Can't live outside you: a thematic issue on obligate intracellular bacterial pathogens

To establish their niches for microbial replication, obligate intracellular bacterial pathogens co-opt numerous host cell resources and hijack metabolic pathways. Because of their unique evolutionary strategies of host adaptation, species belonging to the bacterial genera Chlamydia, Rickettsia, Anaplasma, Ehrlichia and Orientia are confined to an intracellular lifestyle and can currently not be cultured axenically. In this thematic issue, we compiled a collection of review and primary research articles covering diverse facets of the biology of obligate intracellular bacterial pathogens, as well as methodological aspects including disease modeling and advances in applying tractable genetic tools to the study of these fastidious and fascinating microbes.

The advent of high throughput next-generation sequencing has accelerated research in many fields but has been particularly fruitful in overcoming technical challenges unique to the study of obligate intracellular pathogens. Genome sequencing and comparative genomics studies as performed by Dimond and Hefty (2021) for the ubiquitous swine pathogen Chlamydia suis provide insights into evolutionary dynamics of the Chlamydia genus and identify gene sets specific to certain Chlamydia species as potential host tropism determinants. Deep sequencing combined with traditional chemical mutagenesis is amongst the many newly developed genetic approaches that have revolutionized Chlamydia research over the last few years, as expertly reviewed by Banerjee and Nelson (2021). Progress in developing genetic tools has also been made for Rickettsia and these advance are being discussed as part of a comprehensive review of Rickettsia biology (McGinn and Lamason 2021). The only obligate intracellular bacterial pathogen for which no genetic manipulation methods have been developed so far is the genus Orientia in the family of Rickettsiaceae. As one of the first steps towards the development of such genetic tools, Hunt and Carlyon functionally assessed a series of putative promoters found in the genome of Orientia tsutsugamushi (Hunt and Carlyon 2021), the causative agent of scrub typhus. These types of incremental advances are needed to pave the way towards the development of genetic systems that can be applied to study O. tsutsugamushi pathogenesis.

Mutagenesis combined with functional screens is a powerful approach to identify specific bacterial factors that co-opt host cellular processes, such as energy metabolism, or undermine cell-intrinsic immune responses. While this approach is limited in so far as many bacterial genes are essential for culturing obligate intracellular bacteria in cell-based systems, the distribution of recoverable mutations can be used to predict the repertoire of essential genes. Munderloh and colleagues took this approach to predict bacterial genes essential for growth of Anaplasma phagocytophilum inside human cells (O'Conor et al. 2021). Ultimately, studying the function of essential genes requires more sophisticated genetic toolkits such as the design of conditional gene deletion alleles, or alternatively, the establishment of axenic culture system, a feat that was accomplished for the formerly unculturable bacterial genus Coxiella more than a decade ago (Omsland et al. 2009). Employing this axenic culture system, Sanchez and Omsland (2021) report here how the loss of specific metabolic enzymes in Coxiella provides a fitness advantage that may account for some of the pseudogenization and genome contraction often observed in obligate intracellular bacteria. In this context, the article goes on to discuss the diversity of energy metabolism among intracellular bacterial pathogen more broadly.

Applying genetic tools to the study of obligate intracellular bacterial pathogens has been particularly successful in characterizing the specific functions of bacterial virulence factors or ‘effectors’ that are being delivered into host cell by designated bacterial secretion systems. A total of three excellent review articles provide overviews on how Chlamydia, Ehrlichia or Rickettsia, respectively, employ their arsenal of effectors to usurp host cell processes for the benefit of these bacterial invaders (Andersen et al. 2021, Byerly, Patterson and McBride 2021, Voss and Rahman 2021). An additional review examined the various strategies used by obligate intracellular pathogens to maintain host viability during infection, a process necessary for maintenance of the intracellular replicative niche (Loterio, Zamboni and Newton 2021).

Complementary to the study of bacterial effectors are approaches that interrogate the function of host factors manipulated by bacterial effectors. One powerful approach to monitor host membrane protein dynamics is the use of proximity ligation systems. However, as Rucks and colleagues report, potential caveats in applying this methodology during infection studies need to be considered (Jorgenson, Olson-Wood MG and Rucks 2021). In a separate study, Carlyon and colleagues demonstrated that host acid sphingomyelinase (ASM), previously shown to be critical for A. phagocytophilum growth inside human neutrophils and productive infections of immune-competent mice (Cockburn et al. 2019), is not strictly required for bacterial colonization of an immunocompromised rodent host lacking an adaptive immune system (Naimi et al. 2021). Their work, therefore, suggests possible functional links between the parasitic exploitation of ASM and the adaptive immune response during A. phagocytophilum infections.

As the aforementioned study already illustrates, discoveries made in cell culture systems need to be assessed in more complex in vivo systems to determine their physiological relevance. The use of mouse models has been instrumental in forming our understanding of the role innate and adaptive immunity plays in providing host protection and promoting immunopathogenesis of genital Chlamydia infections, as summarized in two review articles (Dockterman and Coers 2021, Helble and Starnbach 2021). Mouse infection models are also increasingly being used to assess the role of individual bacterial genes in chlamydial diseases. For example, Liu and colleagues report that the plasmid-encoded enzyme glycogen synthase A, required for bacterial glycogen storage, promotes vaginal bacterial shedding and gross pathology during Chlamydia muridarum infections (Hu et al. 2020). As alternatives to mouse models, Dragan and Voth (2021) review primary human lung tissue models for the study of human pulmonary pathogens such as Coxiella burnetii. Taking their work to the bedside, Greub and colleagues report detection of the pulmonary pathogen Chlamydia pneumoniae at relatively high frequency in cystic fibrosis pediatric patients (Pittet et al. 2021), unveiling an unexplored potential relationship between cystic fibrosis and susceptibility to pulmonary Chlamydia infections that awaits further investigation.

The obligate intracellular bacterial pathogens Orientia, Ehrlichia, Anaplasma and Rickettsia transmit to their mammalian hosts via arthropod vectors. As the Lone Star tick has expanded its geographic distribution northwards within North America, so have incidences of febrile illness of unknown etiology. Serological data implicate Lone Star-transmitted Rickettsia amblyommatis as the possible causative agent of disease but evidence for a causal relationship has been limited. Using bioinformatics, cell culture and a novel in vivo mouse model, Kim and colleagues demonstrate the virulence potential of R. amblyommatis (Yen et al. 2021), underscoring its possible role in human disease. Whereas R. amblyommatis is transmitted by ticks, the emerging human pathogen Rickettsia felis transmits to its mammalian host via flea bites. Exploring the relationship between R. felis and its arthropod vector, Macaluso and colleagues profiled gene expression in the salivary glands of R. felis-infected and uninfected fleas, and discovered the Rickettsia-induced expression of several antimicrobial peptides (Danchenko, Laukaitis and Macaluso 2021). Future studies will need to explore the functional role of these antimicrobial peptides, and specifically determine whether fleas use these antimicrobials as a defense against skin-resident microbes it comes into contact with during blood feeding, as it has been reported for an antimicrobial toxin produced by ticks (Hayes et al. 2020). These and other foundational principles of arthropod-microbe interactions are exemplified by the relationship between the bacterial genus Ananplasma and its tick vector, as expertly summarized by Pedra and colleagues (O'Neal et al. 2021).

In summation, this thematic issue provides many novel insights and perspectives into the biology of obligate intracellular bacterial pathogens. Study of these pathogens comes with significant technical hurdles. Yet, they are clearly a class of microbes underlying many threats to public health and deserving of our investigative attention.

Conflicts of Interest

None declared.