the wild-type and Aurli_150841 knockout mutants in features such as growth rate and biomass yield, lipid content, survival in stationary phase and response to oxidative stress are being evaluated under growth conditions that induce different amounts of carotenoid accumulation in the wild-type.

Access Microbiol. 2019 Apr 8;1(1A):15.

The genomic (re)definition of EPEC

Eva Heinz ^1,^2,^*, Alison Mather ^3,², Nicola Petty ^4,⁵, Teemu Kallonen ^6,⁵, Torsten Semmler ⁷, Lothar Wieler ⁷, Waldir Elias ⁸, Adriana Regua-Mangia ⁹, Jukka Corander ^6,⁵, Tânia Gomez ¹⁰, Claire Jenkins ¹¹, Gad Frankel ¹², Nicholas Thomson ^5,¹³

Abstract

Enteropathogenic E. coli (EPEC) were the first E. coli strains linked to human disease (1945), and pose a serious health threat. The burden for public health changed over the past century as it did for other diarrheal diseases; whilst it is still endemic in large parts of South America, cases in Europe are commonly associated with recent travel. Despite its long-standing importance for global health and its significant impact on child mortality in Low- and Middle Income Countries (LMICs), we still have very limited understanding of what defines an EPEC beyond the diagnostic LEE island, or indeed if there are any shared characteristics, as most in-depth analyses of their pathogenic determinants are confined to few model strains. We present a study analysing ∼1300 whole-genome sequences combining published and newly sequenced datasets of EPEC and non-EPEC strains to map the evolutionary history and molecular determinants. Importantly, we have expanded the published data with whole-genome sequences of 300 historical and contemporary EPEC strains from a large collection of clinical isolates, mainly from Brazil and England, which enables us to compare the epidemiology in a high-income with a LMIC over an extended time frame. We furthermore present a molecular definition of EPEC, including mapping of phage islands and de-novo prediction of effectors in this large-scale dataset, as well as investigating the patterns of adhesins and other secretion systems, thus characterising different EPEC lineages which have emerged numerous times during the evolution of E. coli.

Access Microbiol. 2019 Apr 8;1(1A):15.

Uncovering the molecular basis of viable but non culturable (VBNC) cells

Sariqa Wagley ^1,^*, Richard Titball ¹, Clive Butler ¹

Abstract

Viable but non-culturable (VBNC) cells are cells that are metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can ‘resuscitate’ restoring their ability to grow on media. Currently, over 80 bacterial species are reported to enter the VBNC state. The ability of VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in pathogens may pose a risk to human health and thus warrants further investigation. This research project has investigated the ability of the human pathogen Vibrio parahaemolyticus to form VBNC cells when exposed to stressful conditions. V. parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide and often results in watery/bloody diarrhea and vomiting. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and report that different sub populations of VBNC cells can occur based upon their metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella. Using mass spectrophotometry we have identified several proteins which may play roles in VBNC formation and resuscitation in V. parahaemolyticus.

Access Microbiol. 2019 Apr 8;1(1A):15.

Developing genetic manipulation platforms for Naegleria gruberi

Eleanna Kazana ¹, Tobias von der Haar ¹, Anastasios D Tsaousis ^1,^*

Abstract

Naegleria gruberi, is a free-living microbial eukaryote, which belongs to the group of Excavates. The organism is widely distributed especially in aquatic environments and it is famous for its ability to transform from an amoeba to flagellate and cyst forms depending on its surroundings. In silico examination of the published Naegleria gruberi genome opened up the possibility of functional exploration of the organism by molecular cell biology. Despite this, several attempts to genetically transfect or genetically manipulate the organism have been unsuccessful so far due to the unique morphological and cellular adaptations of the organisms, but also due to its resistance to certain basic antibiotics. Using a series of protocols and combination of cell biological tools, we attempted to genetic manipulate Naegleria using both CRISPR/Cas9 and traditional genetic transformation protocols. Preliminarily data from these investigations will be discussed. This work is going to provide traits found in the last eukaryotic common ancestor and provide a model for investigating the cell biology of other free-living eukaryotes.

Access Microbiol. 2019 Apr 8;1(1A):15.

Clinical management of low-level hepatitis B surface antigen in haemodialysis patients with a recent history of HBV vaccination – the results of a UK-wide survey

Megan Goddard ^1,^*, Judith Timms ¹

Abstract

Identifying transient low-level hepatitis B (HBV) surface antigen (sAg) post-vaccination is a well-established phenomenon in haemodialysis patients. These results create a clinical conundrum; should patients be deemed potentially infectious, requiring further testing and heightened infection control precautions, or should negligible risk be assumed? National guidance covering this scenario is lacking; therefore we conducted a survey of the clinical practice of UK virology departments. Thirty-six laboratories were contacted and 17 responses received (47 %). Eleven responders had observed sAg positivity post-vaccination, for a maximum duration of 3 weeks. Clinical management was highly variable. Only 3 hospitals had specific written policies, with others following an internal consensus or a standard protocol for sAg positive patients without recent vaccine. Further testing consisted of HBV serology (3/11), HBV PCR (1/11) or PCR and serology (7/11). Three departments were highly risk averse, recommending universal isolation with dedicated dialysis machines. Two departments did not advise additional precautions and 5 based decisions on risk factors e.g. foreign travel. Heightened precautions were stopped on the basis of negative serology (1/11), PCR (5/11) or serology and PCR (2/11). To summarise, vaccine-derived sAg positivity is commonly encountered on haemodialysis units. Recommendations on the interval between vaccination and testing would alleviate the issue, with this study suggesting a minimum of 3 weeks. Infection control precautions varied considerably. In most cases, patients are risk assessed for isolation as a minimum; however this may depend on local expertise and the availability of isolation facilities. It is clear that national guidance would standardise patient care.

Access Microbiol. 2019 Apr 8;1(1A):15.

A large-scale investigation of stress response mechanisms in the industrial yeast Kluyveromyces marxianus

Noemi Montini ^1,^*, Tyler Doughty ², Ivan Dmenzain Del Castillo Cerecer ², Javier Varela ¹, Darren Anthony Fenton ¹, Pavel Baranov ², Jens Nielsen ², John Morrissey ¹, Verena Siewers ²

Abstract

Microbial production strains need to operate under sub- optimal growth conditions such as low pH, high osmolarity and thermal stress. The capacity to carry out industrial fermentations at higher temperatures reduces the risk of bacterial contamination and lowers cooling costs. We want to understand the basis of thermotolerance in the industrial yeast Kluyveromyces marxianus. As part of the EU-funded project, CHASSY, K. marxianus was grown in chemostat cultures under different stress conditions and a multi-omics analysis performed to study a range of stress responses, including elevated temperature (40 °C). Transcriptomes were generated from steady state cultures growing at identical growth rates under different stress conditions and gene set enrichment analysis (GSEA) performed. A range of functions were identified as being specifically expressed at higher temperatures and these are now being further investigated. One example is the temperature-specific expression of two putative hexose transport genes. Subsequent mutational inactivation using CRISPR and heterologous complementation established that at least one of these two genes is required for growth at (40 °C). We are now trying to determine the substrates for, and the precise function of, these genes. We also developed a ribosome profiling pipeline for K. marxianus and are using this to investigate the translational response to temperature stress. The combined study of both transcription and translation at steady state and as a culture responds to a temperature shift will give a comprehensive view of the basis of thermotolerance in K. marxianus and should identify strategies to exploit this in biotechnological processes.

Access Microbiol. 2019 Apr 8;1(1A):15.

Effects of an RNA chaperone on mutation tolerance

Valerie Soo ^1,^*, Tobias Warnecke ¹

Abstract

Due to their intrinsic thermodynamic properties, RNA can misfold easily in cells. One way to mitigate RNA misfolding is through the actions of RNA chaperones, which bind and unwind structured RNA molecules and thereby offer opportunities for these misfolded species to refold properly. Such rescue activity has implications for the fitness effects of individual mutations-- at least mutations that compromise RNA folding or structure might be buffered by RNA chaperones. However, little is known about the rules governing such mutation buffering. Here, we describe how a model RNA chaperone, the DEAD-box RNA helicase CYT-19, affects the fitness effects of mutations in a model structured RNA, the Tetrahymenagroup I intron, whose self-splicing activity is dependent on its structure. We performed deep mutational scanning on the P1ex region of the intron which is critical for its self-splicing activity, and assayed differential splicing activity of all possible P1ex mutants in the presence and absence of CYT-19 to identify mutations that are buffered by RNA chaperone activity. I will discuss the properties of the chaperone-dependent and chaperone-independent mutation pools. Our results highlight that, to understand RNA robustness in vivo, we need to consider how mutational fitness effects are modulated by RNA chaperones and other trans-acting factors.

Access Microbiol. 2019 Apr 8;1(1A):15.

The biocide triclosan triggers multiple regulatory systems in Staphylococcus aureus to induce antibiotic tolerance

Dean Walsh ^1,^*, Jonathan Aylott ¹, Kim Hardie ¹

Abstract

The biocide triclosan is used extensively in household and hospital settings, resulting in chronic exposure to the biocide in individuals that use triclosan-containing products. Triclosan is thought to induce antibiotic tolerance and alter biofilm formation, although the underlying mechanisms causing these changes are yet to be elucidated. If true, the widely used biocide may contribute to antibiotic treatment failures, and therefore requires investigation. To determine how triclosan induces antibiotic tolerance, Staphylococcus aureus was pre-treated with triclosan prior to treatment with the clinically relevant antibiotics ciprofloxacin, rifampicin, and vancomycin. Planktonic S. aureus cultures pre-treated with triclosan had 1000 fold higher viable counts compared to non triclosan pre-treated cultures. Inspection of biofilms by live/dead staining found that triclosan pre-treatment protected S. aureus biofilms from treatment with otherwise lethal doses of ciprofloxacin, rifampicin, or vancomycin. Biofilms of mutant strains with a defective stringent response were not protected from antibiotic treatment, even in the presence of triclosan. Interestingly, stringent response mutants still exhibited triclosan-induced antibiotic tolerance in planktonic culture, but mutants with a defective agr quorum sensing system did not. Confocal laser scanning microscopy revealed that incubation of S. aureus with triclosan altered biofilm structure, resulting in increased proportions of polysaccharide in the biofilm matrix that could potentially mediate protection against antibiotics. Neither the stringent response mutants nor agr mutants influenced triclosan-induced biofilm changes, suggesting another, currently uncharacterised response. We suggest that triclosan triggers multiple global regulatory systems in S. aureus, subsequently inducing tolerance to multiple antibiotic classes and altering biofilm structure.

Access Microbiol. 2019 Apr 8;1(1A):15.

Using quantitative proteomics to analyse HCMV manipulation of dendritic cells following cell-cell transfer

Lauren Kerr ^1,^*, Lior Soday ², Michael Weekes ², Chris Von Ruhland ¹, Edward Wang ¹, Richard Stanton ¹

Abstract

Human cytomegalovirus (HCMV) rapidly mutates during in vitro passage, and this strongly alters the way the virus spreads. In vivo HCMV spreads by direct cell-cell contact, as do recent clinical isolates. In contrast, passaged strains spread via cell-free virions. Because of this, cell-cell spread remains largely uncharacterised. We have developed a strain (Merlin) that retains a full length, wildtype genome. As a result, it mimics clinical HCMV and spreads by direct cell-cell contact, a method of spread that is more resistant to neutralising antibodies, and innate and intrinsic immunity. We now show that each cell-cell transfer is equivalent to an extremely high MOI infection, with up to 300 genomes delivered to each cell, potentially providing an explanation for the ‘immune-evasive’ properties of cell-cell transfer. Furthermore, infectious virions accumulate at cell-cell contacts between cells. This may represent a ‘virological synapse’ that protects virions from neutralising antibodies. Not only does Merlin enable us to characterise cell-cell spread, but it enables us to infect a wide range of clinically relevant cells with a virus expressing the complete complement of virus genes. In vivo, HCMV infects dendritic cells (DCs), but is never cleared, implying that it is able to subvert DC function. Therefore, we performed quantitative proteomic analysis of infected primary immature DCs, following cell-cell transfer. This quantified 7992 intracellular proteins, and 703 plasma membrane proteins. Over 99 proteins were downregulated following infection. Many of these are DC-specific, and have roles in regulating adaptive immunity. These viral-manipulations may therefore dramatically impact DC function.

Access Microbiol. 2019 Apr 8;1(1A):15.

Multi-scale variability analysis of Arctic soil microbial communities

Lucie Malard ^1,^*, David Pearce ¹

Abstract

Understanding distribution patterns at various spatial scales is a central issue in microbial ecology. Beyond the lone identification of biogeographical patterns, understanding the environmental drivers behind community diversity and structure is key. While many studies identify pH as a major parameter structuring microbial communities at large spatial scales, many other variables impact distribution patterns on smaller scales. Here, we investigated the biogeographical patterns of Arctic soil microbial communities from 1 m to 500 m, within Adventdalen, Svalbard, using 16S sequencing, gravimetric measurements and X-ray fluorescence. Multivariate analyses identified key environmental variables shaping microbial communities and revealed the importance of soil moisture, organic carbon and elements such as aluminium, calcium and potassium in structuring distribution patterns. The indicator species analyses identified key associations between environmental variables and OTUs. Using geostatistical kriging, we mapped the biodiversity and distribution of key OTUs across the landscape. Overall, our results highlight the spatial heterogeneity in Arctic soils and identifies the sampling scale needed to characterize microbial communities within an area of interest with seemingly homogeneous landscape.

Access Microbiol. 2019 Apr 8;1(1A):15.

Flavivirus membrane (M) proteins as potential ion channel antiviral targets

Emma Brown ^1,^*, Hannah Beaumont ¹, Daniella Lefteri ¹, Matthew Bentham ¹, Richard Foster ¹, Clive McKimmie ¹, Antreas Kalli ¹, Stephen Griffin ¹

Abstract

The Flavivirus small integral membrane (M) protein resides within mature infectious particles yet, unlike the envelope (E) protein which mediates membrane fusion upon encountering low pH within the acidifying endosome, the function of M within this context is unknown. We are investigating whether Zika virus (ZIKV) M protein exhibits channel activity, acting as a viroporin playing a role mediating virus entry and uncoating. Importantly, ZIKV entry was blocked in a dose-dependent fashion by the prototypic channel blocker rimantadine and the drug also prevented virus spread in mouse models of ZIKV infection. Molecular dynamics simulations supported that M protein is able to oligomerise into a hexameric viroporin channel, opening of which is promoted in an acidified environment via protonation of a conserved histidine residue. Rimantadine is predicted to bind onto this structure in silico at a lumenal binding site, against which we are currently designing improved small molecule inhibitors that could form the basis of novel M protein targeted drug discovery. Importantly, drugs targeting M might either prevent or reduce the severity of ZIKV infections, including those crossing the placenta, and could also be translated for use against other Flaviviruses.

Access Microbiol. 2019 Apr 8;1(1A):15.

The ‘missing’ gastric microbe; the impact of gastric cancer-associated microbiota on Helicobacter pylori growth in vitro and its implications in gastric carcinogenesis

Dana Alfawaz ¹, Paidamoyo Katsande ¹, Helen Jordan ¹, Sarah Kuehne ¹, Richard Ferrero ², Amanda Rossiter ^1,^*

Abstract

In recent years, multiple studies have examined the bacterial communities present in the gastric microbiota during the progression to gastric cancer (GC). Although Helicobacter pyloriis the biggest risk factor for GC, the microbiota of GC comprises a decreased load of H. pylori and an enrichment of bacteria, such as Prevotella spp., Veillonella spp., Actinomyces spp. However, interactions between H. pylori and these cancer-associated bacteria is hugely understudied. Here, we have used hypoxic growth conditions (5 % O₂) to investigate polymicrobial interactions between cancer-associated bacteria and H. pylori in vitro. We found that whilst the co-culture of H. pylori with Prevotella spp. and Veillonella spp. had no effect on growth of either bacteria, Actinomyces oris completely inhibited the growth of H. pylori. Moreover, A. oris did not inhibit the growth of other Gram-negative pathogens such as Salmonella Typhimurium and E. coli, whilst there was a slight growth inhibition of Campylobacter jejuni. Furthermore, ultrafiltration of A. oris culture supernatants revealed that inhibition is mediated by a secreted factor larger than 5 kDa, which can be heat inactivated. Interestingly, Actinomyces viscosus can also specifically kill H. pylori suggesting that this inhibition could be conserved across the Actinomyces genus. We are currently identifying the inhibitory factor responsible for inhibiting H. pylori growth. Furthermore, we are investigating whether A. oris can clear gastric H. pylori infection in a mouse model of infection and the implications of this on gastric carcinogenesis. In conclusion, whilst data-rich microbiota studies continue to thrive, it is imperative that we understand the mechanisms underpinning changes to the gastric microbiota and whether these bacteria are drivers or ‘passengers’ of gastric carcinogenesis.

Access Microbiol. 2019 Apr 8;1(1A):15.

Proteomic analysis reveals vector-virus interactions between Zika virus and Aedes aegypti mosquito cells

Rommel Gestuveo ^1,^2,^*, Margus Varjak ¹, Alain Kohl ¹

Abstract

Zika virus (ZIKV) is an arbovirus (family Flaviviridae) mainly transmitted by Aedes mosquitoes causing febrile illness and Zika congenital syndrome in infants if mothers were infected during pregnancy. ZIKV manipulates its host’s cellular machinery in order to facilitate infection and evade antiviral responses. The identification of host and vector proteins involved in these processes may lead to novel antiviral strategies. In this study, Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C) or anchored capsid (AC) proteins were developed to investigate virus-vector protein interactions. To identify interaction partners, immunoprecipitation (IP) of V5-tagged C or AC was performed and subjected to proteomic analyses using nLC-MS/MS under label-free quantification conditions. A total of 148 and 53 mosquito protein interactors unique to C and AC were identified, respectively. Protein network and gene ontology analyses showed biological processes possibly important for ZIKV infection. To investigate further the role of these proteins during infection, 25 were chosen for dsRNA-based knockdown screen and infection with reporter virus (ZIKV-Nluc) in AF5 cells. Significant reduction in reporter virus signal was observed during knockdown of 6 interactors suggesting a pro-viral role for these proteins during infection. This was corroborated by conducting the same knockdown experiments but infecting with a clinical isolate of ZIKV (PE243), which showed reduced virus RNA levels and titre. Interestingly, three of the six proteins are part of the ubiquitin-proteasome pathway (UPP). Currently, functional experiments are underway to investigate the role of UPP during ZIKV infection.

Access Microbiol. 2019 Apr 8;1(1A):15.

Mastitis and animal husbandry – high-throughput sequencing as a support tool

Daniel Blake ^1,^2,^*, Edward Haynes ¹, Ilias Kyriazakis ²

Abstract

Mastitis is a disease of the mammary gland which affects most mammals and is one of the costliest ongoing challenges in modern dairy farming. The most significant challenges in mastitis management are the speed and accuracy of diagnosis and the use of antibiotics. Current mastitis diagnosis in veterinary practices in the United Kingdom utilises culture-based techniques. However, this approach has some limitations including; processing time, species selection biases and culture failure. This ongoing PhD project seeks to assess the utility of high-throughput sequencing technology in addressing the current challenges in mastitis diagnosis. Samples were obtained from cattle from farms in North Yorkshire, United Kingdom. Cattle were selected from monthly somatic cell count records as mastitis positive cases with a cell count between 250 000 and 550 000 cells ml⁻¹ or control animals with a count below 200 000 cells. Mastitis positive cases were further split into bacteriology positive and negative groups. During sampling a cow-side somatic cell test (California Milk Test) was performed to confirm the monthly database readings. All farms sampled were conventional dairy farms with comparable management systems. Samples were collected by trained veterinarians following a pre-defined protocol designed to limit sample contamination. Microbial community diversity, richness and composition will be compared between sample groups to survey for variations which may explain why culture fails in culture negative subclinical cases. 131 sample animals have been assigned to study groups with samples collected, DNA extracted and prepared for sequencing on the Illumina MiSeq platform with results expected in January 2019.

Access Microbiol. 2019 Apr 8;1(1A):15.

Unravelling a co-nsP-iracy: the role of chikungunya virus non-structural protein 3 in replication and pathogenesis

Siu Yi Lee ^1,^*, Nicola Stonehouse ¹, Mark Harris ¹

Abstract

Chikungunya virus (CHIKV) is a member of the Alphavirus genus, transmitted to humans by mosquitoes of the Aedesgenera. Infection with CHIKV causes chikungunya fever, which in many cases can lead to chronic joint disease, leaving patients with reduced ambulation. Despite its rising potential as a threat to global health, no effective vaccine or antiviral agent for protection or treatment are available. The CHIKV non-structural protein 3 (nsP3) is essential to the virus lifecycle and is believed to be a component of the genome replication complex. However, to date, the exact role of this protein has yet been determined. Although a conserved polyproline motif in the C-terminal hypervariable domain of nsP3 has been reported to interact with cellular SH3 domains, the function of this motif remains enigmatic. To address this question we generated a panel of mutations in this motif and tested the phenotype in the context of both a subgenomic replicon and full-length infectious virus, in both mammalian and mosquito-derived cell lines. Most of the mutations were well tolerated in the sub-genomic replicon, however, a subset either attenuated or completely abolished production of infectious CHIKV. These results suggest that as well as its role in genome replication, nsP3 also functions during assembly and release of infectious virus particles and that the C-terminal polyproline motif is a critical determinant of this function.

Access Microbiol. 2019 Apr 8;1(1A):15.

From Antarctic DNA to stress tolerant crop plants – exploiting the why protein domain

Jasmin Mertens ^1,^*, Dominique Anderson ², Eloy Ferreras ¹, Don Cowan ¹

Abstract

Screening of an Antarctic soil functional fosmid metagenomic library identified a novel bacterial gene, homologous to known Water Hypersensitivity (WHy) domains. The WHy domain is a typical component of Late Embryogenesis Abundant (LEA) proteins which occurs widely in both prokaryotes and in plant eukaryotes and are expressed under various stress conditions [1]. A phylogenetic analysis of multiple WHy homologues from different species suggested that the ancestral origin of this protein gene lies within the ancient archaea [1]. Our previous studies have shown that this bacterial protein elicits significant protection against freeze and cold stress in recombinant E. coli [2]. Expression of the WHy gene in Arabidopsis resulted in a wide range of statistically significant stress-tolerant phenotypic properties. These included an increase of up to 6-fold higher germination efficiency of transgenic recombinant seeds compared to the WT, and a 100 % survival rate of WHy gene-expressing plants compared to 0 % survival of adult WT plants after freeze shock. Similar improvements in survival rates were observed for recombinant plants in drought stress experiments.

References

Mertens J, Aliyu H, Cowan DA (2018). Applied and environmental microbiology, AEM-00539.

Anderson D, Ferreras E, Trindade M, Cowan D (2015). FEMS Microbiology Letters, 362(15):fnv110.

Access Microbiol. 2019 Apr 8;1(1A):15.

Characterisation of bovine dendritic cells following FMDV infection

Amina Yasmin ^1,^2,^*, Katy Moffat ¹, Elizabeth Reid ¹, Bryan Charleston ¹, Simon Milling ², Julian Seago ¹

Abstract

Dendritic cells (DCs) are the sentinels of the immune system, responsible for recognising invading pathogens and priming the adaptive immune system for appropriate responses. Hence they are considered potential targets for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Little is known of the events of FMDV replication in bovine moDCs. Present work therefore sought to characterize FMDV and its immune complex (IC) replication in bovine moDCs in vitro. A chimeric heparin sulphate FMDV (O1M) was used in this study. Immuno-fluorescence microscopy (IFM) and quantitative RT-PCR was used to analyse viral replication at 0–6, 8, 16 and 24 hpi. Plaque assays were used to investigate the yields of live virus produced in moDCs at 0, 4, 8 and 24 hpi. FMDV and IC FMDV could infect moDC. In moDC infected with FMDV alone, or with immune-complexed (IC) FMDV, replication was observed by IFM between 2–4 and 1–16 hpi. In contrast, for both FMDV and FMDV IC infections RT-PCR analyses showed viral replication peaked at 4 hpi and then decreased between 8 to 24 hpi. Plaque assays using supernatants of the infected moDC showed no evidence of an increase in viral titre at 24 hpi. The detection of viral nsp (3AB and derivatives) suggests replication of FMDV persists for longer in moDCs when entry is mediated by IC. However, the lack of increase in virus yield suggests replication is abortive. One possible explanation for this difference could be that bovine moDCs are able to recognise non-immune complexed FMDV more rapidly.

Access Microbiol. 2019 Apr 8;1(1A):15.

Using a metabolic model of Acetobacterium woodii for insights into its utility for biotechnological purposes

Noah Mesfin ^1,^*, David Fell ¹, Noah Mesfin ^1,^*

Abstract

Acetogens are microbes which produce acetate as a fermentation by-product. They have diverse phylogeny but a metabolic feature in common called the Woods-Ljungdahl Pathway (WLP), which confers the ability to fix carbon dioxide via a non-photosynthetic route. Electrons for this process are derived from diverse substrates including molecular hydrogen and carbon monoxide. The ability of acetogens to utilise components of syngas (H₂, CO, CO₂) make them an attractive target for metabolic engineering for industrially relevant products. We have previously reported the construction of a genome-scale metabolic model of the model acetogen Acetobacterium woodii using a sequenced and annotated genome of strain DSM1030. The model consists of 836 metabolites, 909 reactions and 84 transporters and can account for growth on diverse substrates reported in the literature. We identified the reactions used to catabolise fifteen single substrates and 121 substrate pair combinations, and used this to construct a sub-model representing a core set of energy producing catabolic pathways. We then introduced heterologous reactions to allow for the production of chemical of interest. Elementary modes analysis of this extended sub-model was applied to further decompose it into unique sets of the smallest functioning sub-networks. With CO₂ and H₂ as substrates, we find routes for the production of several chemicals where small amounts of excess ATP are produced simultaneously. Repeated analysis with alternative renewable feedstocks such as methanol and formate, indicate a wider potential in producing compounds of interest while also maintaining energy generation and co-factor conservation.

Access Microbiol. 2019 Apr 8;1(1A):15.

Phylogenetics and vector competence of a bovine ephemeral fever virus strain from Israel

Mar Fernandez de Marco ¹, Luis Hernandez-Triana ¹, Daniel Dorey-Robinson ¹, Lorraine McElhinney ¹, Chris Sanders ², Simon Carpenter ², Anthony Fooks ¹, Olga Zalensky ³, Boris Gelman ³, Oran Erster ³, Nicholas Johnson ^1,^*

Abstract

Bovine ephemeral fever virus (BEFV) [Family Rhabdoviridae: genus Ephemerovirus] causes a transient febrile illness in cattle that results in economic losses and morbidity, with occasional mortality. Epidemics of the disease have been particularly costly in countries of the Middle East, including Israel. The virus is considered a vector-borne pathogen although the exact relationship with a number of blood-feeding arthropods has not been established. In order to improve developments in diagnostic detection, phylogeographic investigations and virus-vector relationships of BEFV we have derived the first genome of this virus from an isolate from Israel and used this to assess recent outbreaks of disease. We have also investigated the vector competence of BEFV with a number of target arthropod species. The complete genome sequence of BEFV (Israel strain 1) is 14 850 base pairs in length and shows over 95 % identity with the only other Middle Eastern genome for BEFV from Turkey. A wider phylogeny shows that these viruses form a clade, previously described as cluster II, which is clearly distinct from BEFV strains derived from China, Japan and Australia. However, there is a distinct separation of those viruses from Israel to others in the Middle East. Preliminary vector competence studies suggest that at least three species of mosquito that are potential transmission vectors of the virus are incapable of infection with, or transmission of, BEFV. Current studies are ongoing to assess other blood-feeding insect species as potential vectors.

Access Microbiol. 2019 Apr 8;1(1A):15.

Characterisation of new environmental bacteriophages targeting the Escherichia coli LamB outer membrane porin

Ziyue Zeng ^1,^*

Abstract

Bacteriophages are viral parasites of bacteria. A successful infection starts with the adsorption of the bacteriophage to a specific receptor on the host cell surface. Most bacteriophages are thought to have a narrow host range but this can be extended in certain cases. One strategy forextending host range is to first express a known functional bacteriophage receptor protein in bacteria previously non-susceptible to the bacteriophage, thereby enabling adsorption and potential infection by viruses that target the specific receptor. To investigate the feasibility of this approach, a plasmid (pMUT13) encoding the Escherichia coli LamB porin, the receptor for bacteriophage Lambda, was transferred into three different enterobacterial genera, namely Citrobacter, Yersinia, and Serratia. Over 100 environmental bacteriophages were isolated that infected these pMUT13-containing strains, and some bacteriophages were shown to infect their respective hosts in a LamB-dependent way. The host ranges of the environmental bacteriophages were cross-tested across the heterologous genera and surface adsorption kinetics investigated. Unlike bacteriophage Lambda, which is a member of the Siphoviridae, these newly-isolated LamB-dependent bacteriophages were more commonly members of the Myoviridae, based on transmission electron microscopy and whole genome sequences. Furthermore, an interesting selection of evolved bacteriophage mutants with broader host range were isolated, and the key mutations involved in their evolution to adapt to new hosts were investigated by genome analysis.

Access Microbiol. 2019 Apr 8;1(1A):15.

The role of SARM in the control of immune response driven by Klebsiella pneumonia infection

Claudia Feriotti ^1,^*, Jose Bengoechea ¹

Abstract

Introduction

Klebsiella pneumonia is a Gram-negative, capsulated bacteria, which is an important cause of community-acquired and nosocomial pneumonia. Klebsiella co-opts cellular functions dedicated to control immune balance to limit the activation of inflammatory responses. SARM (Sterile α-and armadillo-motif containing protein), the fifth identified member of the TIR (Toll-interleukin 1 receptor (1LR)) adaptor family, negatively regulates IRF and NF-kB activation by affecting TLR4 and TLR3 TRIF-dependent signalling. It is currently unclear the role, if any, of SARM in bacterial infections. Here, we aim to dissect the contribution of SARM in Klebsiella infections, and, specifically, to investigate whether Klebsiella may exploit SARM as part of the pathogen’s portfolio immune evasion strategies.

Results

SARM contributed to Klebsiella anti-inflammation strategies in macrophages through by increasing AKT phosphorylation (to limit phagosome-lysosome fusion), and preventing the activation of NF-kB (to control inflammatory responses). SARM also negatively regulated type I IFN regulatory factor (IRF3) by decreasing IRF3 phosphorylation. Notably, SARM played a role as inflammasome inhibitor, as observed by increased IL-1β secretion in the supernatants of infected sarm-/-. SARM also inhibited ASC oligomerization in Klebsiella-infected macrophages as seen by the increased ASC monomers release by sarm-/- BMDMs. SARM was also required for pyroptosis following Klebsiella infection. Interestingly, Klebsiella induced the expression of SARM in a TLR4-TRAM-TRIF-IRF3-IFNAR dependent manner, demonstrating that Klebsiella exploits type I IFN to trigger SARM to control inflammasome activation, and the activation of inflammatory responses.

Conclusions

These findings have uncovered how Klebsiella manipulates the TLR adaptor SARM to dampen the activation of host defences.

Access Microbiol. 2019 Apr 8;1(1A):15.

Evolutionary strategies of Bdellovibrio bacteriovorus predators and prey

Kim Summers ^1,^*, Jan-Ulrich Kreft ¹

Abstract

Antibiotic resistance is a serious threat to human health and new treatments for bacterial infections are urgently needed. Bacteriophages, first used at the beginning of the 20th century, and the predatory bacterium Bdellovibrio bacteriovorus (discovered in 1962) are potential alternatives to antibiotics. We developed a mathematical predator prey model to explore the effects of Bdellovibrio and bacteriophage on prey bacterial numbers. Our system has an abiotic resource that is consumed by the E. coli prey following Monod kinetics and up to two predator species with Holling type I or type II functional responses. As Bdellovibrio spends considerable time in the periplasm of its prey as a ‘bdelloplast’, this stage is also modelled, giving a delay between prey removal and ‘birth’ of predators. We used the model to examine the effects of Bdellovibrio and a bacteriophage on prey populations and found a distinct difference in effectiveness between Bdellovibrio and bacteriophages. We also looked at how various biological factors change predation effeciency. We found that there is an optimal predator:prey ratio for the predator. We also discovered that there is an optimal attack rate and an optimal mortality for the predator.

Access Microbiol. 2019 Apr 8;1(1A):15.

Generation, lyophilisation and epitope modification of high titre filovirus pseudotyped lentiviruses for use in antibody neutralisation assays and ELISA

Martin Mayora-Neto ^1,^*, Emma Bentley ², Edward Wright ³, Nigel Temperton ¹, Ivo Ploemen ⁴, Peter Soema ⁴, Rimko ten Have ⁴, Stuart Masterson ⁵, Martin Michaelis ⁵, Mark Wass ⁵, Simon Scott ¹

Abstract

The 2014–2016 Ebola outbreak in West Africa highlighted the need for improved diagnostics, surveillance and therapeutics for filoviruses. The need for high containment virus handling facilities creates a bottleneck hindering research efforts. A safe alternative to working with native viruses are pseudotyped viruses (PV) which are non-replicating particles bearing surface glycoprotein(s) that can be used for antibody detection. The aim of this study was to create a diagnostic tool to distinguish between genera and species of pathogenic filoviruses (e.g. neutralization tests and ELISA), avoiding the cross reactivity currently seen. High titre PVs bearing the receptor glycoprotein (GP) of different filovirus species, plus specific epitope chimeras, were successfully generated. Next, lyophilisation studies to assess particle stability/degradation transportation and long-term storage were conducted. Filoviruses maintained their titres for at least 1.5 years after lyophilisation when kept in temperatures of up to 4 °C, with all filovirus genera following a similar trend. At higher temperatures, PVs degraded to unworkable titres. Reconstituted PVs also performed well in neutralisation assays. A chimeric cuevavirus GP bearing ebolavirus (Zaire sp.) epitopes KZ52 and 1 H3 retained infectivity, with average titres of approximately 1×10 7 RLU ml⁻¹, similar to wild type, indicating its structure was not compromised. These chimeras are now being assessed in neutralisation tests using specific monoclonal antibodies and incorporated into ELISA with PVs as antigens. The data suggests lyophilised PVs are amenable to long-term storage, and their GPs can be modified to create artificial antigens for diagnostics and serosurveillance.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of putative packaging signals in the RNA of foot-and-mouth disease virus (FMDV)

Chris Neil ^1,^*, Grace Logan ¹, Joseph Newman ¹, Joseph Ward ², David Rowlands ², Nic Stonehouse ², Toby Tuthill ¹

Abstract

Viruses in the picornavirus family comprise a single molecule of positive sense RNA contained within a simple non-enveloped capsid. The mechanism for RNA packaging is not well understood. We have developed a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. To further test the function of these putative packaging signals (PPS), we have developed a trans-encapsidation assay using subgenomic replicons expressing GFP, helper virus and flow cytometry. The results of these studies provide evidence for the involvement of predicted RNA structures in picornavirus packaging and offer readily transferable methodologies for identifying packaging requirements in many other viruses.

Access Microbiol. 2019 Apr 8;1(1A):15.

Discovering the biology behind the organism while developing genetic tools for Corallochytrium limacisporum

Aleksandra Kozyczkowska ¹, Sebastian Najle ¹, Iñaki Ruiz-Trillo ¹, Elena Casacuberta ^1,^*

Abstract

To address biological questions that cannot be answered by current model organisms, we need to develop genetic tools in the specific taxa that can provide the best answers. Such is the case of the origin of animals in which, genetic tools need to be developed among the closest unicellular relatives of animals. To fill this gap, we are developing genetic tools in Corallochytrium limacisporum, a close unicellular relative of animals that also has a fascinating biology. Corallochytrium is a marine free-living walled saprotroph that develops through a choenocyte. Moreover, because of the basal phylogenetic position of Corallochrytria together with Ichthyosporea, this lineage is especially informative to fill the void of information between yeast and metazoans. We have successfully developed transient and a stably transfection protocols by introducing the resistance gene to puromycin, allowing us to select individual transformants. Deep characterization of the established transformed lines has revealed important biological features of this organism such as the plasticity of its genome, the mode of plasmid integration, and some differences between the two known strains (Hawaii and India). Currently we are better characterizing these features and, in parallel, developing genome-editing technologies. Progress and the potential implications of our research will be presented and further discussed.

Access Microbiol. 2019 Apr 8;1(1A):15.

The African horse sickness virus NS4 counteracts the antiviral response and is a determinant of viral virulence

Yi Jin ¹, Siddharth Bakshi ¹, Marco Caporale ², Aislynn Taggart ¹, Meredith Stewart ^1,^*, Massimo Palmarini ¹

Abstract

African horse sickness is a major infectious disease of equids and is caused by African horse sickness virus (AHSV), a dsRNA virus with 10 genome segments encoding for 7 structural and 4/5 non-structural proteins. Here, we focused on the characterisation of the AHSV NS4, the latest protein found to be expressed by this virus. In silico analysis of available sequences confirmed the existence of two phylogenetically distinct AHSV clades: NS4-I and NS4-II. NS4-II is further divided into three subtypes (a, b and g). Confocal microscopy demonstrated that all AHSV NS4 types localised in the cytoplasm of infected cells, unlike the BTV NS4 which, has a strong nucleolar localisation. The replication kinetics of reverse genetics derived AHSV NS4 deletion mutants (AHSVDNS4) were similar to their wild type counterparts in insect (Kc) or interferon incompetent (BSR) cells. However, replication of AHSVDNS4 mutants in primary horse endothelial cells was restricted, in comparison to wild-type viruses. Importantly, primary cells restriction to AHSV replication was dependent on the JAK/STAT pathway. Furthermore, AHSVDNS4 mutants were not able to efficiently suppress the secretion of anti-viral cytokines from primary cells, while the wild-type viruses suppressed this response to varying degrees. Importantly, AHSVDNS4 mutants were less virulent than their wild type counterparts in a murine model of AHSV infection. These results indicate that AHSV NS4 has a role in interferon IFN antagonism and a determinant of viral virulence. We are currently carrying out mass spectrometry analyses to identify the cellular proteins interacting with the AHSV NS4.

Access Microbiol. 2019 Apr 8;1(1A):15.

The co-occurrence and co-exclusion of evolving objects in prokaryotes

Fiona J Whelan ^1,^*, Martin Rusilowicz ², James O McInerney ¹

Abstract

Throughout evolution, evolving objects (domains, genes, operons etc.) have continuously combined, forming new proteins, gene clusters, and genomes. Horizontal gene transfer, particularly among prokaryotes, has facilitated this combinatorial process. Thus, evolving objects that interact positively or synergistically with each other are expected to co-occur more often than by chance; conversely, evolving objects may avoid co-occurrence, indicating an antagonistic or redundant functionality between objects. In this work, we use methods adapted from graph theory to understand patterns of co-occurrence and exclusion in prokaryotes. We have implemented multi-level graph models in which each node (vertex) is a gene or species connected by an edge (relationship) to another node to display these coincidence relationships. Our method incorporates the phylogenetic distribution and synthenic distances of these genes, and we demonstrate how these concepts can be used to identify conserved clusters of vertical and horizontally inherited units of selection. We apply these multi-level graph models to a variety of datasets including prokaryotic pangenomes, and metagenomic sequencing datasets from human-associated microbial communities. We find evidence for genes that significantly co-occur with each other within each of these datasets; these genetic clusters include objects from characterized biological pathways but also include genes with unknown functions. Further, we identify genes that exclude each other, indicating evolving objects with antagonistic or redundant biological functions. This work represents a different approach to understanding the evolution of prokaryotes and allows us to draw novel hypotheses as to the potential role of these genetic clusters in prokaryote biology.

Access Microbiol. 2019 Apr 8;1(1A):15.

Human papillomavirus E6 regulates the trafficking of gap junction protein Cx43

Li Dong ^1,^*, Scott Johnstone ², Sheila Graham ¹

Abstract

High-risk human papillomavirus (HR-HPV) infects epithelial cells and is the major cause of anogenital and oropharyngeal cancers. HR-HPV oncogenic activity is through E6 control of p53, but E6 binds and degrades PDZ proteins such as the tumour suppress protein hDlg (human homologue of DrosophilaDiscs Large). The E6/hDlg complex also contains Connexin 43 (Cx43) (MacDonald, Sun et al. 2012), the major building block of gap junctions that allow intercellular molecular communication. In HPV16-positive non-tumour cervical keratinocytes (W12NT: low E6 levels) Cx43/hDlg is on the plasma membrane but in the cytoplasm in W12T tumour epithelial cells (W12T: high E6 levels) correlating with loss of gap junction cell-cell communication. E6 siRNA depletion in W12T cells restored Cx43 to the cell membrane, while overexpressing E6 in HPV-negative cervical cancer cells C33a resulted in Cx43 moving to the cytoplasm. E6 could control Cx43 trafficking through controlling hDlg or by altering cell signalling. In the absence of E6 in HEK293, HaCaT and normal immortalised keratinocytes (NIKS), Cx43 and hDlg could be co-immunoprecipitated and they co-localised on the plasma membrane. Thus, the Cx43/hDlg interaction is not carcinoma cell-specific, is not dependent on HR-HPV E6, and may have a functional role in non-cancer cells. siRNA depletion of hDlg, led to reduction in Cx43 protein levels and some relocation to the cytoplasm. This indicates that HPV E6 controls Cx43 through interaction with and degradation of hDlg. However, we cannot discount that E6 itself may have additional effects on Cx43 levels and trafficking besides via hDlg.

Access Microbiol. 2019 Apr 8;1(1A):15.

Cross-sectional study of respiratory Aspergillus spp. colonization or infection in patients with various stages of chronic obstructive pulmonary disease (COPD) using culture vs non-culture based technique

Sarmad Waqas ^1,^2,^*,, Katie Dunne ¹, Alida Fe Talento ², Graham Wilson ², Ignacio Martin-Loeches ², Joseph Keane ^1,², Thomas R Rogers ^1,²

Abstract

Background

COPD patients are now recognized to be at increased risk of colonization by Aspergillus spp. which may progress to invasive pulmonary aspergillosis (IA). Published data on the frequency of Aspergillus detection in COPD are limited.

Methods

A cross-sectional study was undertaken to determine Aspergillus colonization or infection in COPD patients undergoing bronchoscopy for any indication. Culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL).

Results

One hundred and fifty patients were included (44.7 % female, mean age 68.2 years). 21.3 % were inpatients, 74.7 % outpatients and 4 % were ICU patients. Investigation of lung masses was the most common indication (43.3 %) for bronchoscopy. Most patients (81.3 %) were either GOLD stage 1 or 2 COPD. Cancer was the most frequent co-morbidity (60.48 %). 12 % and 48.7 % were on systemic and inhaled steroids respectively. Lung mass was the most common (28.43 %) CT imaging finding. Seventeen patients (11.3 %) had a positive result for Aspergillus (Culture+Galactomannan+PCR). 76.4 % out of these seventeen were in the early stages (GOLD stage 1 or 2) of COPD.

Conclusion

Aspergillus sp. was detected in 3.3 % of patients by culture, which increased to 11.3 % if culture was combined with either a positive GM or PCR result. Overall the frequency of Aspergillus detection in this population of COPD patients was low which may reflect the predominance of Gold stages 1 and 2 among the study population.

Access Microbiol. 2019 Apr 8;1(1A):15.

Modification of the ADP-ribose-1"-monophosphatase domain in recombinant infectious bronchitis virus affects viral replication in vitro and attenuates the virus in vivo

Giulia Dowgier ^1,^*, Sarah Keep ¹, Paul Britton ¹, Erica Bickerton ¹

Abstract

The gammacoronavirus infectious bronchitis virus (IBV) is responsible for an acute respiratory disease in domestic fowl, which has high economic impact and welfare implications in the poultry industry. The IBV non-structural protein, nsp3, is a multifunctional protein containing several putative domains, including an ADP-ribose-1’-monophosphatase (ADRP) domain conserved among coronaviruses. Inactivation of the ADRP domain in alpha- and betacoronaviruses is associated with reduced pathogenicity in vivo and altered interferon response and cytokine profiles in the host, without affecting viral replication in vitro. Therefore, recombinant viruses lacking ADRP functions have been proposed as ideal candidates for live attenuated vaccines. A recombinant IBV (rIBV) was generated in the backbone of the pathogenic M41-K strain containing a mutation in the ADRP domain catalytic core, known to abolish ADRP function in other coronaviruses. The ADRP-defective rIBV was characterised in vitro and in vivo; conversely to previously described ADRP-defective coronaviruses, in vitro analysis showed a reduction of viral replication, and the rIBV displayed a distinctive plaque phenotype. No reversion of the mutation occurred after serial passages of the virus in primary avian cell culture, nor in ex vivotracheal organ cultures which wereutilisedas a surrogate for in vivo stability testing. Pathogenicity experiments conducted in vivo resulted in a reduction in clinical signs in comparison to M41-K-infected birds, and tracheal ciliary activity, a marker for pathogenicity, was comparable to mock infected birds. These data support the role of ADRP as a pathogenic determinant and demonstrate the potential of ADRP-defective rIBV as a promising candidate vaccine.

Access Microbiol. 2019 Apr 8;1(1A):15.

Marine Streptomyces spp. isolates with synthetic polyesters-degrading activity

Eduardo Leao de Almeida ^1,^*, Andrés Felipe Carrillo Rincón ¹, Seija Elizabeth Nevalainen ¹, Stephen Jackson ¹, Niall O’Leary ^1,², Alan Dobson ^1,²

Abstract

The rapid expansion of global plastic production in the last number of decades (>355 million tonnes in 2017), coupled with poor waste management, has resulted in an estimated 5–12 million metric tonnes of plastic waste entering our oceans. Packaging applications account for much of the current waste production, and commonly include polyethylene- and polyethylene terephthalate (PET)-based materials that are resistant to natural degradation processes, particularly in marine environments. In response to this global marine pollution issue and the continuing demand for effective treatment of such plastics in terrestrial environments (e.g. 27.1 million tons of annual, post-consumer plastic waste in Europe alone), researchers have focused on addressing the biodegradation of recalcitrant plastic waste such as PET. To this end, we screened 20 Streptomyces spp. strains isolated from marine sponges for polycaprolactone (PCL)-degrading activity, which is considered a model substrate for PET. Although the Streptomyces genus is commonly explored for natural products discovery, little is still known about its potential for polymer degradation. Genomic analysis of two of the Streptomyces isolates which screened positive for PCL-degrading activity were found to have PET-hydrolase gene homologs that shared 41 % identity to the well-characterised PETase from Ideonella sakaiensis 201 F6. One of these genes was subsequently heterologous expressed in E. coli in order to further characterise the enzymatic activity and other biochemical properties of the enzyme.

Access Microbiol. 2019 Apr 8;1(1A):15.

Microbiology in primary school teaching

Lisa Crossman ^1,^2,^*

Abstract

Within a large-scale report commissioned by the Wellcome Trust1, primary school teachers were described as facing barriers in teaching science. The top barriers described were the lack of budget and resources, lack of time and curricular importance as well as other issues such as a lack of subject knowledge or confidence and concerns relating to space and resource access. Teaching science is just one part of a primary teacher’s complex role and is a subject in which most primary teachers do not have a degree or A level qualification2,3. There is little Microbiology content within the National Curriculum, however, schools can introduce additional scientific content within the Primary Key Stages. Within this context, a Microbiology, Genomics and Bioinformatics researcher in association with Key Stage 2 classes in a Norfolk Junior School carried out a joint project for Microbiology-related science in conjunction with Norwich Research Park facilities in May 2017 and the Microbiology Society. Here we report on the findings from teacher and pupil’s perspectives and consider how Microbiology/Hygiene could be presented to this age group in a classroom setting.

1. ‘State of the Nation’ report of UK primary science education. S. Leonardi et al. CFE Research, Leicester LE1 5TE. September 2017.

2. The Royal Society (2010) Science and mathematics education 5–14. A ‘state of the nation’ report. London: the Royal Society

3. ASE Guide to Primary Science Education (Serret and Earle, 2018), reviewed in https://tdtrust.org/cpd-primaryscience

Access Microbiol. 2019 Apr 8;1(1A):15.

SNP based transmission study of badgers infected with Mycobacterium bovis in the edge risk area of England

Colman O’Cathail ^1,^*, Elsa Sandoval Barron ¹, Ben Swift ², Malcolm Bennett ¹, Richard Emes ¹

Abstract

Mycobacterium bovisis the causative agent of bovine tuberculosis (bTB), one of the most costly and persistent agricultural infectious diseases still widespread in England today. However, not all parts of England are created equally with respect to the incidence of bTB. In 2012 the Department for the Environment, Food and Rural Affairs (DEFRA) divided the country into three risk areas; the high, low and edge risk areas. The perpetuation of the bTB epidemic is often blamed on the known wildlife host of M. bovis, the European badger (Meles meles). Despite this, no large scale studies examining both the prevalence and transmission patterns of M. bovis in this species has been undertaken. Here we describe the results of a major survey of 600 road-kill badgers from 6 counties within the edge risk area of England. Mycobacterium like colonies were isolated from over 80 carcasses of which, 65 were confirmed to be tuberculosis complex positive by PCR. These isolates were spoligotyped, VNTR typed and then subsequently whole-genome sequenced. We describe a SNP based transmission analysis of the sequenced isolates that provides a higher degree of resolution between the badgers compared to the described molecular typing methods.

Access Microbiol. 2019 Apr 8;1(1A):15.

The persistence and dynamics of commensal poultry gut flora within a broiler rearing house

Helen Brown ^1,^*, Sara Healy ¹, Isabella Pursley ¹, Daniel Parker ², Grant Hayes ², Daniel Horton ¹, Roberto La Ragione ¹, David Neilson ³

Abstract

Commercial poultry rearing systems often house successive flocks of birds with limited between-flock cleaning of the poultry houses. Previous research focused on opportunities for successive flocks to become colonised with pathogenic bacterial species. However, there is a paucity of information regarding the transfer and persistence of commensal bacterial between flocks, and if this might confer health benefits on subsequent flocks. The work presented here utilised 16S community sequencing to characterise the development of the microbial flora of commercially reared broiler chickens and turkeys to determine microbial environmental persistence. DNA was isolated from caecal contents, faeces, and various bedding samples collected from nine sites over a nine month period. Samples were taken from houses used for rearing chickens or turkeys or where alternating chicken and turkey flocks were reared. Measures of alpha diversity for the different samples suggested that both chickens and turkeys had a similar microbiota. Unsurprisingly, members of the microflora could also be found in the environmental samples tested, although survival was dependent on the phyla and bedding material. Further analysis of the samples is currently underway, in order to determine the extent, if any, of microbial transfer between flocks, with a particular focus on potential poultry microbiota species differences. This study demonstrated how commensal microbes are able to persist within poultry rearing sheds and if this transfer impacts on subsequent flock performance. Alongside increasing understanding of microbial environmental persistence, the work also shows how effective current biosecurity methods are in controlling the transfer of all microbes, including pathogens.

Access Microbiol. 2019 Apr 8;1(1A):15.

Of microscopes and microbes; novel applications of optical microscopy to microbiology

Liam Rooney ^1,^*, Paul Hoskisson ¹, Gail McConnell ¹

Abstract

The development of optical microscopy has been traditionally driven by the needs of eukaryotic cell biology. Therefore, there are a number of unmet requirements for the application of advanced microscopy techniques to the field of microbiology. Conventional techniques, such as widefield epi-fluorescence and confocal laser scanning microscopy, are common-place in most laboratories, however these methods have trade-offs in terms of their attainable resolution and limited imaging volume. Here we present the application of several optical microscopy methods with the aim addressing the unmet needs of the field; increasing spatial resolution and sampling volume. We demonstrate the use of Interference Reflection Microscopy (IRM) for investigating the morphology and gliding motility of Myxococcus xanthus. This label-free technique provides super-resolution in the axial plane where changes in cell shape on the order of 100 nm can be detected in live cells. Using IRM we show novel insights into the gliding behaviour of these bacteria. We also present the application of the Mesolens to microbiology. The Mesolens is a large optical microscope with the unique combination of a low magnification and a high numerical aperture which results in an imaging volume >100 mm 3 with isotropic sub-cellular resolution. We demonstrate the use of the Mesolens to image live bacterial communities at multiple spatial scales simultaneously and offer new insights for bacterial community dynamics and biofilm architecture. Our work details novel applications of advanced microscopy to the field, and in doing so fills the technology gap which has previously restricted the study of complex microbial behaviours.

Access Microbiol. 2019 Apr 8;1(1A):15.

Role of BPIFA1 in the pathogenesis and immune response against Influenza A virus in mice

Sanaria Al Katy ^1,^2,^*, Anja Kipar ³, James Stewart ¹

Abstract

The epithelial layer of the respiratory system has a critical role in the defense against microbes and secretes a number of proteins that function in host defense. BPIFA1 is secreted by the epithelium of the respiratory tract and we have shown previously that it inhibits binding and entry of Influenza A Virus (IAV) into respiratory epithelium (Akram et al. 2018), Mucosal Immunol (11, 71). However, its precise biological functions remain unclear. The aim of this study was to assess the influence of BPIFA1 in antibody production during IAV infection. BPIFA1 KO and wild type C57BL/6J mice were infected with IAV using different virus doses and blood, broncho-alveolar lavage and nasal washes were collected at several time points for analysis of IAV-specific antibodies. The results showed that BPIFA1 has role in the efficient generation of virus-specific IgA in the respiratory tract. Thus, BPIFA1 has an important role not only in innate defense but also in the adaptive immune response against IAV.

Access Microbiol. 2019 Apr 8;1(1A):15.

PorA-Loop4 derived peptides of Neisseria meningitidis cause a G1 cell cycle arrest through the Akt signalling pathway in human brain microvascular endothelial cells

Rininta Firdaus ^1,^2,^*, Neil J Oldfield ¹, Karl G Wooldridge ¹

Abstract

Neisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.

Access Microbiol. 2019 Apr 8;1(1A):15.

Discovery of novel highly divergent RNA viruses in European rodents and rabbits

Theocharis Tsoleridis ^1,^*, Joseph Chappell ¹, Okechukwu Onianwa ¹, Elodie Monchatre-Leroy ², Gérald Umhang ², Mang Shi ³, Rachael Tarlinton ⁴, Patrick McClure ¹, Edward Holmes ³, Jonathan Ball ¹

Abstract

Although RNA viruses are likely to exist in every species of cellular life, our knowledge of their biodiversity and evolution has been focused on those that can cause disease in humans and in economically important animals and plants. Recently published studies, however, have shown that every healthy organism can carry viruses. In this study, we present the unbiased discovery of highly divergent RNA viruses in European rodents and rabbits using Next Generation Sequencing (NGS). Tissue samples from different organs were collected from UK rodents and French rabbits and were initially screened for viruses with PCR. Following up on preliminary data, the positive samples were sent for NGS to acquire full genomes and perform unbiased virus discovery. Our findings encompass a number of novel viruses including astrovirus, rotavirus A, hantavirus, picornavirus, coronavirus, paramyxovirus etc. Among the novel viruses, the picornavirus interestingly showed 50 % and 34 % similarity to the closest relative for the non-structural and the structural protein respectively at amino acid level, suggesting a potentially novel genus within the family. This study shows the presence of highly divergent RNA viruses in European rodents and rabbits enriching thus our current knowledge of their origin and evolution. Finally, this study shows that these animal species can be the reservoirs of RNA viruses notorious for cross-species transmission, increasing the risk of a spill-over to humans or livestock.

Access Microbiol. 2019 Apr 8;1(1A):15.

Waste not, want not: enhancing the ability of yeast to utilise its own leftovers from the brewing industry to fuel the transport industry with ethanol

Ainsley Beaton ^1,^*, Nicholas Tucker ¹, Franck Escalettes ², Leonardo Magneschi ²

Abstract

The global brewing industry produces a large amount of waste, 85 % of this is composed of spent brewers’ grain. One use for this waste product is in the bioethanol industry where the yeast, S. cerevisiae uses the spent grain as a feedstock. Due to the nature of the feedstock, there is a lack of utilisable carbon for S. cerevisiae. To obtain optimum utilisation of the waste product in conjunction with high process efficiency, enhanced carbon metabolism of the production strain is required. As well as expanded nutrient utilisation there is also a requirement to maintain high ethanol production and ethanol tolerance that industrial strains have acquired in a preferred growth medium. We are using high-throughput phenotypic arrays to rapidly identify strains best able to grow in a wide range of conditions, including various carbon and nitrogen sources and multiple stress inducing conditions. This method has shown small but measurable differences between production strains in industrially relevant growth conditions. In collaboration with an industrial partner, both targeted and random chromosomal integration of transgenes have been made to multiple candidate production strains to improve recycled feedstock utilisation and process efficiency. In addition, whole genome sequencing is being utilised to interrogate the genetic basis for phenotypic differences between production strains. It has been found that some important null phenotypes are at the transcription level, this information is now in use to drive future rounds of genetic manipulation.

Access Microbiol. 2019 Apr 8;1(1A):15.

Repurposing histone deacetylase inhibitors (HDACi) to treat Candida glabrata infections

Callum J O’Kane ¹, Andrew Marshall ¹, Mary J O’Connell ², Edel M Hyland ^1,^*

Abstract

Candida glabrata currently accounts for 25 % of all fungal cases in UK hospitals, second only to C. albicans. This number is expected to rise given the intrinsic anti-fungal resistance of this species and the difficulty in treating it. In an effort to identify novel-anti fungal targets in C. glabrata, we used comparative genomics within Saccharomycotina yeast to predict which genes are under positive selection in this species specifically. Such genes are predicted to have influenced the adaptation of C. glabrata from a free-living microbe to a human pathogen, potentially due to functional shift(s) of the proteins they encode. Our analysis predicts that histone acetylation pathways are under positive selection in C. glabrata. Thereforewe hypothesised that we could use histone deacetylase inhibitors (HDACi) to interfere with histone acetylation levels and impact C. glabrata virulence. By treating C. glabrata withbroad spectrum HDACis we show it has a reduced capacity to form biofilms, it is less well adapted to high salt conditions typically found within a human host, and most importantly, it reverts to a more anti-fungal sensitive state. RNAseq analysis indicates that HDACi treatment interferes with the C. glabrata transcriptional response to anti-fungal treatment, rendering it incapable of combating against these drugs. Furthermore, using an in vivo worm model of candidiasis, we show that HDACi treatment in conjunction with the anti-fungal fluconazole, can increase the survival rate of individuals with C. glabrata infections. Taken together our data suggest that the health threat posed by C. glabrata might be addressed by repurposing HDACi to treat this infection.

Access Microbiol. 2019 Apr 8;1(1A):15.

Influence of gestational and developmental age on human airway epithelial innate immune responses to Respiratory Syncytial Virus (RSV) in early life

Helen Groves ^1,^*, Lindsay Broadbent ¹, Hong Guo-Parke ¹, Mike Shields ¹, Ultan Power ¹

Abstract

Background

RSV is a major respiratory pathogen in infants, with preterm and young infants being at increased risk of severe disease. Airway epithelial cell (AEC) innate immune responses represent an important first line of defence. Development of these responses in early life is poorly understood.

Methods

Well differentiated cultures were generated using nasal AECs harvested from term and preterm infants at birth and from the same infants at one-year old. Cultures were infected with RSV and resulting cytopathology and innate immune responses studied.

Results

RSV growth kinetics were similar between preterm and term newborn cohorts at birth and in the one-year-old repeat cohort. Secretion of interferon lambda-1 (IFN-l1), CXCL10 (IP-10), CCL5 (RANTES) and CXCL8 (IL-8) were similar between RSV-infected preterm and term newborn AECs. Following RSV infection, significantly higher secretion of IFN-l1 (P=0.0034), CXCL10 (P<0.05) and CCL5 (P<0.05) was noted in one-year-derived AECs compared to newborn AECs. We also identified differential expression of a novel endogenously expressed anti-RSV protein, pleiotrophin (PTN), which interacts with nucleolin, a cofactor for RSV entry. PTN expression/secretion was significantly decreased in preterm versus term AECs (P=0.014) and in newborn compared to one-year WD-PNECs (P=0.0008).

Conclusion

These exciting findings represent the first description of age- and prematurity-related differences in AEC innate immune responses, demonstrating greater RSV-induced expression of pro-inflammatory chemokines with increasing age and developmental differences in expression of the novel anti-RSV protein PTN. These findings may, in part, explain the increased susceptibility of preterm and very young infants to severe RSV disease.

Access Microbiol. 2019 Apr 8;1(1A):15.

St. Abb’s Head phlebovirus – a separate virus species or a strain of Uukuniemi phlebovirus?

Agnieszka M Szemiel ^1,^*, Brian J Willett ¹

Abstract

St. Abb’s Head virus (SAHV), a member of the genus Phlebovirus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments. SAHV was isolated from a pool of seabird ticks (Ixodes uriae) collected at a seabird colony in St. Abb’s Head National Nature Reserve, Berwickshire, Scotland in 1979. Antigenically, SAHV appeared to be related to the Uukuniemi serogroup of phleboviruses. Similarly, the proteins of SAHV shared similar biochemical properties to Uukuniemi phlebovirus. Here, we describe an in depth molecular characterisation of SAHV. Using next generation sequencing technology, we demonstrate that SAHV is very closely related to the Uukuniemi phlebovirus (UUKV). We examine the growth of SAHV in mammalian, avian and tick celllines and define its target cell tropism.

Access Microbiol. 2019 Apr 8;1(1A):15.

Modified time-temperature combinations reduce beef carcass contamination but will this benefit be seen thoughout the food chain?

Leonard Koolman ^1,^*, Siobhan McSharry ^1,², Paul Whyte ², Declan Bolton ¹

Abstract

The overall value of Irelands beef exports is worth approximately €2.5bn which is an annual increase of 5 % in 2017. However, the beef industry faces many challenges to export products to distant markets including a short shelf life and other economic losses that are mainly caused by microbial contamination. One of the simplest approaches to limit this contamination on the surfaces of beef carcasses is to use alternative carcass chill regimes. Carcasses underwent an industry standard chill process (10 °C for 10 h followed by 0 °C for 38 h) and were compared with carcasses that underwent a more rapidly chilled process (0 °C for 5 h and −2 °C for 3 h). Bacterial concentrations (mesophilic and psychrophilic total viable counts, total Enterobacteriaceae counts, Lactic Acid Bacteria, Pseudomonas spp., Brochothrix thermosphacta, Clostridium spp.), physiochemical (pH, temperature, water activity (aw)) and organoleptic (colour, odour, texture) changes were monitored throughout the entire beef food chain (carcass → primal → retail steak) until end of shelf life. Rapidly chilled carcasses had significantly (P<0.05) less bacterial surface contamination compared to conventionally chilled carcasses. There was also significantly (P<0.05) less evaporative loss on carcasses and primals which will have a positive economic impact on the beef industry. This longitudinal study is one of the largest trials ever performed on beef shelf life extension.

Access Microbiol. 2019 Apr 8;1(1A):15.

Transcriptomic analysis indicates the mode of action of the novel antibiotic MGB-BP-3 against Staphylococcus aureus

Leena Nieminen ¹, Kimon Lemonidis ², Douglas Browning ³, Iain Hunter ⁴, Colin Suckling ⁴, Nicholas Tucker ^4,^*

Abstract

MGB-BP-3 (MGB) is a novel synthetic antibiotic inspired by Distamycin – a natural product that is capable of binding to the minor groove of DNA. MGB has a high bactericidal activity against a broad range of Gram-positive bacteria without the toxicity associated with the natural products that it was inspired by. Its oral formulation, developedfor the treatment of Clostridium difficile infections, is currently progressing through a phase 2 clinical trial. This study investigatesthe mode of action of this novel antibiotic. To allow better understanding of MGB’s mode of action, RNA-Seq analysis was undertaken on S. aureus following challenge with 0.5 x MIC (0.1 µg ml⁻¹) MGB-BP-3. Triplicate samples of RNA were extracted at 10 min after challenge. RNA-Seq analysis identified 698 transcripts showing significant changes in expression profile, which were confirmed by quantitative RT-PCR. Amongst these, 62 essential genes showed transcriptional arrest. Glycolysis, pentose phosphate pathway and the TCA cycle were affected. In addition, biosynthesis of nucleotides and certain amino acids were altered and Biolog phenotype arrays were performed in the presence of MGB to confirm this. DNA binding assays demonstrated MGB binding to intergenic regions upstream of strongly down-regulated essential genes (mraY and dnaD). Attempts to evolve resistance to MGB have so far been unsuccessful unlike with the rifampicin control. In conclusion our findings are consistent with a bactericidal mode of action of MGB at the transcriptional level of multiple essential genes.

Access Microbiol. 2019 Apr 8;1(1A):15.

Genomics for rational autogenous vaccine design to control Campylobacter infection in poultry

Jessica Calland ^1,^*, Evangelos Mourkas ¹, Ben Pascoe ¹, Sam Sheppard ¹

Abstract

Campylobacter is the leading cause of food-borne gastroenteritis worldwide and the most common cause of infection in humans have been linked to the consumption of contaminated poultry meat. Previous poultry vaccines have provided short term reduction in Campylobacter intestinal load in chickens but have limited commercial efficacy because of high diversity and rapid evolution of strains. Genome-wide association study (GWAS) of isolates from humans and chicken have identified genes associated with survival of Campylobacter through the poultry processing chain. These genes represent targets for vaccine design that would be selectively neutral within the chicken gut and therefore could be sustained in the population. We designed an autogenous vaccine based on isolates with survival-associated genes and monitored the efficacy in the chicken and through processing. First, we sampled for Campylobacter across five broiler farms feeding into one abattoir in Norfolk and characterized genomic diversity using next generation sequencing (NGS), including the identification of survival genes. These data were then used in a predictive model to identify the minimum number of strains to be used in vaccine design based on known immunogenicity of the strains. Second, the vaccine was developed and given at two time-points to a whole farm of breeder chickens. Third, vaccine efficacy was monitored at three time points post-vaccination in vaccinated breeders and their progeny. The cfu/g reduction in the ceacum and neck skin were determined. This study demonstrates the potential for NGS in autogenous vaccine design to reduce harmful strains of bacteria that are carried commensally in livestock.

Access Microbiol. 2019 Apr 8;1(1A):15.

Combating and detecting Histomonas meleagridis, the causative agent of black head disease, through the administration of novel antimicrobial peptides derived from microbiomes

James Pickup ^1,^*, Linda Oyama ¹, Cliff Nixey ², Sharon Huws ¹

Abstract

Histomonas meleagridis is a protozoan parasite that causes mortality and morbidity in a wide range of gallinaceous fowl. It most notably affects turkeys, causing 80–100 % mortality in a flock. There is no commercial treatment for this parasite at the moment and attempts at a vaccine have failed. In this study we investigated the potential efficacy of novel rumen and chicken gastrointestinal tract microbiome-derived antimicrobial peptides (AMPs) against H.meleagridis. H. meleagridis was cultivated in growth flasks in Dwyer media. The concentration of the parasite was determined using a Haemocytometer, the cells were counted under the microscope. The AMP challenges were carried out in 96-well plates. The starting concentration of the AMPs were 1024 µg ml⁻¹ down to 2 µg ml⁻¹. The cell densities of H. meleagridis were checked at 24 h. The protozoal cell densities were checked by extracting DNA and performing qPCRs targeted against FeHyD and Rpb1 genes. Fluorescent microscopy was also used to check parasite densities as described above. Five Chicken and three Rumen AMPs resulted in decreases in protozoal cell densities following microscopy, suggesting that the peptides show potential therapeutic application for blackhead disease. Nonetheless, the qPCR primers were non-specific and resulted in bacterial DNA amplification. Consequently, in order to understand the biological function of H. meleagridis and design new qPCR primers allowing quantification, we isolated the macronuclei of a H. meleagridis strain followed by sequencing using the illumine Hi-Seq 2500 and paired ends. Future work will focus on confirming how well the AMPs work agent H. meleagridis by employing electron microscopy to observe how the AMP affect the cells.

Access Microbiol. 2019 Apr 8;1(1A):15.

The bacterial microbiome of in vitro cultures of Paramoeba perurans

David MacPhail ^1,^*, Rhea Koppenstein ², Matt Longshaw ³, Fiona Henriquez ⁴

Abstract

Amoebic Gill Disease (AGD) is a major problem in the aquaculture industry, as it is responsible for substantial losses of farmed Atlantic salmon in various worldwide locations. The disease is caused by the usually free-living Paramoeba perurans compromising the gills through the resulting development of hyperplastic lesions and lamellar fusion. These structural changes result in a reduction in the functional surface area of the gill tissues. Recent research has focused on identifying bacteria present within a culture of P. perurans, through performing isolation and identification of bacteria present in the cultures using 16S sequencing. Further NGS sequencing was performed from various culture conditions to provide insight into the changes of the bacterial microbiome during amoeba culture. As attempts to isolate the amoeba from the bacterial contamination has been unsuccessful, consideration into a possible symbiotic relationship between the amoeba and bacteria was considered. A filtering method was used to attempt to identify the genera of bacteria present within the amoeba. The isolation and 16S sequencing identified the presence of various marine bacteria, including those of the Pseudoalteromonas, Halomonas, Cellulophaga and Mesonia genera. The NGS sequencing identified a substantial proportion of sequences to match the Vibrio genus and suggests an association between this genus and the amoeba. If symbiotic relationships between specific bacteria and amoeba can be confirmed, the bacteria could potentially be used as an indicator organism for the risk of AGD outbreak. It may also provide an indirect target for the control and treatment of AGD.

Access Microbiol. 2019 Apr 8;1(1A):15.

Novel insights into human cytomegalovirus gene function from a multiplexed proteomic screen of multiple block deletion viruses

Katie Nightingale ^1,^*, Ceri Fielding ², Colin Davies ¹, Eddie Wang ², Robin Antrobus ¹, Andrew Davison ³, Gavin Wilkinson ², Richard Stanton ², Peter Tomasec ², Michael Weekes ¹

Abstract

Human Cytomegalovirus (HCMV) is a master immune regulator, encoding multiple proteins that modulate a variety of immune signalling pathways. We previously performed a systematic proteomic analysis of temporal changes in host and viral proteins throughout the course of infection and determined that HCMV downregulates >900 host proteins. HCMV is the largest human herpesvirus, potentially encoding hundreds of ORFs. Identification of which individual gene targets a given cellular factor can therefore be challenging. To facilitate the mapping of viral gene functions, we employed a panel of HCMV mutants, each deleted in contiguous gene blocks dispensable for virus replication in vitro. Three proteomic screens of these mutants were performed, with each mutant represented in at least duplicate. From these data we have defined the genetic loci responsible for targeting >250 host proteins. Bioinformatic enrichment analysis on the targets of each mutant virus enabled attribution of novel functions to blocks of uncharacterised genes. Our approach was validated from analysis of the US1-11 genetic locus, which confirmed that the major function of US1-11 genes is the regulation of MHC class I molecules and other cell surface receptors. The data also suggests that the major functions of the poorly characterised blocks RL1-6 and US29-34A are the regulation of secreted proteins and the regulation of a family of cell surface adhesion molecules respectively. Overall this approach can be used to gain global insights into HCMV gene function, the study of which has previously been only been possible on a single gene basis.

Access Microbiol. 2019 Apr 8;1(1A):15.

Rational design of TFDs as novel oligonucleotide antimicrobials to treat Gram-negative infections

Sandeep Bahia ¹, Christopher Morris ¹, Louis Laurence ², Michael McArthur ^2,^3,^*

Abstract

We have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.

Access Microbiol. 2019 Apr 8;1(1A):15.

Molecular typing of human enteroviruses: reanalysis of previously untypable EV isolates and the rapid detection of EV-D68

Hannah Howson-Wells ^1,^*, Patrick McClure ², Gemma Clark ¹, William Irving ^2,¹, I Michael Kidd ^1,³

Abstract

Molecular diagnostics have superseded cell culture and neutralisation as the gold standard for enterovirus (EV) diagnosis and serotyping in resource rich countries. EV positive isolates from Nottingham University Hospitals NHS Trust are referred to the Enteric Virus Unit (EVU) for typing; performed through amplification and sequencing of viral capsid protein-1 (VP1). An audit of local EVU typing data (2013–2017 showed a significant (P<0.005) increase in untypable EV strains isolated from clinical specimens. Following a literature review of published EV typing assays and in silico analyses of 2201 EV genomes, an end point RT-snPCR VP1-typing assay was derived from a widely cited protocol and successfully employed to produce a simple, reliable and cost-effective typing assay. Twenty-three previously untypable isolates were identified, with phylogenetic analyses of the VP1 region showing broad distribution across EV-A and B species. This assay detected serotypes across EV species A-D, including EV-D68, responsible for severe neurological and respiratory disease. Following an increase in severe EV infections, including six cases of suspected acute flaccid myelitis, this RT-snPCR was employed locally as a rapid typing assay. In combination with 5’UTR PCR and an EV-D68 specific PCR, designed to amplify the complete VP1 region, EV-D68 was detected in respiratory and neurological specimens, including the CSF of a patient with brainstem encephalitis.

Access Microbiol. 2019 Apr 8;1(1A):15.

Wombling surplus diagnostic nucleic acid for novel pathogenesis and genetic epidemiology of viral infections

Patrick McClure ^1,^*, Gemma Clark ², Akhil Chellapuri ¹, Matthew Smitheman ¹, Arwa Bagasi ¹, Terry Akagha ¹, Tasneem Khandaker ¹, Hannah Howson-Wells ², Joseph Chappell ¹, Theocharis Tsoleridis ¹, Jonathan Ball ¹, William Irving ^1,²

Abstract

Nottingham University Hospitals Trust receives circa 13 000 samples for diagnosis of respiratory and neurological viruses per annum, however positive results are achieved in approximately 50 % of respiratory and 20 % of neurological investigations. We therefore aimed to retrospectively extend the diagnostic spectrum for these samples by applying a battery of degenerate PCR assays to surplus diagnostic nucleic acids.218 previously negative respiratory specimens collected in 2016 from children under 5 years old, identified positivity of 11 % for Human Bocavirus, 5 % for Human Enteroviruses (including 3 cases of Enterovirus D68), 4 % for Human Coronaviruses and one individual positive for Trichodysplasia Spinulosa Polyomavirus. Complementary investigation of 1730 previously negative specimens from children and adults with neurological symptoms yielded positive results for Hepatitis E, BK Polyomavirus and Astroviruses in addition to Entero- and Parechoviruses apparently missed by standard diagnostic assays. Our extensive archive further allowed us to investigate relatively rare viral infections in significant numbers. Therefore we also studied the genetic and clinical epidemiology of the human Rubulavirus pathogens Parainfluenza 2 and 4 in 121 and 237 patients respectively between 2013 and 2017. This indicated co-circulation of three clusters of Parainfluenza 4 in Nottingham with greater presentation of subtype 4b than 4a and 2 clades of Parainfluenza 2. 5 fatalities were recorded in the Parainfluenza 2 cohort. In summary, surplus nucleic acid from viral diagnostic laboratories represents a valuable resource for both service development and clinical research. Coronavirus, Bocavirus and Enterovirus testing have since been implemented in the routine diagnostic panel for respiratory investigations.

Access Microbiol. 2019 Apr 8;1(1A):15.

High susceptibility, viral dynamics and persistence of South American Zika virus in New World monkeys

Neil Berry ^1,^*, Claire Ham ¹, Jo Hall ¹, Adrian Jenkins ¹, Elaine Giles ¹, Sarah Kempster ¹, Nicola Rose ¹, Debbie Ferguson ¹, Neil Almond ¹

Abstract

South American Zika virus (ZIKV) emerged as a novel enzootic pathogen linked with neonatal brain defects and acute neuropathies, its natural history characterised by a sylvatic transmission cycle with arboreal mosquitoes and African primates. Yet infectivity studies in New World primates indigenous to Zika-endemic regions including host range, susceptibility, replication dynamics, tissue tropism and virus persistence are currently lacking. New World species Callithrix jacchus (marmosets) and Saguinus labiatus (red-bellied tamarins) were highly susceptible to sub-cutaneous challenge with South American ZIKVPRVABC59 inducing rapid, high, acute viraemia in each species. Differences in acute phase vRNA in blood were highly statistically significant at day 3 between these New and two Old World species Macaca mulatta (rhesus macaque) and Macaca fascicularis (cynomolgus macaque), Mann-Whitney U testp=0.00058. Comparative quantitative RT-PCR and RNAscope in situ hybridisation analysis of tissue viral copy number and cellular localisation patterns with Old World NHP revealed early, widespread distribution across multiple skin biopsies distant to the inoculation site, lymphoid organs, reproductive sites and the brain. While viraemia was cleared from blood by day 42 in all individuals, ZIKV persistence in most tissues was identified 100 days post-inoculation. Early neuroinvasion and persistence, following acutely resolved infection characterises ZIKV susceptibility in multiple hosts, especially New World species. Establishment of a ZIKV sylvatic cycle in the Americas may provide persistent animal reservoirs for future outbreak resurgence. New World monkeys represent viable models to understand ZIKV pathogenesis, potential therapeutic interventions and vaccine development strategies.

Access Microbiol. 2019 Apr 8;1(1A):15.

Determining the function of proteins degraded by Human Cytomegalovirus

Alice Fletcher-Etherington ^1,^*, Katie Nightingale ¹, Kai-Min Lin ¹, Benjamin Ravenhill ¹, Luis Nobre ¹, Michael Weekes ¹

Abstract

Human Cytomegalovirus (HCMV) is associated with significant morbidity and mortality in the immunocompromised, and is a leading cause of congenital infection. Only three drugs are available for treatment, all with significant toxicities. New therapies and a vaccine are urgently required. Susceptibility to viral infection and disease is determined in part by antiviral restriction factors (ARFs) and the viral proteins that have evolved to degrade them. Small-molecule disruption of the interaction between an ARF and a viral antagonist can inhibit viral replication and may be utilised for antiviral therapies. To identify novel restriction factors against HCMV, we previously developed a multiplexed proteomic approach to identify proteins that are actively degraded early during HCMV infection. We reasoned that these would be enriched in known and novel ARFs that the virus must degrade in order to replicate. 35 proteins were shown to be degraded according to stringent statistical criteria, which included the known anti-HCMV restriction factors Sp100 and MORC3, and a novel ARF, HLTF. Here, we present preliminary results from a combination of viral replication assays to identify other novel ARFs. These include a new ‘two-colour’ approach to characterise HCMV restriction, which aims to eliminate variability in cell density by mixing populations of cells in a single cell culture well. Preliminary studies have identified a number of proteins that inhibit virus replication, as well as a novel dependency factor which the virus may degrade in order to attenuate cellular immune signalling or help establish latency.

Access Microbiol. 2019 Apr 8;1(1A):15.

Industrially useful microbial species for production of biofuels and chemicals from municipal solid waste

Aritha Dornau ^1,^*, Gavin Thomas ¹, Simon McQueen-Mason ¹

Abstract

Municipal solid waste (MSW) production is projected to reach 3.4 billion tonnes per annum by 2050. The majority of MSW produced globally is incinerated or diverted to landfill, both methods which pollute the environment and contribute substantially to climate change. The organic fraction of MSW (OMSW) typically comprises ∼50 % lignocellulosic material and presents an abundant renewable feedstock for producing biofuels and chemicals. An important step toward OMSW valorisation is the identification of suitable microorganisms capable of fermenting this highly inconsistent, heterogeneous and complex feedstock. We have characterised the fermentation performance of eight biotechnologically relevant microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis) on enzymatic hydrolysate of OMSW fibre produced by a commercial autoclave pre-treatment. S. cerevisiaewas the most efficient ethanol producer, followed closely by Z. mobilis. Both species produced ethanol to high titre within 24 h, but neither could ferment xylose. The most effective performance was demonstrated by R. opacus, which consumed all available glucose and xylose concurrently over 72 h and produced a remarkably large yield of triacylglycerol (a biodiesel and aviation fuel precursor). This work demonstrates that OMSW is a promising renewable feedstock capable of supporting the growth several industrially useful microorganisms to high product titres. The best performing species identified here are interesting candidates to study further for application in a MSW biorefinery.

Access Microbiol. 2019 Apr 8;1(1A):15.

Enterobacteriaceae-selective modification of the intestinal microbiota using oligonucleotide antimicrobials

Nichola Wong ¹, Michael McArthur ^1,^2,^*

Abstract

The human gut microbiota is complex, dense, and hugely influential to health. Imbalances in the microbiota have been associated with numerous disease states, in many cases due to the overgrowth of Enterobacteriaceae, such as Escherichia coli. We describe an oligonucleotide antimicrobial that selectively reduces levels of Enterobacteriaceae in vitro, in a model of the human colonic microbiota, and in vivo in a murine study, whilst leaving the core microbiota intact. The antimicrobials are Transcription Factor Decoys (TFDs) that bind to and competitively inhibit an identified transcription factor necessary for growth in the intestine. This is highly conserved amongst Enterobacteriaceae and controls anaerobic respiration and response to nitrosative stress caused by the innate immune response of the host. A nanoparticulate formulation delivers the TFDs to the cytoplasm of E. coli, as visualized by confocal microscopy, and rapidly kills the bacteria under microaerobic conditions. When applied to anin vitro model of the human intestinal microbiota the TFD produced a decrease up to log10 6 c.f.u. ml⁻¹ in coliforms within the Enterobacteriaceae family while other families remain intact. When delivered orally to the intestines of mice similar results were seen: Enterobacteriaceae were decreased or cleared from the wild-type intestinal microbiota while the remaining bacteria were unaffected. This demonstrates that TFDs can be used to make precise changes to the microbiota and has utility in testing associations between dysbiosis and disease and developing microbiota targeted therapeutics.

Access Microbiol. 2019 Apr 8;1(1A):15.

Evaluating the epizootic risk to poultry of a novel Chinese H7N9 virus variant with increased pathogenicity in turkeys

Joe James ^1,^*, Saumya Thomas ¹, Sahar Mahmood ¹, Amanda Seekings ¹, Marek Slomka ¹, Ian Brown ¹, Sharon Brookes ¹

Abstract

Previously we successful infected turkeys with the China-origin H7N9 low pathogenicity avian influenza virus (LPAIV, A/Anhui/1/13, referred to as ‘wild-type’ (Wt)) which successfully transmitted to contact turkeys with virulent outcomes, highly unusual for LPAIV infection, particularly as the LPAIV cleavage site remained unchanged in all experiments. Sequencing of progeny viruses revealed consistent emergence of the L226Q polymorphism in the HA gene, termed the ‘turkey-adapted’ (ty-ad) virus. Ty-ad and Wt were used to compare the epizootic risk posed by both H7N9 LPAIVs in turkeys and to explore the mechanisms which underpins any differences. The Wt and ty-ad viruses robustly infected inoculated and contact turkeys, producing similar shedding titres. However, the ty-ad virus was more pathogenic than the Wt virus in directly-infected and contact turkeys, causing 100 % (Wt) compared to 16 % (ty-ad) survival. The ty-ad virus was detected in broader range of turkey organs, and at higher titre, compared to the Wt variant. This contrasted with pathogenicity and tissue-tropism observations for both viruses in chickens. The wt and ty-ad viruses did not replicate without trypsin in vitro, affirming a typical LPAIV phenotype. The L226Q polymorphism is known to alter receptor binding, with key differences in receptor distribution between turkey and chicken tissues observed. Replication kinetics differences in a range of avian cells will be reported for both viruses. Consequently, if this ty-ad variant were to arise more frequently in nature, it would pose an increased virulent risk to turkeys. It is therefore important to maintain surveillance and understanding of China-origin H7N9 viruses.

Access Microbiol. 2019 Apr 8;1(1A):15.

The chitin attenuator: the Ca2+/calcineurin pathway maintains the viability of Candida albicans cells with supra-normal chitin levels

Alessandra da Silva Dantas ¹, Neil Gow ^1,^*, Louise Walker ²

Abstract

Chitin is an essential structural polysaccharide component of the cell walls and septa of fungi. Recent reports have suggested that Candida cells can resist killing by echinocandins by up-regulation of chitin synthesis thereby sustaining cell wall integrity both in vitro and in vivo (Lee et al. 2012). This increase in chitin content seen in C. albicans cells that are less susceptible to caspofungin is coordinated simultaneously by the PKC, Ca2+/calcineurin and HOG pathways (Munro et al. 2007, Walker et al. 2008). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of caspofungin often have supra-normal chitin levels rather than low chitin levels, and therefore that having too much chitin in the cell wall may be detrimental for viability. Chitin content may therefore need to be clamped at levels that enable cells to survive cell wall stresses but are not so high that they negatively affect cell viability.

Access Microbiol. 2019 Apr 8;1(1A):15.

The secretome profiling of a pediatric airway epithelium infected with human respiratory syncytial virus (hRSV) identified aberrant apical/basolateral trafficking and novel immune modulating (CXCL6, CXCL16, CSF3) and antiviral (CEACAM1) proteins

Olivier Touzelet ^1,^2,^*, Lindsay Broadbent ¹, Stuart Armstrong ^2,³, Waleed Aljabr ^2,⁴, Elaine Cloutman-Green ⁵, Ultan Power ¹, Julian Hiscox ^2,^3,⁶

Abstract

Rationale

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remains poorly understood.

Objective

To investigate the impact of hRSV on the secretome of pediatric respiratory epithelium.

Methods

A proteomics approach was combined with an ex-vivo pediatric airway epithelial model (HAE) (n=3 donors) of hRSV infection to identify the apical and basolateral secretome of hRSV-infected cultures.

Measurements and main results

Following hRSV infection, many host proteins lost their apical- or basolateral-restricted secretion or displayed altered apical/basolateral abundance ratios. Fifty three proteins were specifically associated with RSV infection, including modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) that were never previously associated with hRSV. Importantly, CXCL6, CXCL16, CSF3 was also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not non-infected controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells.

Conclusions

hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome. It also specifically induced immune modulating (CXCL6, CXCL16, CSF3) and an antiviral protein (CEACAM1) that are new to hRSV infection or disease. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

Access Microbiol. 2019 Apr 8;1(1A):15.

Clinical evaluation of herPHEgen^®: a herpesvirus genotype-to-phenotype antiviral resistance database and testing service

Hodan Mohamed ¹, David Bibby ¹, Eileen Gallagher ¹, Renata Piorkowska ¹, Andros Gavriel ¹, Carmen Manso ¹, Nigel Wallis ¹, Chris Parry ¹, Mark Zuckerman ², Patricia Cane ¹, Daniel Bradshaw ^1,^*, Jean Mbisa ¹

Abstract

Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) infections may be life-threatening in immunocompromised patients. Antiviral treatment is available but resistance can develop. The gold standard for resistance testing is cell culture-based phenotyping which is slow (3–4 weeks) and has a high (50 %) failure rate. Faster (<10 working days) genotypic testing of viral thymidine kinase (TK) and DNA polymerase (DNApol) can be used with >95 % success rate. However, a reference database for interpreting drug susceptibility from genetic data is lacking. We developed an HSV genotypic test and genotype-to-phenotype drug resistance database. We evaluated the service using 325 clinical samples, previously characterised by phenotypic susceptibility testing, from 248 treatment-experienced patients. The median age was 42.5 years [IQR30.0–51.0] and 50.8 % (n=126) were female. Clinical details were as follows: 42.3 % (n=105) haemato-oncology patients; 12.1 % (n=30) HIV-infected; 4.4 % (n=11) unspecified immunosuppression; 2.4 % (n=6) congenital infection; 1.2 % (n=3) solid organ transplant; 37.5 % (n=93) unknown. HSV-1 was identified in 58.2 % (n=189) samples. Phenotypic testing identified resistance in 63.7 % (n=207) samples. Genotypic testing identified a resistance-associated mutation (RAM) in TK and/or DNApol in 200/207 samples, a positive percent agreement of 96.6 %, whereas a RAM was detected in 1/118 susceptible samples, a negative percent agreement of 99.2 %. Most RAM occurred in TK (n=195; 97.0 %) with few in DNApol (n=32; 15.9 %). In summary, through herPHEgen we have developed a robust HSV resistance testing service, providing clinicians with timely and accurate results. This will improve clinical decision-making, optimising treatment efficacy and minimising toxicity in immunocompromised patients with HSV.

Access Microbiol. 2019 Apr 8;1(1A):15.

Direct proteolytic control of an extracytoplasmic function RNA polymerase sigma factor

Bohdan Bilyk ^1,^*, Sora Kim ², Tania Baker ², Ryan Seipke ¹

Abstract

Streptomyces species are well known for harbouring a large number of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors; nearly all of regulated genes required for morphological differentiation and/or response to environmental stress. The activity of ECF σ factors is typically modulated by a cognate anti-σ factor protein co-encoded at the same locus. In previous work, we identified σAntA as a cluster-situated regulator of starter unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is controlled by a cognate anti-σ factor, σAntA is an orphan, which raises intriguing questions about how its activity is controlled. Inspection of the σAntA amino acid sequence revealed a carboxyterminal di-alanine, which is the only motif of the Clp-protease recognition signal obligately required for proteolysis. Here, we show by immunoblotting that the abundance of 3xFLAG-σAntA in vivo is enhanced by alteration of the C-terminal Ala-Ala to Asp-Asp and that abundance of both variants is elevated in the absence of genes encoding the Clp-protease and its regulatory subunits ClpX and ClpA. We also show that (His)6-SUMO-σAntA, but not a variant lacking the C-terminal di-alanine motif, is degraded by the ClpXP protease in vitro. These data unambiguously establish direct proteolysis as an alternative control strategy for ECF RNA polymerase σ factors and expands the paradigmatic understanding microbial signal transduction regulation.

Access Microbiol. 2019 Apr 8;1(1A):15.

Use of Nano- SIMS at the single cell-level to evaluate drug penetration into mycobacterial biofilms

Winifred Akwani ^1,^2,^*, Suzie Hingley-Wilson ¹, Mark Chambers ¹, Paulina Rakowska ², Greg McMahon ²

Abstract

Non-tuberculous mycobacteria (NTMs), such as Mycobacterium abscessus and chimaera, can cause high mortality and morbidity amongst patients who are immunocompromised or have chronic lung diseases, such as cystic fibrosis. Mycobacterial biofilms can form in the alveolar walls of such patients following inhalation from environmental reservoirs. Recently, pathogenic strains have been isolated from shower heads and hospital air conditioning units. Biofilms are notoriously difficult to eradicate and are associated with the development of increased antimicrobial resistance (AMR). Treatment for M. abscessus and chimaera infections often requires 2–3 antibiotics over 2 years. The question we want to address is whether the increased AMR and treatment time in NTM biofilm formation is due to lack of antibiotic penetration, resulting in non-therapeutic and AMR-generative levels, or the development of phenotypic and/or genetic resistance. In this project we will use Nano-SIMS (nano-scale secondary ion mass spectrometry) to measure penetration of the antibiotic bedaquiline (BDQ), used to treat NTM infections, into individual cells of NTM biofilms (M. abscessus and M. chimaera). Nano-SIMS maps the relative abundance of different ions down to the nano-scale and can be used to measure in profile through the biofilm. In addition, the minimum inhibitory concentration (MIC) and minimum duration for killing (MDK) of BDK will be measured to determine antibiotic susceptibility in biofilms and a planktonic growth control. Understanding the AMR generation and prolonged treatment in NTM biofilms could lead to improved mortality and morbidity. The development of novel treatment strategies could enhance treatment efficacy, reduce treatment duration and AMR development.

Access Microbiol. 2019 Apr 8;1(1A):15.

The Candida albicans arginase family encodes enzymes with diverse catabolic activities that differentially influence host-fungus interactions

Katja Schaefer ¹, Steven Bates ¹, Ryan Ames ¹, Stella Christou ², Neil Gow ^1,^*

Abstract

In the blood stream, arginine is an essential amino acid that is required by phagocytes to synthesize iNOS. Previously we showed that the fungus Candida albicans induces host arginase production that diverts arginine from the pathway that leads to the production of nitrite oxide. We therefore investigated whether C. albicans arginase activity also contributed to the protection of the fungus by competing for arginine during infections. Three C. albicans genes had been annotated as putative arginase encoding genes. Heterologous expression of these genes suggested all three had some arginase activity and one gene product (Car1) encoded a bone fide arginase that was required for growth on arginine. However, single and double mutations in the two other genes (AGM1 and GBU1) did not affect growth on arginine as a single nitrogen source and were found instead to encode agmatinase and guanidinobutyrase respectively that participate in two other pathways related to arginine metabolism. This family of three enzymes therefore exhibits mixed biochemical activities and collectively participate in the catabolism of exogenous and endogenous sources of arginine. Virulence of the triple mutant lacking all three genes was reduced in a Galleria infection model, but single or double mutants were fully virulent. None of the single or multiple mutants affected host NO production suggesting they do not influence the oxidative burst of phagocytes. In addition, CAR1 expression was required for hyphal growth. This family of enzymes therefore represent a novel enzyme set that is essential for growth in vivo and indirectly for fungal virulence.

Access Microbiol. 2019 Apr 8;1(1A):15.

Single cell and single molecule resolution of herpesvirus genome transport, condensation state and transcriptional output

Eiki Sekine ¹, Peter O’ Hare ^1,^*

Abstract

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.

Access Microbiol. 2019 Apr 8;1(1A):15.

Host response of Agaricus bisporus to mushroom virus X infection

Eoin O’Connor ^1,^*, David Fitzpatrick ¹, Helen Grogan ²

Abstract

Commercial mushroom crops (Agaricus bisporus) are susceptible to a disease causedby a complex of 18 viruses known collectively as mushroom virus X (MVX). Symptoms of MVX infection vary from bare patches in crops to mushroom discolouration (browning). To understand the dynamic interaction between A. bisporus and MVX, we have studied five strains; including the globally-cultivated commercial strain, one wild strain, and one commercial-wild hybrid strain. Our transmission experiments using ‘healthy’ mycelium challenged with MVX-infected mycelium, detected MVX in the first day of hyphal anastomosis in all five strains. However, our commercial-scale crop experiment revealed varying degrees of disease tolerance in the fruiting body, the commercial strain being most susceptible and the hybrid strain most resistant to MVX. LC-MS/MS proteomics and RNA-seq analyses have elucidated key differences in response to both early and late crop inoculation of MVX. Quantitative shotgun proteomics of the susceptible commercial strain at late MVX inoculation revealed striking levels of proteins relating to mechanical membrane damage via detection of myo-inositol-associated biosynthetic proteins. Defense proteins relating to chalcone isomerase activity were also detected exclusively in the MVX-infected commercial strain. MVX-infected wild strain isolates showed significantly greater abundance of proteins related to fundamental cellular processes such as RNA polymerase activity and cell-redox homeostasis. Our findings show that although vegetative transmission of MVX is prevalent in all five strains, the fruiting body may oppose the infection in certain strains. Our findings of dynamic host response of A. bisporus to MVX, provides novel insights for this economically important, globally cultivated crop.

Access Microbiol. 2019 Apr 8;1(1A):15.

The effect of dietary fatty acid supplementation on gut microbiome development in weaning piglets

Robyn McKenna ^1,^2,^*, Paul McAdam ², Victoria Smyth ², Mark Robinson ¹, Louise Cosby ^2,¹

Abstract

The gastro-intestinal tract hosts a complex microbial ecosystem that helps regulate the physiological, immunological and nutritional functions of the pig so disturbances within this microbiota can have profound effects on porcine health and disease. The gut microbiota is shaped by the environment, immune pressures and diet. The weaning transition represents a critical time-point that can interfere with intestinal development and cause dramatic shifts in the gut microbiota. Previously, in-feed antibiotics were used to counteract the adverse effects of weaning but an EU-wide ban since 2006 has propelled the search for safe and sustainable alternatives within the livestock industry. Studies have indicated that dietary fatty acids are efficient in terms of minimising weaning disorders including elevated incidence of enteric disease and immunodepression and thus represent a promising alternative to antibiotics. Furthermore, previous research has indicated that including fatty acid mixtures in the porcine diet has a positive effect on overall animal performance, increasing growth rate and enhancing feed conversion ratios. It is thought that these health benefits and production gains stem from the ability of fatty acids to mediate the effects of gut microbiota on intestinal immune function through modifying the proportions of microorganisms present. The aim of this research was to use deep sequencing and metagenomic methods to explicitly explore the influence fatty acid supplementation has on shaping the intestinal microbiome during weaning. Results have indicated that fatty acids play a role in modifying the intestinal microflora, establishing an environment that favours the growth of commensal species such as Lactobacillus.

Access Microbiol. 2019 Apr 8;1(1A):15.

An investigation of the molecular and cellular mechanisms of in vitro phage therapy in human cells

Christian Møller-Olsen ^1,^*, Siu Ho ¹, Toby Ross ¹, Antonia Sagona ¹

Abstract

As the consequences of increasing bacterial resistance to traditional antibiotics become evident, the importance of research and development of therapeutic alternatives is apparent. Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host. However, whether and how bacteriophages can kill intracellular bacteria in a human cell environment remains elusive. E. coli strains displaying the K1 polysaccharide capsule virulence factor, are nosocomial pathogens responsible for urinary tract infections (UTIs), neonatal meningitis and potentialprecursors for septicaemia. These different types of infections were modelled in vitro by infecting human bladder epithelial cells (T24 HTB-4) and humancerebral endothelial cells (hCMEC/D3) with E. coli EV36, a strain displaying the K1 capsule. The infected human cells then received in vitro phage therapy using bacteriophage K1F that specifically targets E. coli strains displaying the K1 capsule. The rate of bacterial infection and the molecular and cellular mechanisms of in vitro phage therapy was analysed by means of flow cytometry, confocal and live microscopy. We show that rfp-tagged E. coli EV36-RFP and gfp-tagged bacteriophage K1F-GFP, enter the human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP can efficiently kill intracellular E. coli EV36-RFP in human urinary bladder epithelial cells and humancerebral endothelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy. Collectively this data contribute evidence-based knowledge for the ongoing development of phage therapy.

Access Microbiol. 2019 Apr 8;1(1A):15.

Development of Bacteroides thetaiotaomicron outer membrane vesicles as a universal, mucosal vaccine for Influenza A virus

Eleanor Bentley ^1,^*, Sanaria Al Katy ^1,², Anja Kipar ³, Regis Stenz ⁴, Simon R Carding ^4,⁵, James Stewart ¹

Abstract

Influenza A Virus (IAV) is a zoonotic pathogen which causes seasonal epidemics worldwide, resulting in significant morbidity and mortality. Due to rapid virus evolution, available vaccines/antiviral drugs must be re-formulated and re-administered in most years. There is therefore an unmet need for a ‘universal’ vaccine to provide long-lasting, adaptive immunity to multiple strains of IAV. The aim of this study was to assess outer membrane vesicles (OMVs) produced by Bacteroides thetaiotaomicron (Bt) containing IAV antigens as a candidate universal mucosal vaccine. First, using intranasally-delivered fluorescently-labelled OMVs and FACS we demonstrated good uptake of OMVs by dendritic cells in both secondary and tertiary lymphoid tissue, showing OMVs are efficiently trafficked from the mucosa to the lymphatic system. Immunohistology confirmed this and showed expansion of lymphoid tissues and lymphoid follicles after OMV delivery. The stem/stalk of IAV HA is less variable and will generate antibodies that are cross-protective. Thus, we produced OMVs (termed H5F) that contained the stalk region of HA from strain A/VietNam/1203/04 (H5N1). We inoculated mice intranasally with H5F expressing OMVs or wild type OMVs and assessed antibody production and protection from challenge using a lethal dose of a different strain of influenza (PR8; H1N1). OMV-H5F-inoculted mice produced IgG and IgA in the respiratory tract that recognised both H5 HA and H1 HA. OMV-H5F inoculated mice were also protected from lethal challenge and supported lower virus titres. Our results indicate a strong potential for this approach to generate a universal IAV vaccine.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating the role of low-oxygen-activated (lxa) encoded proteins in the pathogenesis of Burkholderia cepacia complex and their contribution to chronic infection in cystic fibrosis

Andrew O’Connor ^1,^*, Siobhán McClean ¹

Abstract

Burkholderia cepacia complex (Bcc) is a group of 22 closely related species of Gram-negative bacteria that cause chronic infections in people with cystic fibrosis (CF) that are rarely eradicated. The high-level antibiotic resistance and poorly understood mechanisms by which Bcc survive and persist during chronic infection mean that combatting these chronic infections is particularly challenging. We have previously found that a group of 20 low-oxygen-activated (lxa) encoded proteins were consistently upregulated in sequential Bcc isolates in two chronically infected CF patients over time of infection. Many of these proteins have not previously been studied in Bcc but the consistent upregulation over time of infection suggests that they play an important role during chronic infection. Two particular proteins of interest within the lxalocus that were consistently upregulated were a universal stress protein (USP) and a phospholipid binding protein (PBP). Single gene deletion mutants (Δusp and Δpbp) in the wildtype Bcc strain K56-2 (WT) both showed a 90 % reduction in attachment to CFBE41o- cells compared to WT. There was also a 5-fold reduction in the virulence of Δpbp in the acute infection model Galleria mellonella (P<0.005) and an increased sensitivity of Δusp to peroxide-induced oxidative stress (P<0.0001) and low pH (P<0.05) relative to WT. A reduction in both uptake and survival of Δusp in U937 macrophage-like cell line compared to WT, suggests the USP plays a role in the intra-macrophage survival of Bcc. Overall, these proteins, previously associated with low-oxygen conditions may play a considerable role in Bcc pathogenesis and its adaption during chronic infection.

Access Microbiol. 2019 Apr 8;1(1A):15.

Deciphering adaptation through mobile element pangenome composition

Nicholas Waters ^1,^2,^*, Ashleigh Holmees ², Florence Abram ¹, Leighton Pritchard ², Fiona Brennan ^1,³

Abstract

Pangenome analysis can help reveal functional and physiological differences between organisms by linking genes present in their genomes with phenotypic characteristics. While this works well for closely-related organisms and large numbers of isolates, analysis of species with a large pangenome may be problematic as optimal sample size increases with intraspecies diversity. Organisms such as E. coli exhibit high intraspecies diversity, resulting in an exceptionally large pangenome, pushing the limits of what constitutes a species. However, the phylogeny of the mobile elements can differ strongly from the phylogeny of the rest of the genome, reflecting how rapidly these elements can be gained or lost. We suggest when studying adaptations on a short genetic time-scale (ie, within species or strain), the mobile regions of the genome may be more informative than the more stable parts of the genome. To this end, we developed a pipeline, Horizontally Acquired Partial Pangenome of Inserted Elements (or ‘happie’), which allows researchers to study the mobile pangenome. Thus, happie could be used as a proxy to detect genes associated with a particular trait amidst the noise of a diverse genetic background. We aim to use happie to compare a panel of known soil-persistent E. coli isolates and a curated panel of non-soil-persistent isolates. This will allow us to determine whether specific mobile elements (or genes carried on those elements) are associated with soil persistence, which would indicate that the cost of harbouring the mobile element was less than the advantage conferred.

Access Microbiol. 2019 Apr 8;1(1A):15.

Low prevalence of resistance mutations to integrase inhibitors (INI) in patients living with HIV-1 at baseline using next generation sequencing (NGS)

Jon Hubb ^1,^*, Mark Hopkins ¹, Jane Deayton ¹, Sophie May ¹

Abstract

Current guidelines indicate that baseline INI resistance testing is not required; however, increased prescribing may lead to increased resistance mutation prevalence. NGS allows the detection of minority variants (MVs) not be detected by conventional methods. Barts Health NHS Trust patient records were reviewed to identify INI naïve patients prescribed INI between March 2014 and March 2017. Baseline plasma samples were extracted for nucleic acid and amplified in a single RT-PCR. Libraries were prepared using the Nextera XT library preparation kit and sequencing performed on an Illumina Miseq. Sequencing reads were assembled into consensus sequences and resistances determined using the Stanford HIV resistance database (HIVdb). MVs were variants <20 % of the overall population. Of 248 patients prescribed INI, 128 patients were excluded from the study due to an undetectable viral load or lack of sample. Of the remaining 121 patients, integrase sequences were successfully obtained from 91. No major INI mutations were detected. Six patients were identified with an HIVdb INI score ≥10: E157Q (4 patients) and T97A (2 patients). Only 1 MV accessory mutations was observed (A128T). Of patients with resistance mutations, 5 received raltegravir- and 2 received dolutegravir-based regimes. No treatment failures were observed. Prevalence of baseline INI mutations increased in recent years: 0/12 (0 %) patients in 2014–2015, 2/32 (6.3 %) patients in 2015–2016 and 5/47 (10.6 %) patients in 2016–2017. Resistance MVs to INI were extremely rare. Although this study supports current baseline INI resistance testing guidelines, increasing mutation prevalence’s may require future revision of guidelines.

Access Microbiol. 2019 Apr 8;1(1A):15.

Mycovirus induced hypervirulence of Leptosphaeria biglobosa enhances systemic acquired resistance to Leptosphaeria maculans in Brassica napus

Unnati Shah ¹, Ioly Kotta-Loizou ^2,^*, Bruce Fitt ¹, Robert Coutts ³

Abstract

Phoma stem canker is one of the most important diseases of winter oil seed rape (Brassica napus) world-wide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and Leptosphaeria biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double stranded (ds) RNA virus in L. biglobosa and no viruses in L. maculans. The virus forms isometric particles ca. 40–45 nm in diameter and has four genomic segments, each possessing a single open reading frame flanked by untranslated regions. Phylogenetic analysis revealed modest similarities to known and suspected members of the family Quadriviridaeand therefore the virus was nominated Leptosphaeria biglobosa quadrivirus-1. Following eradication of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance towards L. maculans in B. napus following lower leaf pre-inoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defences. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of phoma stem canker later in the growing season.

Access Microbiol. 2019 Apr 8;1(1A):15.

A CRISPR-associated Rossmann fold (CARF) domain regulates transcription of an RNA repair system in Escherichia coli

Ioly Kotta-Loizou ^1,^*, Milija Jovanovic ¹, Jorrit Schaefer ¹, Christoph Engl ², Nan Zhang ³, Martin Buck ¹

Abstract

CRISPR-associated Rossmann fold (CARF) domain signalling underpins the modulation of CRISPR-Cas systems. Unlike the majority of known CARF domains associated with nuclease activity in CRISPR-Cas systems, the CARF domain of the transcriptional regulator RtcR modulates the opposite function by activating an RNA end sealing system. The Rtc RNA repair system in Escherichia coli consists ofthe universally conserved RNA cyclase RtcA and RNA ligase RtcB, and is known to be induced by antibiotics and oxidative stress. We aim to investigate the CARF domain mediated transcriptional regulation of the Rtc system in vivo and in vitro. A reporter based assay confirmed that the RtcR CARF domain has a negative regulatory effect on RtcR activity and subsequent induction of the rtcBA operon. Both predictive modelling and site-directed mutagenesis suggest the Rtc induction is not due to oxidation of cysteines present in the RtcR regulatory CARF domain. Interestingly, the enzymes RtcA and RtcB, products of the actions of RtcR as a transcription regulator, are required for RtcR to stimulate expression of the rtcBA operon and directly bind to the RtcR CARF domain as shown in vivo by two-hybrid and in vitro by gel filtration. Given that CARF domains are known to be activated by RNA binding, a range of RNA molecules linked to the Rtc system or CARF domains in general are currently being investigated as potential inducers in an in vitro transcription system. Together, our data indicate an expanded range for CARF domain signalling, including via protein-protein interactions.

Access Microbiol. 2019 Apr 8;1(1A):15.

Use of Bacteroides-derived microvesicles for mucosal vaccines

Ariadna Miquel-Clopes ^1,^*, Ana L Carvalho ¹, Udo Wegmann ¹, Eleanor G Bentley ², Sanaria HM Al-Katy ², Janine L Coombes ², Anja Kipar ³, Regis Stentz ¹, James P Stewart ², Simon R Carding ^1,⁴

Abstract

The vast majority of infectious agents enter the body via mucosal sites yet there are very few licensed mucosal vaccines able to generate protective immunity at the sites of pathogen entry. A major obstacle in developing mucosal vaccine is delivering biologically active vaccine antigens (Ag) to mucosa-associated lymphoid tissues to prime protective immune responses. To address these issues we have developed a drug delivery technology platform to deliver intact antigens to the respiratory and gastrointestinal tract using outer membrane vesicles (OMV) naturally produced by Bacteroides thetaiotaomicron (Bt), a non-pathogenic human commensal gut bacterium. We have developed the capability of engineering Bt to express antigens of interest in their OMVs which we have shown are stable for long periods of time across a wide temperatures range. They also have inherent adjuvanticity as shown by the ability of native OMVs to elicit the formation of organised lymphoid follicles comprising dendritic cells and T and B cells in both the upper and lower respiratory tract after intranasal administration. The intransal administration of Bt OMVs expressing the pre-fusion headless hemagglutinin mini-stem protein of influenza type A virus (IAV) subtype, H5N1, induced high titre antigen-specific antibodies in the respiratory mucosa (IgA) and serum (IgG) that conferred heterotypic protection to infection by a H1N1 IAV. Collectively, our data demonstrates the feasibility of using Bt OMVs in mucosal vaccine formulations for respiratory infections.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of niche-specific virulence factors via experimental evolution of Streptococcus pneumoniae

Angharad Green ^1,^*, Daniel Neill ¹, Jay Hinton ¹

Abstract

Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, adept at colonising various ecological niches within the host. Colonisation of the nasopharynx, followed by asymptomatic carriage and non-inflammatory clearance is the predominant outcome of infection, but diverse disease manifestations including pneumonia, septicaemia and meningitis occur in a minority of individuals. Through experimental evolution of pneumococci in mouse disease models, we are investigating the genetic basis of the niche adaptations that enable pneumococci to switch from a commensal lifestyle in the nasopharynx, to a pathogenic phenotype in the lungs, brain or blood. Experimental evolution was carried out via serial passage of pneumococci separately through pneumonia and nasopharyngeal carriage mouse models, to generate lineages adapted to the lung and nasopharyngeal environments, respectively. Starting from a non-passaged (lab adapted) isolate, ten independently-evolved lineages of lung-adapted pneumococci have been generated, each having been passaged 20-times through a mouse pneumonia model. Pneumococci recovered from the infected lungs were used to inoculate further mice for the next passage round. We will present the results from whole genome sequencing and phenotypic analysis of lung-passaged bacterial isolates including growth characteristics, toxin production, adherence and invasion with lung epithelial cells. We will also describe how the acquired pneumococcal adaptations, which facilitate survival in the lung environment, can influence bacterial gene expression during exponential growth. These studies will provide insight into genetic changes associated with pneumococcal commensal to pathogen switch. Identifying such genetic determinants of virulence will be valuable for the development of vaccine candidates and targets for therapeutic intervention.

Access Microbiol. 2019 Apr 8;1(1A):15.

Using cryo-electron tomography to elucidate the structural basis of the endosomal K+ requirement during Bunyavirus entry

Samantha Hover ^1,^*, Emma Punch ¹, Juan Fontana ¹, John Barr ¹, Jamel Mankouri ¹

Abstract

The Bunyavirales order are the largest group of negative stranded RNA viruses, infecting humans as well as a bewildering array of animals and plants, in which select members cause severe or fatal disease. To enter host cells, bunyaviruses undergo endosomal transport to specific cellular destinations and exploit the changing environment of maturing endocytic vesicles to mediate genome release. Several virus-endosome fusion triggers have previously been identified, including endosomal potassium (K+) recently identified by our group. Specifically, we demonstrated a role for K+ channels and endosomal K+ concentration ([K+]) in the ‘priming’ of virions for fusion and uncoating events. Interestingly for Bunyamwera virus (BUNV), both a reduced pH and elevated [K+] were required to permit endosomal escape of the virus. To understand the molecular basis for this requirement we have used cryo-electron tomography to study the changes in virion structure upon K+ and pH treatment. These studies reveal why endosomal [K+] and K+ channels are required for bunyavirus entry, highlighting the potential of K+channels as druggable anti-viral targets.

Access Microbiol. 2019 Apr 8;1(1A):15.

Developing a universal strategy for cloning and assembly of the genomes of diverse Epstein – Barr virus strains

Amr Bayoumy ^1,^*, Mohammed Ba Abdullah ¹, Rob White ¹

Abstract

Epstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.

Access Microbiol. 2019 Apr 8;1(1A):15.

Characterising the genomes and transcriptomes of hyper ammonia producing bacteria from the rumen

Jess Friedersdorff ^1,^*, Chris Creevey ², Alison Kingston-Smith ¹

Abstract

Ruminants depend on the highly diverse microbial community that resides in the rumen, the first and largest chamber of their digestive system, to gain nutrients from their herbivorous diet. The Hyper Ammonia Producers (HAPs) are obligate amino acid fermenting bacteria found in low numbers in this community. This break down of amino acids and peptides results in excessive ammonia production, as well as hydrogen and carbon dioxide, resulting in loss of nitrogen from the host and contribution to environmental emissions from enteric fermentation. Despite their large impact, little is known about the genomic underpinnings of the HAP phenotype. Our study employed comparative genomics and transcriptomics approaches to address this question. A phylogenetic tree of 498 rumen prokaryotic microbial genomes from the Hungate 1000 project (including 12 known HAPs) identified the HAP phenotype as polyphyletic, indicating independent origins or a result of horizontal gene transfer (HGT). However, following construction of sequence similarity networks for all genomes, few uniquely shared homologous genes families were apparent in the HAPs, suggesting that HGT did not drive their evolution. Instead, independent evolution of the phenotype is supported by similar functional analog profiles in the genomes of organisms with the HAP phenotype. Genome-wide characterisation and expression of functional analogs in known HAPS will allow in silico prediction of novel HAPs from the rumen which can be confirmed in vitro.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating evolution of the paediatric cystic fibrosis lung microbiota using induced sputum sampling and culture-independent techniques

Rebecca Weiser ^1,^*, Juliette Oakley ², Cerith Jones ³, Eshwar Mahenthiralingam ¹, Julian Forton ^2,⁴

Abstract

Background

Children with cystic fibrosis are frequently non-productive of sputum even during exacerbation. Current routine sampling methods are either invasive (Bronchoalveolar lavage; BAL) or insensitive (cough swab) which makes pathogen surveillance challenging. We investigated induced sputum (IS) as a promising complementary sampling technique, looking at both its comparison to BAL and the evolution of the lung microbiota across children aged 0.5–18 years.

Methods

BAL and IS samples were collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). DNA was extracted from samples and Illumina NextSeq sequencing of the 16S rRNA gene V4 region was performed. Bioinformatics data processing was carried out using Mothur. Microsoft Excel and R statistical software were used for downstream analyses.

Results

Comparison of the microbiota of 30 BAL-IS matched patient samples indicated that at a presence/absence level, IS captured >80 % of the pathogens observed in BAL samples. These findings validated the expansion of the study to 136 un-matched IS sputum samples, and we found that as patient age increased, bacterial diversity decreased, and changes in the abundance of key genera occurred over time. Both Neisseria and Haemophilus decreased with age, whilst Pseudomonas and a sub-group of Prevotella increased.

Conclusions

Here we uniquely demonstrate using culture-independent techniques that IS captures the majority of the bacterial diversity observed in BAL samples. The age associated decline in lung microbiota diversity, previously documented using BAL samples, can also be captured using IS samples. This suggests that surveillance of microbiota evolution may be possible using this method.

Access Microbiol. 2019 Apr 8;1(1A):15.

African Horse Sickness virus: pathogenicity in an IFNAR(-/-) mouse model of infection

Luke Jones ^1,^*, Pippa Hawes ², Javier Salguero ³, Javier Castillo-Olivares ^4,⁵

Abstract

African Horse Sickness (AHS) is a highly lethal, vector-borne viral disease of equids, endemic to sub-Saharan Africa but with a history of outbreaks into Europe. The pathogenesis of the virus is not fully understood; some studies have shown that certain proteins are linked to virulence, and that the outcome of infection is highly dependent on viral factors. Disease can also manifest with several different presentations in the equine host (fever form, cardiac form, pulmonary form and mixed form) but the mechanism underlying this variation is not fully understood. Pathogenicity and virulence studies of AHSV are difficult to perform in horses for logistical, ethical and financial reasons. As an alternative, Interferon-alpha receptor knockout (IFNAR-/-) mice have previously been used in AHSV vaccinology studies. Full pathology characterization of the AHSV infection in this model is the primary objective of our work, which will address questions regarding pathogenesis of AHSV. Here we present data collected from experimental infection of IFNAR (-/-) mice with a strain of AHSV serotype 4, including: clinical signs; histopathology; immuno-histochemical analysis of immune response to infection; antigen detection in tissues; and transmission electron microscopy of AHSV infected tissues. In addition, we will show data from experimental infection in this animal model comparing the pathogenicity of different AHSV strains. Results obtained indicate AHSV-4 infection is correlated with oedema and pneumonia in the lungs, inflammation in the liver and meningitis plus perivascular cuffing in the cerebrum. Other data shows that different strains of AHSV differ in terms of their pathogenicity and tropism.

Access Microbiol. 2019 Apr 8;1(1A):15.

Applying Next Generation Sequencing Technology to Hepatitis C genotyping in a routine diagnostic laboratory

Sophie May ^1,^*

Abstract

Background

To meet the target of Hepatitis C elimination in the UK, a rapid and high throughput method of genotyping is desirable. Current methods target the NS5B region with a nested PCR followed by Sanger sequencing. Drawbacks include insufficient information to guide second line treatment in the event of failure and poor sensitivity compared to assays targeting the 5’UTR.

Method

Previously tested HCV positive patient samples were selected and RNA extracted using an ultra-centrifugation manual extraction method. A two-step reverse transcription long range PCR generated amplicons which were quantified using the Qubit fluorometer and libraries prepared using the Nextera XT library preparation kit. Sequencing was performed using an Illumina Miseq and genomes were assembled using a Linex-based bioinformatics pipeline.

Results

A total of 150 samples were tested, with full HCV genomes obtained for genotypes 1–6 and the sensitivity of the assay corresponded to around 1000 IU ml⁻¹. Where previous sequence data for resistance associated variants (RAVs) was available, mutations were consistent. Furthermore, the assay identified a 2 k/1b recombinant and allowed the typing of previously unassigned genotypes, demonstrating its utility in the diverse local population.

Conclusion

This study displayed the utility of a whole genome sequencing approach to HCV. A major benefit of whole genome analysis was the inclusion of regions targeted by antiviral therapy such as NS5A and NS3. This allowed the identification of RAVs in patients experiencing treatment failure. Its application to dried blood spots is currently being investigated as part of a wider screening programme.

Access Microbiol. 2019 Apr 8;1(1A):15.

Identification of novel factors associated with severe respiratory syncytial virus disease in infants

Hong Guo Parke ¹, Helen Groves ¹, Lindsay Broadbent ¹, Olivier Touzelet ¹, Isobel Douglas ², Guillermo Lopez Campos ¹, Paul Canning ¹, Jeremy D Lyons ², Peter Coyle ³, Liam Heaney ¹, Michael D Shields ¹, Ultan F Power ^1,^*

Abstract

Background

Almost all infants are infected with RSV by 2 years. 1–3 % of RSV-infected infants are hospitalised with severe disease. Reasons for susceptibility to severe disease remain obscure. We aimed to identify factors that might explain such susceptibility.

Methods

We generated well-differentiated primary nasal epithelial cell (WD-PNEC) cultures from infants with histories of severe or mild RSV disease. Following infection with RSV BT2a (clinical isolate), virus growth kinetics, cytopathogenesis, chemokines, and IL-29/IFNλ1 responses and differential gene expression were determined. In an exciting development, 1 differentially expressed gene, ptn, encodes pleiotrophin (PTN), which interacts with nucleolin (NCL), an RSV entry co-factor. The PTN antiviral activity was determined in BEAS-2B cells and WD-PBECs (where B=bronchial).

Results

Viral growth kinetics, cell tropism, IP-10/CXCL10, TRAIL and RANTES/CCL5 responses did not differ significantly between cohorts. However, apical cell sloughing and IL-29/IFNλ1 were diminished in severe WD-PNECs following infection, while expression of isg15, ifi6, irf9, duox2 and tap1 was also reduced. Interestingly, diminished ptn expression was evident in severe WD-PNECs, irrespective of RSV infection. Furthermore, pre-treatment with PTN blocked RSV infection in BEAS-2B cells and WD-PBECs, while neutralisation of PTN with either antibodies or siRNAs resulted in increased RSV replication.

Conclusions

Diminished apical cell sloughing and expression/secretion of IL-29/IFNλ1 and specific interferon stimulated genes in WD-PNECs were associated with severe RSV. Importantly, PTN was identified as a novel endogenously expressed RSV antiviral protein in human airway epithelium. Lower expression of PTN in paediatric airway epithelium may explain, in part, increased susceptibility to severe disease.

Access Microbiol. 2019 Apr 8;1(1A):15.

Characterising the insect-borne transmission of the cattle poxvirus lumpy skin disease virus

Beatriz Sanz-Bernardo ^1,^*, Najith Wijesiriwardana ¹, Ismar Haga ¹, Simon Gubbins ¹, Pippa Hawes ¹, Anthony Wilson ¹, Christopher Sanders ¹, Sanjay Basu ¹, Luke Alphey ¹, Zoe Langlands ¹, Karin Darpel ¹, Pip Beard ¹

Abstract

Lumpy skin disease (LSD) is an emerging poxviral disease of cattle caused by the Capripoxvirus lumpy skin disease virus (LSDV) which generates widespread cutaneous lesions in affected animals. LSD is recognised as a transboundary high consequence disease in Africa where it contributes to rural poverty and food insecurity. In 2015 LSDV spread to southeastern Europe and currently poses a threat to cattle in neighbouring regions. Previous research indicates that LSDV is most likely transmitted by insect vectors however details of transmission pathways are unclear. This study was designed to identify the risk of transmission of LSDV posed by different insect vectors. A bovine experimental model of LSD was established in the high containment facilities at The Pirbright Institute. Potential insect vectors (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus) were fed on LSDV-infected cattle then incubated for up to eight days. Cattle and insects were regularly sampled to quantify LSDV present in different tissues and vector species. This data was then used to model the dynamics of LSDV infection and transmission. All four species of vector successfully acquired LSDV from infected cattle and maintained the virus up to eight days post feeding. The outputs of this research will now be used to design more effective LSD control programmes.

Access Microbiol. 2019 Apr 8;1(1A):15.

The long isoform of ZAP widely restricts Paramyxoviruses

Patricia A Thibault ^1,^*, Andrew P Ryan ², Chuan-tien Hung ¹, Matthew Daugherty ², Benhur Lee ¹

Abstract

Paramyxoviruses (PVs) are negative-sense RNA viruses that are important in human and animal health, and can cause severe zoonotic diseases. We screened a wide range of animal and human PV matrix proteins for host interactions, and found that the long (L) isoform of the host protein ZAP (zinc-finger antiviral protein) interacted with all the tested viral matrixes. ZAP-L is constitutively expressed, has a prenlyation motif for membrane-localization, and primarily mediates antiviral activity by binding and degrading viral RNAs through the exosome complex. However, ZAP restriction of PVs has not been demonstrated in the literature. We found that knockdown of ZAP-L results in increased replication of a panel of five human and animal PVs. Overexpression of ZAP-L (but not ZAP-short) restricts replication of PVs in a reciprocal pattern – with the notable exception of Sendai virus (SeV) – and as with other viruses, mutating the prenylation motif of ZAP-L abolishes restriction. RT-qPCR of PV RNAs and pulled-down RNAs does not indicate specific targeting of a viral transcript by ZAP-L, although overall genome abundance is reduced. Finally, immunoprecipitation of ZAP shows an additional RNA-independent interaction between ZAP-L and SeV-nucleocapsid not found with HPIV3-nucleocapsid, a closely-related PV. Thus, we have observed that ZAP-L interacts with the matrix of, and restricts replication of, a wide range of paramyxoviruses. A PV that is not restricted (SeV), has an additional interaction with ZAP via its nucleocapsid protein that may ameliorate ZAP restriction. Investigating ZAP-related restriction differences between closely-related PVs may shed light on anti-viral mechanisms of ZAP.

Access Microbiol. 2019 Apr 8;1(1A):15.

Tracking plasmid-mediated antibiotic resistance from environmental reservoirs to the food chain

Sinéad Murphy ^1,^*, Aoife Joyce ¹, Vera Vollenweider ², David Drissner ², Fiona Walsh ¹

Abstract

It has been well documented that antibiotic resistance (AR) is a clinical concern that affects both human and animal health but AR in the environment and food-chain is not as well understood. AR bacteria can occur naturally in soil, water and organic fertilizers used in agriculture so there is a risk that AR can pass to humans via the food-chain. This study focuses on lettuce cultivation undergoing four treatments (Normal irrigation water+normal soil, normal irrigation water+manure, UV irrigation water+normal soil, UV irrigation water+manure)to determine the mechanisms by which the AR is transferred to the plants over the growth period of the lettuce (7 time-points – week 0 to week 6). Plasmids (n=318) have been isolated from irrigation water (n=36), soil (n=45) and lettuce (n=42) samples using the exogenous isolation method for week 0 and week 6 initially. Antibiotic susceptibility testingto amikacin, cefotaxime, ciprofloxacin, imipenem, kanamycin, tetracycline has been carried out. Multi-drug resistance profiles were established for soil taken at timepoint 0 and lettuce taken at timepoint 6. Extracted plasmid DNA was sent for metagenomic analysis to determine which genes are involved in the transfer of AR at the interfaces. The results of the sequencing showed that there are multiple AR genespresent, including Tet, Sme, Cmy, Oxa and ANT(4’)-Ib, that confer resistance to bacteria. The identification of multi-drug resistance in soil and lettuce samples is concerning and highlights the need to determine the mechanisms leading to antibiotic resistance in food.

Access Microbiol. 2019 Apr 8;1(1A):15.

Spatiotemporal dynamics of host cell modification caused by herpesvirus infection

Katharina M Scherer ^1,^*, Timothy Soh ², James D Manton ³, Luca Mascheroni ¹, Colin M Crump ¹, Clemens F Kaminski ¹

Abstract

Herpesviruses are large and complex DNA viruses that are composed of an icosahedral capsid, a proteinaceous layer termed the tegument, and a glycoprotein rich lipid envelope. One important area of host-pathogen interaction that is still poorly understood is the extensive change to intracellular organelles and cellular morphology that occur within the infected cell during active virus replication. In order to characterise the spatiotemporal dynamics of host cell remodelling caused by herpesvirus infection, we use novel multiparametric fluorescence microscopy methods compatible with live-cell imaging. In addition, we apply expansion microscopy to map 3D rearrangement in great detail. The remodelling of the host cell is correlated to the stage of virus replication which can highly vary between individual cells. Therefore, we have constructed a recombinant reporter virus that expresses eYFP-tagged ICP0, a multifunctional immediate early tegument protein, as well as mCherry-tagged glycoprotein C (gC), a late protein that is a major component of the viral envelope. The sequential expression of these two viral proteins provides us with an intrinsic time stamp for the stage of virus infection in each cell. With this fluorescent reporter virus, we are able to describe the remodeling of- the three-dimensional architecture of microtubules and the actin network,- compartments of the secretory and endocytic pathways which are intimately linked to viral envelope protein synthesis, maturation and transport,- and key antiviral and inflammatory signalling platforms (mitochondria and peroxisomes).

Access Microbiol. 2019 Apr 8;1(1A):15.

Cellular cholesterol abundance regulates potassium accumulation within endosomes and is an important determinant in Bunyavirus entry

Frank Charlton ^1,^*, Samantha Hover ¹, Jack Fuller ¹, Roger Hewson ², Juan Fontana ³, John Barr ³, Jamel Mankouri ³

Abstract

The Bunyavirales order of segmented negative sense RNA viruses includes over 500 isolates that infect insects, animals, and plants, and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must transport their genomes from outside the cell into the cytosol, achieved by transit through the endocytic network. We have previously shown that the model bunyaviruses Bunyamwera virus (BUNV) and Hazara virus (HAZV) exploit the changing potassium concentration ([K+]) of maturing endosomes to release their genomes at the appropriate endosomal location. K+ was identified as a biochemical cue to activate the viral fusion machinery, promoting fusion between viral and cellular membranes, consequently permitting genome release. In this study, we further define the biochemical prerequisites for BUNV and HAZV entry and their K+ dependence. We report four major findings: (1) BUNV and HAZV require cellular cholesterol during virus infection; (2) cholesterol is required during BUNV endosomal escape; (3) cholesterol depletion from host cells impairs their ability to accumulate K+ in maturing endosomes, revealing new insights into endosomal K+ homeostasis; (4) ‘priming’ BUNV virions with K+ prior to infection alleviates BUNV cholesterol requirement, revealing the mechanism of cholesterol dependence. Taken together, we provide a new model in which cholesterol abundance influences K+ endosomal homeostasis and consequently the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol lowering drugs offers new options for future anti-bunyavirus interventions for pathogenic family members.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigation and genome characterisation of tatenale hantavirus in wild rodent populations in the United Kingdom

Joseph G Chappell ^1,^*, Theocharis Tsoleridis ¹, C Patrick McClure ¹, Malcolm Bennett ¹, Rachael E Tarlinton ¹, Jonathan K Ball ¹

Abstract

Hantaviruses are a diverse group of single-stranded, negative sense RNA viruses, belonging to the Bunyaviridae family. They are primarily rodent-borne and transmitted into humans through the inhalation of aerosolised excreta of infected animals. Each hantavirus is typically associated with a single reservoir species, resulting in a persistent, yet asymptomatic infection. In humans, however, in addition to asymptomatic infection two disease syndromes are associated with hantavirus infection, hantavirus haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Tatenale Hantavirus (TATV) was first isolated from field voles in NW England in 2013 and has since been detected at other sites in Northern England. However, only two small fragments of the L and S segments have been sequenced, precluding full phylogenetic characterisation of the virus. Field voles were sampled at two sites in Leicestershire (N=104) and Cheshire (N=12), and their lungs subjected to a degenerate pan-hantavirus RT-PCR assay, targeting a section of the polymerase gene. Eleven Cheshire and one Leicestershire sample were TATV positive, and were 94 % homologous across the amplicon. Illumina Hi-Seq sequencing revealed the complete coding regions of the Leicestershire isolate. Analysis of the sequence showed that TATV is most closely related to Khabarovsk virus, with a homology of 91%/88%/89 % (AA) across the L/M/S Segments, respectively. ICTV species demarcation requires an AA difference of >7 % across the S and M, TATV meets the requirement of a novel species. Retrieval of full coding sequence will allow further investigation into TATV, and its potential to infect humans through pseudotyping of the glycoproteins.

Access Microbiol. 2019 Apr 8;1(1A):15.

High-throughput sequencing of patients with symptoms of unknown Etiology

Joseph G Chappell ^1,^*, Theocharis Tsoleridis ¹, C Patrick McClure ¹, Rachael Tarlinton ¹, Gemma Clark ², William L Irving ^1,², Jonathan K Ball ¹

Abstract

Routine clinical diagnosis of patients with a suspected viral infection involves screening with multiple assays, often limited to only a single genus or species. This approach, however, may fail to detect novel species, atypically presenting viruses (i.e a ‘respiratory’ virus causing neurological symptoms) and viruses that are imported from other countries; often due to clinicians focusing on the ‘likeliest’ candidates. High-throughput sequencing (HTS) allows for the identification viruses present within a sample, by sequencing all viral genomes present. Without the inherent bias of limiting screening targeted assays, these unusual viruses are more likely to be detected. Samples taken from patients with an illness of unknown etiology, were grouped into 5 pools; 2 Respiratory, 2 CSF and an EDTA blood pool. The CSF Pools each contained 200 samples, the Respiratory pools 100 and the EDTA blood pool 80. HTS libraries were created from each of these pools and an additional CSF sample from a single patient with encephalitis and were then sequenced using an Illumina HiSeq platform. Human Pegivirus was detected in both CSF Pools, the EDTA pool and a single respiratory pool. Picobirnavirus was detected in a respiratory pool. RT-PCR was used to screen individual samples compromising these pools. BK Polyomavirus and Mastadenovirus C were detected in the CSF of a patient who had presented with encephalitis. Coinfection of these viruses typically cause neurological symptoms only in immune-compromised patients, so this exemplifies the advantage of using HTS for the detection of atypically presenting viruses.

Access Microbiol. 2019 Apr 8;1(1A):15.

The Savage Dawn Peptide: an antibiotic woven from from 12th century Welsh poetry

Benjamin Thomas ^1,^2,^*, Jurnorain Gani ³, Chris Creevey ², Kai Hilpert ³

Abstract

Bacterial antibiotic resistance is widely regarded to be one of the most pressing threats facing humanity. Finding new antibiotics is a vital research area and can now be supported by a vast reservoir of readily available ‘omic data on the back of the explosion of low cost sequencing technologies. Antimicrobial Peptides (AMPs): endogenous peptides that provide a fast and effective means of defence against pathogens as part of the innate immune response. The detection of AMPs in metagenomic data is a tantalising low-hanging fruit for computational biologists. Large reservoirs of existing sequences exist and are well annotated and understood. Post-computational wet-lab work is relatively cheap with spot synthesis of peptides cheaply available from a wide array of third party companies. A well organised screening program can screen 100 s of prospects a day against model bacterial organisms to test for activity and is one of the few areas of biological science that can scale to meet the data output from computational prediction toolkits. AMPLY, an in-house tool designed at Aberystwyth University, supported by Life Science Wales and working in collaboration with Tika Diagnostics at St. George’s Hospital (London) and Queen’s University (Belfast) is part of a next wave of computational drug discovery platforms and is already uncovering a treasure trove of novel AMPs in diverse microbial environments. However, AMPLY’s abilities extend beyond the analysis of ‘omic data alone and its predictive modelling has extracted a therapeutically viable novel AMP built from a string of ancient Welsh poetry.

Access Microbiol. 2019 Apr 8;1(1A):15.

Induction of inflammasome-dependent signalling in the human monocytic cell line THP-1 by Campylobacter lipooligosaccharides

Amber Hameed ¹, Lee Machado ¹, Alexandra Woodacre ^1,^*, Gemma Marsden ^2,¹

Abstract

The Campylobacter lipooligosaccharides (LOS) can stimulate membrane-bound innate immune receptors in human macrophages. However, the association of Campylobacter LOS in the stimulation of cytosolic receptors or the inflammasome remains poorly characterised. Therefore, the aim of this study was to determine the role of Campylobacter LOS in the activation of NLRP3 inflammasome-dependent signalling in a human monocytic cell line. The induction of NLRP3 inflammasome-mediated IL-1β and Caspase-1 secretion in THP-1 supernatants was quantified using ELISA following co-culture of THP-1 cells with LOS extracts from wild type C. jejuni 11168, mutant C. jejuni 11 168 with reduced LOS and two wild type C. coli strains (RM1875 and 76339). Our results demonstrate that LOS purified from both C. jejuni and C. coli can induce Caspase-1 and IL-1β production in human macrophages. However, C. jejuni 11 168 mutant LOS with modified lipid A and lack of core oligosaccharides stimulated significantly reduced Caspase-1 and IL-1β. This result was also replicated in co-culture of live wild type and mutant C. jejuni with THP-1 cells. This study provides new insight into the interaction of Campylobacter with human macrophages and suggests that variation in LOS structure may alter NLRP3 inflammasome activation.

Access Microbiol. 2019 Apr 8;1(1A):15.

Investigating the interferon antagonistic abilities of louping ill virus, a neglected animal pathogen endemic to the UK

Jordan Clark ^1,^2,^*, Giel Goertz ³, Esther Schnettler ⁴, Colin McInnes ², Roman Biek ⁵, Alain Kohl ¹

Abstract

Louping ill virus (LIV; Flavivirus, Flaviviridae) is an important—but poorly characterized—animal pathogen of significant economic concern within the UK. Transmitted by ticks, LIV predominantly causes disease in ruminants and grouse, resulting in heavy losses. LIV is closely related to another flavivirus—tick-borne encephalitis virus (TBEV) which, unlike LIV, is a significant human pathogen. The molecular mechanisms that underpin host restriction in these viruses are poorly understood however, previously it has been shown that the TBEV non-structural (NS) proteins do not act as type-I interferon (IFN) antagonists, unlike many other Flavivirus NS proteins. Therefore, to facilitate comparison with LIV we investigated the possible antagonistic actions of the LIV NS proteins using a luciferase-based IFN reporter assay. Utilising this assay we identified six LIV NS proteins that function as antagonists throughout the IFN induction cascade. We also identified and modelled a subgenomic flavivirus RNA (sfRNA) that is produced during LIV infection and is similar in structure to TBEV sfRNA. We found that the LIV and TBEV sfRNAs antagonise RIG-I, indicating that the IFN antagonistic ability of sfRNA is not limited to mosquito-borne flaviviruses. Finally, we established the first LIV reverse genetics system using circular polymerase extension reaction (CPER). This powerful tool can be used to produce chimeric viruses which will allow further investigation into the factors governing host restriction and virulence in tick-borne flaviviruses. Investigating the mechanisms that underlie LIV infection aids our understanding of interferon antagonism in flaviviruses and the molecular determinants of host restriction.

Access Microbiol. 2019 Apr 8;1(1A):15.

Deciphering the secretion mechanism and novel protein-protein interactions of TecA, a Burkholderia cenocepacia toxin

Julia Monjaras-Feria ^1,^*, Fabiana Bisaro ¹, Miguel Valvano ¹

Abstract

Burkholderia cenocepacia (Bc) is an environmental opportunistic pathogen that causes persistent, often severe, lung infections in individuals with cystic fibrosis and other underlying diseases. Rho GTPases are central molecular switches that regulate cytoskeletal dynamics, trafficking, immune responses and cell proliferation in eukaryotic cells. Many microbes produce proteins that target Rho GTPase signalling. Bc employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho GTPases and causing actin cytoskeletal defects. Bc protein TecA is a non-VgrG T6SS effector that is responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces Pyrin inflammasome activation1. Our goal is to determine the detailed TecA secretion mechanism and the interacting partners inside the bacterial cytoplasm and inside the macrophages. In this study, we found by Co-IP/MS analysis that TecA interacts with the T6SS tube protein HcP, the membrane anchored TssM and with the elongation factor Tu. We also found that TecA is secreted in the absence of HcP and the secretion mechanism is discussed. 1. Aubert, D.F., X. Hao, J. Yang, X. Shi, W. Gao, L. Li, F. Bisaro, S. Chen, M.A. Valvano, and F. Shao. 2016. A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome. Cell Host and Microbe 19 : 664–674.

Access Microbiol. 2019 Apr 8;1(1A):15.

Swine influenza A viruses with zoonotic potential – PCR HA/NA typing, and differential detection of pandemic09 reassortants in GB and European pigs

Sharon Brookes ^1,^*, Scott Reid ¹, Nicola Lewis ¹, Sharon Chepkwony ², Christine Russell ¹, Jayne Cooper ¹, Vivien Coward ¹, Helen Everett ¹, Elizabeth Coney ¹, Alexander Byrne ¹, Anna Parys ², Elien Vandoorn ², Dominic Byrne ¹, Susanna Williamson ³, Kristien Van Reeth ², Ian Brown ¹

Abstract

Swine influenza A virus (swIAV) causes respiratory disease and productivity loss in pigs. Swine ‘flu viruses have been known to be both zoonotic and reverse zoonotic and they contain genes of swine, avian(av) and human(hu) origin. Surveillance of swIAV subtypes is important as genotypes/phenotypes are fluid and impact with respect to epidemiology, vaccination, pig welfare, veterinary and public health. Three sub-types (H1avN1, H1N1pdm09, H1huN2) are currently found in pigs from Great Britain (GB), plus H3huN2 in Europe and their reassortants. Screening of candidate samples is carried out by RRT-PCR assays – generic detection of swIAV (M gene) followed by a specific RRT-PCR for H1N1pdm09 (HA gene), a suite of RRT-PCR assays for sub-typing (HA and NA genes) and a (differential) RRT-PCR to specifically identify reassortant swIAVs that incorporate the pandemic 2009 internal gene cassette (NP gene). Subtyping assays, conventional and/or molecular, are carried out on virus isolation-positive and –negative (RNA only) samples from clinical material (respiratory tissue and/or nasal swabs). Since 2009, the number of swIAV has expanded with the H1N1pdm09 isolates reassorting with the traditional subtypes. Many European variants arose (>25) of which some have become established – in GB including H1huN2/pdm (since 2010), and H1avN1/pdm (since 2012), and in Belgium the traditional isolates were detected plus H1pdmN1/pdm and H3huN2/pdm reassortants. PCR subtyping (2012 onwards ∼130 from GB and ∼40 from BE/NL), wholegenome sequencing and bioinformatics analysis of these isolates facilitate further diagnostic improvements and assessment of zoonotic pandemic potential (in silico and in vivo).

Access Microbiol. 2019 Apr 8;1(1A):15.

The effects of APOBEC3 proteins on Hepatitis B virus replication

Ibrahim Elbusifi ^1,^*, Michael McGarvey ¹

Abstract

Hepatitis B virus causes chronic liver infection in 257 million people worldwide. Current treatments against HBV can control, but not cure HBV infection. Therefore, new treatments for chronic HBV infection need to be developed. The APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular restriction factors, which have been shown to restrict viral replication for a number of viruses (e.g. HIV) and for retrotransposons. The aim of this study is to elucidate the role of APOBEC3 proteins in inhibiting Hepatitis B virus replication. Quantitative polymerase chain reaction (qPCR) was used to evaluate the impact of APOBEC3 family members on the Hepatitis B virus replication in HepG2.2.15 cells. The highest inhibition of intracellular capsid associated HBV DNA, extracellular virion associated HBV DNA was induced by APOBEC3DE, APOBEC3F, and APOBEC3G as compared to the other APOBEC3 proteins. However, APOBEC3DE showed no inhibition of HBV total RNA, whereas the highest inhibition of HBV total RNA was induced by APOBEC3F and APOBEC3G. The sub-cellular localisation of APOBEC3 proteins was determined by immunofluorescence using confocal microscopy. It was found that APOBEC3DE, APOBEC3F, and APOBEC3G localise to the cytoplasm, suggesting a crucial role of these proteins in HBV replication in the cytoplasm. Nevertheless, APOBEC3A and APOBEC3B localise to the nucleus. The expression and co-localisation of APOBEC3 proteins and viral and host proteins (Uracil DNA Glycosylase UNG and ATP-dependent RNA helicase DDX3) in HepG2.2.15 and HEK293T cells are being investigated in order to determine the interaction between these proteins.

Access Microbiol. 2019 Apr 8;1(1A):15.

Viral adaptation to alternating hosts and associated allelic variants

Oyeronke Ayansola ^1,^*, Benjamin Dickins ¹, Gareth McVicker ¹, Michael Loughlin ¹

Abstract

The long-term reproductive success of a lineage depends on its ability to tolerate a wide range of environmental conditions and for a virus a substantial part of its environment is the host that it infects. Viruses may overcome limitations of a given host environment by switching to other available hosts. Experiments addressing host switching may pave the way for an improved understanding of the emergence of new viral diseases. Here, a model bacteriophage, φX174, its canonical laboratory bacterial host strain, Escherichia coli, and a novel host, Salmonella Typhimurium were employed. Bacteriophage φX174 adaptation was studied at population level in a bioreactor. We analyzed phenotypes and genotypes arising during continuous evolution of φX174 on alternating hosts for four consecutive periods of 10 days each. The fitness and adsorption of each viral population were measured using qPCR in liquid culture. Deep sequencing analysis of isolates was carried out to determine the genetic basis of pleiotropic costs and to characterize allelic variations occurring during growth. Some alleles specific to one host were lost or reduced in frequency in the alternative host while other alleles (not present in the ancestor) were shared between hosts. The fitness effects of specific alleles were examined in isolation through targeted mutagenesis. This work contributes to a better understanding of some of the general constraints, costs and benefits influencing the evolution of parasite populations as they adapt to the complexities of a novel host environment- a key consideration during the emergence of infectious diseases.

Access Microbiol. 2019 Apr 8;1(1A):15.

Replicative fitness and transmission of G57 lineage and UDL01 like H9N2 viruses in chickens

Sushant Bhat ^1,^*, Holly Shelton ¹, Jean-Remy Sadeyen ¹, Munir Iqbal ¹

Abstract

The low pathogenic H9N2 influenza viruses are a threat to poultry as well as global public health due to their ability to reassort with other avian influenza viruses leading to the emergence of novel reassortant viruses having pandemic potential. The continued inter-subtypic reassortment events between influenza viruses in the Indian sub-continent have led to the replacement of the already existing G1 lineage of H9N2 viruses with the UDL genotype-like (A/chicken/Pakistan UDL-01/08/H9N2) viruses, which are triple reassortants between H9N2 virus (G1 lineage), HPAI H5N1 virus (clade 2.2) and HPAI H7N3 viruses. G1 lineage of H9N2 viruses in China has also been replaced with a fitter G57 lineage which donated internal genes to novel H7N9 viruses in 2013. We assessed and compared the replication, transmission and pathogenic potential of UDL01/2008/H9N2 virus and A/Ck/Vietnam/H7F-14-BN4-315/2015 H9N2 virus of G57 lineage isolated from Vietnam in 2015. Vietnam H9N2 virus was found to be relatively more virulent compared to the UDL genotype-like H9N2 in Chickens. Our in-vitro and in-ovo infection studies also showed that Vietnam/BN4-315/H9N2 virus has greater replication fitness compared to UDL-01/08/H9N2 virus. The UDL-01/08 H9N2 reassortants carrying internal genes of Vietnam/BN4-315 virus also showed improved replication fitness in MDCK cells. It is, therefore predicted that genetic reassortment between dominant strains in the Far East and the Indian subcontinent/Middle East may generate more virulent H9N2 viruses.

Access Microbiol. 2019 Apr 8;1(1A):15.

Aedes Aegypti(Aag2)-derived clonal mosquito cell lines reveal impact of pre-existing persistent co-infection with the insect-specific Bunyavirus Phasi Charoen-Like Virus on Arbovirus replication

Louisa Wallace ¹, Anthony Fredericks ², Dabeiba Bernal-Rubio ², Andrew Davidson ³, Ana Fernandez-Sesma ², Kevin Maringer ^1,^*

Abstract

Aedes aegyptiis a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes, however the impact of persistent insect-specific virus infections that more accurately mimic the situation in nature has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures. We generated single cell-derived clonal cell lines from the commonly used Ae. aegypticell line Aag2. Two of the fourteen Aag2-derived clonal cell lines studied in detail harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus or the rhabdovirus vesicular stomatitis virus. Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide well-characterised Aag2-derived clonal cell lines as a valuable resource to the arbovirus research community.

Access Microbiol. 2019 Apr 8;1(1A):15.

Retrospective and prospective evaluation of chickenpox post-exposure prophylaxis (PEP) in at-risk groups before and after the change in PEP guidelines

Stephanie Harris ^1,^*, Mark Zuckerman ¹

Abstract

Background

Public Health England (PHE) chickenpox post-exposure prophylaxis (PEP) guidelines were updated in August 2018. Varicella Zoster Immunoglobulin (VZIg) was replaced with oral aciclovir or valaciclovir in specific settings.

Method

We retrospectively examined issues of VZIg between August 1 2017 and July 31 2018 at a transplantation centre that also had a large maternity service. In addition, since September 2018, information has been collected prospectively. Additional data were collected on exposure incident, patient risk group and any clinical or serological follow up.

Results

Thirty issues of VZIg were identified retrospectively over 12 months. These included 9 pregnant women and 21 immunosuppressed patients, average age was 7.6 years (median 4.4 years, SD 11.1). Prospective outcome data were collected since September 4 2018 involving 2 pregnant women and 6 immunosuppressed individuals. Clinical follow-up information was available for 2 pregnant women and 16 immunosuppressed patients. One pregnant woman exposed to her own child with chickenpox and 2 immunosuppressed patients developed clinical varicella at an average of 15.3 days post VZIg. Five immunosuppressed patients were VZV IgG negative post-exposure. Follow-up available to date for 2 of 8 exposure incidents after the change in guidance has shown no clinical or serological evidence of varicella infection.

Discussion

Where follow up information was available, 19 % developed attenuated VZV around 2 weeks after receiving VZIg. Given the new guidance on using aciclovir prophylaxis, prospective outcome data are being collected and no VZV infections have been noted to date.

Access Microbiol. 2019 Apr 8;1(1A):15.

In vitro reconstitution of the polymicrobial community associated with cystic fibrosis airway infections

Tom O’Brien ^1,^*, Martin Welch ¹

Abstract

The airways of cystic fibrosis (CF) patients provide a rich and unique environmental niche, prone to lifelong chronic infection by a diverse and dynamic polymicrobial community. Such dense microbial ecosystems have a network of interspecies communication between each member of the community, serving to modulate virulence, impact metabolism and contribute towards antimicrobial resistance (AMR). Currently no models exist which enable the long-term culture of a true polymicrobial community. Most existing animal models are only suitable for short term infection studies, often utilising relatively healthy hosts and which use axenically cultured clonal strains, providing little parallel to the complex biochemical interactions occurring within chronic CF infections. Here we describe a simple in vitro model utilising artificial sputum medium to allow the successful coculture of major CF-associated pathogens and begin to recapitulate and maintain the CF microbiome within a relatively steady-state. An in vitro model confers several advantages for studying widespread community changes and pathogenic interactions. Perhaps most importantly, in vitro models can be easily perturbed through the addition of antibiotics or introduction of new species/strain variants, allowing the impact of external stressors upon the emergence and changes in lifestyles of key pathogens to be effectively studied. A simple, robust and physiologically relevant CF model could be applied to address any number of fundamental biological questions surrounding interspecies interactions occurring within polymicrobial infections.

PERMALINK

ACMI conference posters 2019

Viral hijacking of the nucleolar DNA-damage response machinery: a novel mechanism to regulate host cell biology

Stephen M Rawlinson

Tianyue Zhao

Ashley M Rozario

Christina L Rootes

Paul J McMillan

Anthony W Purcell

Amanda Woon

Glenn A Marsh

Kim G Lieu

Lin-Fa Wang

Hans J Netter

Toby D M Bell

Cameron R Stewart

Gregory W Moseley

Abstract

Microbiological profile and risk factors for in-hospital mortality of infective endocarditis intertiary care hospitals of south Vietnam

Hoang Tran

Abstract

Objectives

Materials and methods

Results

Conclusion

The effect of epinephrine and norepinephrine on the growth and pathogenicity of Campylobacter jejuni

Brendha Truccollo

Paul Whyte

Declan J Bolton

Abstract

In-ovo antiviral assay of methanolic leaf extract of Cymbopogon citratus (Lemon grass) on Newcastle disease virus

Joseph Abraham-Oyiguh

Abigail W Zakka

Joseph Taiwo C Onwuatuegwu

Lanre K Sulaiman

Salamatu Y Muhammad

Abstract

Mathematical modelling to characterise the in vivo dynamics of Salmonella in the naïve and immunised host

Myrto Vlazaki

Olivier Restif

Abstract

Structural elucidation of viral antagonism of innate immunity: the STAT1 interface

Gregory W Moseley

Alamgir Hossain

Florence Larrous

Jingyu Zhan

Ashish Sethi

Youssef Ibrahim

Maria Aloi

Stephen M Rawlinson

Kim G Lieu

Yee-Foong Mok

Michael D W Griffin

Naoto Ito

Toyoyuki Ose

Hervé Bourhy

Paul R Gooley

Abstract

Shining new lights on chytrid cell biology: quantitative live cell imaging of rhizoid development in an early-diverging fungus

Davis Laundon

Glen Wheeler

Thomas Mock

Michael Cunliffe

Abstract

The HamE scaffold positively regulates MpkB phosphorylation to promote development and secondary metabolism in Aspergillus nidulans

Dean Frawley

Betim Karahoda

Ozlem Sarikaya Bayram

Ozgur Bayram

Abstract

Identification of Acinetobacter baumannii type VI secretion system effectors and characterisation of a novel effector/immunity pair

Jessica Lewis

Deanna Deveson Lucas

Marina Harper

John Boyce

Abstract

Inter-species transmission of avian influenza virus to dogs: 10 years experience

Daesub Song

Woonsung Na

Minjoo Yeom