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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2016 Dec 21;174(14):2247–2260. doi: 10.1111/bph.13660

The role of apolipoprotein N‐acyl transferase, Lnt, in the lipidation of factor H binding protein of Neisseria meningitidis strain MC58 and its potential as a drug target

R A G da Silva 1, C P Churchward 1, A V Karlyshev 1, O Eleftheriadou 1, A K Snabaitis 1, M R Longman 1, A Ryan 1, R Griffin 1,✉
PMCID: PMC5481643  PMID: 27784136

Abstract

Background and Purpose

The level of cell surface expression of the meningococcal vaccine antigen, Factor H binding protein (FHbp) varies between and within strains and this limits the breadth of strains that can be targeted by FHbp‐based vaccines. The molecular pathway controlling expression of FHbp at the cell surface, including its lipidation, sorting to the outer membrane and export, and the potential regulation of this pathway have not been investigated until now. This knowledge will aid our evaluation of FHbp vaccines.

Experimental Approach

A meningococcal transposon library was screened by whole cell immuno‐dot blotting using an anti‐FHbp antibody to identify a mutant with reduced binding and the disrupted gene was determined.

Key Results

In a mutant with markedly reduced binding, the transposon was located in the lnt gene which encodes apolipoprotein N‐acyl transferase, Lnt, responsible for the addition of the third fatty acid to apolipoproteins prior to their sorting to the outer membrane. We provide data indicating that in the Lnt mutant, FHbp is diacylated and its expression within the cell is reduced 10 fold, partly due to inhibition of transcription. Furthermore the Lnt mutant showed 64 fold and 16 fold increase in susceptibility to rifampicin and ciprofloxacin respectively.

Conclusion and Implications

We speculate that the inefficient sorting of diacylated FHbp in the meningococcus results in its accumulation in the periplasm inducing an envelope stress response to down‐regulate its expression. We propose Lnt as a potential novel drug target for combination therapy with antibiotics.

Linked Articles

This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc

Abbreviations

DUS

DNA uptake sequence

FHbp

Factor H binding protein

His

Hexahistidine

Lgt

Diacylglyceryl transferase

Lnt

Apolipoprotein N‐acyl transferase

Lol

Lipoprotein outer membrane localization apparatus

Lpp

Braun's lipoprotein

Lsp

Lipoprotein signal peptidase

ME

Mosaic End

MIC

Minimum Inhibitory Concentration

PGN

Peptidoglycan

SBA

serum bactericidal antibody

TBS

Tris buffered saline

Tn

Transposon

WC

whole cell

Tables of Links

TARGETS
Catalytic receptors
TLR1
TLR2
TLR6
LIGANDS
Ceftriaxone
Erythromycin
Penicillin G
Rifampicin
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These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Introduction

Neisseria meningitidis is a leading cause of bacterial meningitis (Beernink and Granoff, 2008). This organism is classified into 13 different serogroups depending on its capsular polysaccharide. Invasive meningococci typically express polysaccharides, A, B, C, W135, X or Y (Jolley et al., 2007). Effective protein‐polysaccharide conjugate vaccines are available against serogroup A, C, W135 and Y meningococci (Snape and Pollard, 2005); however, the capsular polysaccharide of serogroup B strains is poorly immunogenic making this type of vaccine ineffective (Yongye et al., 2008). Thus non‐capsular, conserved, surface antigens such as the lipoprotein, Factor H binding protein (FHbp), have been tested for their ability to protect against organisms expressing the group B capsule.

Lipoproteins are a diverse class of multifunctional, membrane‐associated molecules, which constitute a significant fraction of the outer membrane of Gram‐negative bacteria (Nakayama et al., 2012). Their diverse functions range from maintaining envelope architecture and stability to mediating host‐pathogen interactions (Okuda and Tokuda, 2011, Nakayama et al., 2012, Zuckert, 2014). FHbp, as its name suggests, binds human factor H, which enables the meningococcus to evade killing by human complement (Madico et al., 2006). Importantly immunization with FHbp induces serum bactericidal antibody (SBA) responses that confer protection against the meningococcus (Borrow and Miller, 2006).

FHbp is lipidated by three palmitoyl fatty acids (Mascioni et al., 2010). Non‐lipidated FHbp is part of the Bexsero vaccine (Novartis), which was licenced in Europe in 2013 (see McNeil et al., 2013). Fletcher et al. (2004) demonstrated the immunogenic potency of the lipid moiety of FHbp by directly comparing lipidated and non‐lipidated versions in mice and showing that the lipidated form elicited profoundly greater immunogenicity and breadth of protection compared with the non‐lipidated form. Pfizer then developed a vaccine composed of two common variants of lipidated FHbp, which was licenced in the US in 2014. However, it is uncertain whether, in the very young, these serogroup B vaccines can induce sufficiently robust, broad and sustained immune responses. It is also not clear in adolescents, (an age group clearly having a role in the carriage and transmission of meningococcal disease) whether these vaccines elicit sufficient breadth of coverage and potency of the immune response to interrupt transmission (McNeil et al., 2013). Limitations in the breadth of coverage could be explained by the fact that not all strains express FHbp and different strains express different variants of FHbp, which furthermore are expressed at different levels (Biagini et al., 2016). Importantly Koeberling and co‐workers demonstrated that a ‘critical threshold’ of FHbp expression is required to elicit broad protective SBA responses (Koeberling et al., 2011).

Whilst the influence of environmental factors affecting FHbp expression levels, such as oxygen, temperature and iron availability has been established (Oriente et al., 2010, Sanders et al., 2012, Loh et al., 2013), the molecular pathway for FHbp expression, including transport across the inner membrane followed by lipidation and sorting to the outer membrane and export to the cell surface, is unknown. This pathway inevitably requires a wide variety of proteins whose genes may themselves be subject to regulation subsequently affecting FHbp expression levels. This study explores the molecular pathway governing FHbp expression, which will ultimately improve our understanding of the variability in FHbp expression and aid us in our evaluation of FHbp‐based vaccines.

Methods

Bacterial strains and culture conditions

N. meningitidis MC58, serogroup B:15:P1.7,16, ST‐74; ET‐5 strain was purchased from LGC Standards and L91543 serogroup C:2aP1.2, ST‐11; ET‐37 strain was kindly provided by Professor McFadden (University of Surrey). All meningococcal strains were grown on GC agar plates (Difco) containing Kellogg's glucose and iron supplements (Kellogg et al., 1963) in a moist atmosphere containing 5% CO2, at 37 or at 30°C for transformation experiments.

For each experiment, meningococcal cell suspensions were made by resuspending a loop of cells from a freshly grown overnight plate in PBS (137 mM NaCl, 16 mM phosphate, 2.7 mM KCl, pH 7.4) and adjusting to the required optical density at A 600. Serial dilutions of inocula were plated to verify consistency in colony forming unit (cfu) counts between strains.

Escherichia coli strain JM109 single use competent cells (Promega) were grown on Luria‐Bertani (LB) agar or LB broth (Merck) at 37°C with shaking at 200 rpm.

Antibiotics were added at the following concentrations; kanamycin, 30 and 60 μg·mL−1; erythromycin, 300 and 0.3 μg·mL−1 for E. coli and N. meningitidis respectively.

Molecular methods for DNA manipulations

Genomic DNA was extracted from N. meningitidis using the Gentra Puregene Yeast/Bact Kit (Qiagen) and plasmid DNA was extracted from E. coli using the QiaPrep Spin kit (Qiagen). DNA samples were analysed by agarose gel electrophoresis and visualised by staining with SYBR Safe (Invitrogen). Restriction enzymes (New England Biolabs), T4 DNA ligase (Promega), Antarctic Phosphatase (NEB) and T4 Polymerase (Thermo Scientific) were used according to the manufacturer's recommendations. PCRs were performed using HotStar HiFidelity polymerase kit (Qiagen) in a Perkin‐ MJ Research PTC‐200 Peltier Thermal Cycler. Primers were purchased from Sigma and their sequences are listed in Table S1. PCR products and restriction digested DNA were purified using the PCR Mini Elute kit (Qiagen). E. coli was transformed by heat shock (Froger and Hall, 2007).

Construction of the transposon library in strain MC58

Modification of EZ::Tn5< KAN‐2 > transposon to incorporate DUS

The transposon (Tn) from the EZ::Tn5<KAN‐2> insertion kit (Epicentre) was modified to incorporate the DNA uptake sequence (DUS) known to facilitate the uptake of DNA in Neisseriaceae (Frye et al., 2013). This was achieved by PCR amplification of the EZ::Tn5<KAN‐2> Tn using forward primer HindIIIkan2for, which incorporates a HindIII site and anneals immediately downstream of the 5′ mosaic end (ME) of the Tn and the reverse primer EcoRIDUSkan2rev, which incorporates an EcoRI site and DUS element and anneals downstream of the stop codon and upstream of the 3′ ME.

The PCR product was cloned into the EcoRI, HindIII sites of plasmid pMODTM‐2<MCS> (Epicentre). The ligation mixture was used to transform E. coli and transformants were selected by growth on kanamycin. Clones were verified by PCR using primers PCRFP and PCRRP (Epicentre) and by sequencing (MWG Biotech). The resulting plasmid was designated pMODTM‐2<Tn5KAN‐2DUS>. Phosphorylated primers PCRFP and PCRRP (Sigma) were used to amplify the region encompassed by and including the MEs from plasmid pMODTM‐2<Tn5KAN‐2DUS>.

In vitro transposition and transformation of strain MC58

Approximately 1 μg of DraI digested and purified genomic DNA of strain MC58 was mixed with 0.6 μg of phosphorylated PCR product, Ez‐Tn5 reaction buffer and 4 U of transposase (Epicentre) in a total volume of 40 μL. The reaction was incubated at 37°C for 2 h then stopped according to the manufacturers's instructions. The DNA was purified and 3′ overhangs were repaired by adding 5 U of T4 DNA polymerase to 1 μg of the DNA, 100 μM of each dNTP and T4 DNA polymerase buffer to a total volume of 50 μL. The reaction was incubated at 37°C for 5 min followed by heat inactivation at 75°C for 10 min. The DNA was purified and 6 U of T4 ligase and ligase buffer were added to the DNA to a total volume of 50 μL, and the reaction was incubated at 21°C for 2 h. The DNA was again purified and 250 ng used to transform N. meningitidis as described by Zhang et al. (2010) with selection on LB‐Kanamycin.

Construction of pGCC4SfHbpHis

Adopting the approach of Kurokawa et al. (2012), a truncated version of the FHbp protein, incorporating just the first 100 amino acids (from the cysteine at +1), fused to a Hexahistidine (His) tag was expressed in N. meningitidis strains to differentiate between di‐ and tri‐palmitoylated FHbp. The fHbp N‐terminal region containing the N terminal signal peptide sequence which is predicted by the program DOLOP (Babu et al., 2006) to be MTRSKPVNRTAFCCLSLTTALILTAC was PCR amplified from genomic DNA of strain MC58 with primer BamfHbpfor, which anneals upstream of the signal peptide and incorporates a BamHI site, and primer XhofHbprev, which anneals 298 bp downstream of the signal peptide and incorporates a XhoI site to reduce the size of the translated product from 280 amino acids to 125 amino acids, prior to processing, and 100 amino acids after processing. The PCR product obtained was cloned into the BamHI, XhoI sites of pET28b and transformed into E. coli with selection on kanamycin. The resulting plasmid, pET28bSfHbp was verified by DNA sequencing. The SfHbp‐His region was PCR amplified from plasmid pET28bSfHbp using primers PacfHbpfor incorporating a PacI site and PmepET28brev incorporating a PmeI site. The PCR product was cloned into the PacI, PmeI sites of Neisseria complementation vector, pGCC4 (Addgene) (Mehr and Seifert, 1998) and transformed into E. coli with selection on erythromycin. The resulting plasmid, pGCC4SfHbpHis was verified by DNA sequencing and the plasmid used to transform N. meningitidis strains as described by Zhang et al. (2010) with selection on erythromycin. Transformant strains were verified by PCR then grown on GC agar containing 0.5 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) for expression studies.

Construction of the apolipoprotein N‐acyl transferase, lnt complementation plasmid

The lnt gene of strain MC58 was cloned into the FseI and PacI sites of pGCC4 (Addgene). Briefly, primer lntFseIfor incorporating an FseI site and primer lntPacIrev incorporating a PacI site were used to amplify a region from 158 nucleotides upstream of the start codon to 18 nucleotides downstream of the stop codon from genomic DNA of strain MC58. The PCR product obtained was cloned into the FseI, PacI sites of plasmid pGCC4. Cloning of lnt in the opposite direction to the lacZ promoter of pGCC4 allowed transcription from the native promoter of lnt and avoided over‐expression of lnt by the lacZ promoter. Following transformation of E. coli with selection on kanamycin, clones were verified by PCR and sequencing then correct clones designated pGCC4lnt. The plasmid was linearized with NcoI which cuts pGCC4lnt uniquely in the kanamycin resistance gene then used to transform strain MC58Lnt.

Primary antibodies

The following primary antibodies were used; mouse anti‐FHbp‐monoclonal antibody JAR4 (National Institute for Biological Standards and Control), rabbit anti‐RecA antibody (Abcam) and rabbit anti‐His antibody (Santa Cruz Biotechnology).

Immuno‐dot blotting to screen the transposon library

Cell suspensions of the meningococcal strains were heat killed at 65°C for 30 min and 5 μL were spotted onto a nitrocellulose membrane (Thermo Scientific), dried then blocked in PBS containing 0.05% (v/v) Tween 20 (PBST) and 2% BSA (w/v) (Sigma) with gentle shaking for 1 h. Three 2 min washes in PBST were conducted then the membrane incubated in PBST containing 2% BSA (w/v) and 1 μg·mL−1 of anti‐FHbp antibody, JAR4, with gentle shaking for 3 to 4 h. The washes were repeated and the membrane incubated in anti‐mouse alkaline‐phosphatase conjugated secondary antibody solution (Invitrogen) for 1 to 3 h, washed as before and then developed with 5‐bromo‐4‐chloro‐3‐indolylphosphate, nitro‐blue tetrazolium liquid substrate (Invitrogen).

Locating the transposon in the mutant strain with altered binding to anti‐FHbp antibody

In order to locate the Tn in the selected mutant strain and to verify the insertion of only one Tn in its genome, Ion Torrent genome sequencing was performed. The sequencing library was prepared using 0.5 μg of genomic DNA and the ‘fragmentation and library preparation kit’ (New England Biolabs), according to the manufacturer's instructions. The adapter ligated library fragments were separated by gel electrophoresis followed by extraction of approximately 500 bp fragments from a 2% (w/v) E‐gel (Life Technologies). The gel extracted DNA fragments were PCR amplified using 6 cycles as described in the ‘fragmentation and library preparation kit’.

The size and concentration of the amplified library were checked using the BioAnalyser and a high sensitivity DNA kit (Agilent). The library concentration was adjusted to 20 pM and used for template preparation using the One Touch 2 system (OT2, Life Technology). The ion particles were enriched using an automatic system (OT2, Life Technology), loaded onto 314v2 chip and the sequencing was run using 400 bp sequencing kit and IonTorrent PGM with 850 flows. The reads were assembled using IonTorrent assembler plugin.

The contigs generated were analysed using CLC Genomics Workbench software v.7. The contigs containing the Tn sequence were identified using BLASTn program running the extracted Tn sequence against the IonTorrent assembly. The contig containing the Tn sequence was further analysed using NCBI BLASTx and non‐redundant amino acid sequence database to determine the genes flanking the Tn insertion site.

SDS‐PAGE and Western immunoblotting

Whole cell (WC) lysates were prepared by mixing 600 μL of cell suspension, from each strain, standardized to A 600 1 with 300 μL 3X Laemmli sample buffer and heated to 95°C for 5 min. Cell lysate proteins were then separated by 12% (w/v) SDS‐PAGE and transferred to PVDF or nitrocellulose membranes as appropriate. Membranes were then incubated with Tris‐buffered saline, TBS (50 mM Tris, 150 mM NaCl, pH 7.5) containing 0.1% (v/v) Tween‐20 (TBST) and 5% (w/v) non‐fat dry milk for 2 h at room temperature. Membranes were then incubated with either mouse anti‐FHbp JAR4 (1 μg·mL−1) or rabbit anti‐RecA (Abcam) primary antibody diluted (1:1000) in TBST containing 1% (w/v) non‐fat dry milk overnight at 4°C. Membranes were washed for 60 min with TBST at room temperature and then incubated for 2 h at room temperature with either sheep anti‐mouse or donkey anti‐rabbit HRP‐linked secondary antibodies (GE Healthcare, UK) respectively. Membranes were washed with TBST for 30 min and specific protein bands were detected by enhanced chemiluminescence (GE Healthcare, UK). Band intensity was quantified using a GS‐800 calibrated densitometer and Quantity One® 1‐D analysis software v 4.6.2 (Bio‐Rad).

Immunofluorescence microscopy

Bacterial suspensions from each strain were standardized to A 600 0.5 and 50 μL spotted onto 13 mm circular glass coverslips. Once dry, these were transferred to 24 well plates and cells fixed with 4% (v/v) paraformaldehyde for 20 min. The coverslips were washed in PBS then blocked with 500 μL PBS containing 1% (w/v) BSA for 30 min. The blocking agent was removed and 500 μL PBS containing 1% (w/v) BSA and 5 μg·mL−1 of mouse anti‐FHbp antibody, JAR4 added. This was incubated for 2 h with agitation. The wells were washed three times in PBS then PBS with 1% (w/v) BSA containing 1:500 dilution Alexa Fluor 555 labelled donkey anti‐mouse IgG secondary antibody (Abcam) was added (to fluorescently label the FHbp‐bound JAR4 antibody) and 1:500 dilution of FITC‐labelled rabbit polyclonal IgG raised against WC N. meningitidis (Abcam) to detect meningococcal cells. After incubation for 1 h with agitation, wells were washed as before, and the cover slips dipped in deionised water then mounted onto glass slides using fluoroshield mounting medium (Abcam). Fluorescence microscopy was performed using a Nikon Eclipse i80 and images captured using NIS‐Elements Viewer (version BR 3.00).

Quantitative RT‐PCR (qRT‐PCR)

RNA was extracted from 1 mL cell suspensions of each strain standardized to A 600 0.65 (containing approximately 2 × 108 cells) using the RNeasy Mini kit (Qiagen) with enzymatic lysis and Proteinase K digestion. On‐column DNA digestion was performed using the RNase Free DNase set (Qiagen).

One μg of cDNA was synthesized using the QuantiTect reverse transcription kit (Qiagen) with the initial genomic wipe‐out step included. qRT‐PCR was performed in a 15 μL reaction mixture with Quantinova SYBR Green PCR Master Mix (Qiagen), 22.5 ng of cDNA and 0.7 μM of each primer (Sigma). For amplification of cDNA of fHbp, fHbp forward primer and fHbp reverse primer were used, and for amplification of recA, recA forward primer and recA reverse primer were used. PCR was performed in a Prime Pro 48 Real Time PCR machine with the following thermocycling conditions; 95°C for 2 min followed by 40 cycles of 95°C for 5 s (denaturation) and 60°C for 10 s (combined annealing/extension). Six biological replicates from six independent RNA extractions from each of the three strains were run in duplicate along with the corresponding no reverse transcriptase control for each of these samples and a no RNA control. Relative quantification of gene expression was performed using the comparative CT Method (ΔΔCt) (Livak and Schmittgen, 2001) whereby fHbp expression levels were normalised to the mean levels of control (recA) transcripts.

Antibiotic susceptibility assays by microbroth dilution

Meningococcal strains were compared for their susceptibility to a panel of antibiotics by the microbroth dilution method. For each antibiotic, the concentration range was centred around the minimum inhibitory concentration (MIC) value according to the Clinical and Laboratory Standards Institute (CLSI). The antibiotics tested were tunicamycin, ceftriaxone, penicillin G, chloramphenicol, sulfanilamide, globomycin, ciprofloxacin and rifampicin.

Bacterial suspensions were standardized to A 600 0.4 then 5 μL added to 95 μL of Mueller Hinton broth containing doubling dilutions of antibiotic in a Thermo Scientific sterile 96 well plate (Nunclon Delta surface). Negative controls included no bacterial suspension and no antibiotic. For each MIC assay, all conditions were performed in triplicate and each assay was repeated at least five times. A gas permeable seal was placed over the plate and the plate incubated at 37°C in 5% CO2 with gentle shaking for approximately 20 h.

Data and statistical analysis

The data and statistical analyses in this study comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Data are shown as mean ± SEM. Multiple comparisons among groups were performed by one way ANOVA followed by Tukey's HSD test. A value of P ≤ 0.05 was considered statistically significant. Post hoc tests were only run if F achieved P < 0.05 and there was no significant variance in homogeneity. All statistical analysis tests were performed in IBM SPSS Statistics (v. 23). Blinding and randomisation were not used as these were considered inappropriate for the in vitro experiments on bacteria conducted.

Materials

The antibiotics used in these experiments were supplied as follows: chloramphenicol by Apollo Scientific Limited, Manchester, UK; ciprofloxacin by Tokyo Chemical Industry (TCI), Portland, OR: ceftriaxone, erythromycin, globomycin, kanamycin, penicillin G, rifampicin, sulphanilamide and tunicamycin by Sigma‐Aldrich, St Louis. MO.

Results

Reduced binding to the FHbp‐specific antibody following Tn insertion into lnt

A random Tn library of mutants was constructed in N. meningitidis strain MC58 known to express FHbp strongly at the cell surface (Newcombe et al., 2014). The first 100 mutant strains from the library were individually screened by WC immuno‐dot blot using an anti‐FHbp antibody, JAR4, as a probe for FHbp expression. The level of binding or reactivity with the antibody was compared with that of the parent strain MC58 and with that of strain L91543 known to express FHbp very weakly at the cell surface (Newcombe et al., 2014). Mutant 80 showed a marked reduction in reactivity to the antibody (Figure 1).

Figure 1.

Figure 1

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Immuno‐dot blot of WC suspensions of individual Tn mutants of strain MC58 probed with anti‐FHbp antibody, JAR4. Strains MC58 and L91543 were included as positive and negative control strains for reactivity to JAR4.

To identify which gene had been insertionally inactivated by the presence of the Tn in the mutant with reduced binding to the antibody, and to verify if only one Tn was present in this strain, Ion Torrent whole genome sequencing was performed. The single Tn identified was located at chromosomal position 745 462, in the middle of gene NMB0713, annotated as lnt, which lies at chromosomal position 744 476 to 746 050. The mutant was designated MC58Lnt.

The lnt gene encodes the integral membrane protein, apolipoprotein N‐acyl transferase (Lnt), which transfers the third fatty acid to the amide group of the N terminal conserved cysteine of apolipoproteins in bacteria (Chahales and Thanassi, 2015). In this mature form, lipoproteins that are destined for the outer membrane are then translocated by the lipoprotein outer membrane localization (Lol) apparatus. Given that lnt was found to be the single disrupted gene in the mutant strain that was affected in its ability to bind an FHbp antibody, we predicted that lnt was involved in FHbp acylation specifically in the addition of the third amide‐linked palmitoyl fatty acid to the N terminal cysteine of FHbp.

FHbp is not fully mature in strain MC58Lnt

To test if Lnt is involved in FHbp acylation, truncated FHbp fused to a His reporter was expressed in strains MC58 and MC58Lnt using the approach of Kurokawa et al. (2012) and LoVullo et al. (2015) to generate sufficiently small recombinant lipoprotein to differentiate between diacylated and triacylated forms after separation by SDS‐PAGE and detection by Western immunoblotting. Genomic DNA encoding the first 125 amino acid residues of FHbp, including the N terminal signal peptide, was cloned into pET28b to fuse this sequence to a C terminal His tag then the fusion construct was cloned into Neisseria vector, pGCC4 (Mehr and Seifert, 1998) for transformation into strains MC58 and MC58Lnt. The preprolipoprotein would be cleaved to yield a lipoprotein of 106 amino acids including the His tag, with a MW of 11.4 KDa as calculated by Expasy (Gasteiger et al., 2005) (not accounting for the attached fatty acids).

WC extracts from each strain were separated by SDS‐PAGE and immunoblotted with anti‐His antibody. As shown in Figure 2, the mobility of the reporter protein was greater in the MC58Lnt transformant (lane 4) than in the wild type transformant (lane 3). The increase in mobility of the lipoprotein of the mutant strain was consistent with the expected change in acylation state at the N terminus, as such increased mobility has been shown by mass spectrometry to equate to the loss of a fatty acid (Kurokawa et al., 2012). Thus it appears that FHbp is diacylated in the Lnt mutant strain.

Figure 2.

Figure 2

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Western immunoblot of WC lysates from strains MC58 and MC58Lnt transformed to express His‐tagged truncated FHbp, probed with anti‐His antibody (lanes 3 and 4 respectively). Lanes 1 and 2 are non‐transformed MC58 and MC58Lnt strains. Data are representative of five independent experiments.

Disruption of Lnt strongly reduces FHbp cell surface expression

To confirm if the marked reduction in binding of WCs of strain MC58Lnt to JAR4 (as shown by immuno‐dot blotting) was due solely to the disruption of lnt in this strain, the mutant strain was complemented with an intact copy of the lnt gene from strain MC58. The lnt gene was cloned into vector pGCC4 (Mehr and Seifert, 1998) such that expression was under the control of its own putative promoter to generate native levels of Lnt and used to transform strain MC58Lnt. Transformants were verified by PCR and sequencing and designated MC58LntC.

The expression of FHbp on the cell surface of individual cells of the strains was compared by immunofluorescence microscopy using JAR4. Cells of MC58Lnt showed very low levels of expression compared with that of the parent strain and cells of strain MC58LntC showed restored levels like that of the wild type (Figure 3). We concluded the disruption in Lnt alone was responsible for the poor expression of FHbp at the cell surface.

Figure 3.

Figure 3

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Immunofluorescence microscopy of WCs of strains MC58, MC58Lnt and MC58LntC. To confirm the presence of meningococcal cells, cells were incubated with FITC‐labelled rabbit polyclonal IgG raised against WC N. meningitidis (left and right panel). To compare FHbp cell surface expression between the strains, cells were also incubated with anti‐FHbp antibody JAR4 that was detected by Alexa Fluor 555 labelled donkey anti‐mouse IgG secondary antibody (right panel).

To further assess the level of surface expression of FHbp in strain MC58Lnt, its binding to JAR4 was compared with another meningococcal strain, L91543, known to express FHbp of the same variant 1 group (Masignani et al., 2003) and the same subfamily B (Fletcher et al., 2004; Beernink and Granoff, 2009), as strain MC58, with nucleotide and amino acid identities of 95 and 93% respectively (Karlyshev et al., 2015), but very poorly at the cell surface (Newcombe et al., 2014). An immuno‐dot blot using JAR4 was performed from standardized cell suspensions. Both strains MC58Lnt and L91543 showed weak reactivity to JAR4 but the level of reactivity was significantly lower for L91543 compared with MC58Lnt (Figure 4). Confirming our initial immuno‐dot blot and our immunofluorescence microscopy results, the level of antibody binding of strain MC58Lnt was significantly lower compared with the parent strain and was restored in the complemented mutant strain. Together the results confirm that strain MC58Lnt expresses markedly reduced levels of FHbp at the cell surface compared to the parent strain and importantly reveal this strain expresses a higher level of FHbp at the cell surface compared with strain L91543 (Figure 4). This finding suggests that although FHbp is diacylated in strain MC58Lnt, it still sorts to the outer membrane to be exported to the cell surface although the quantity of the exported apo‐ (diacylated) form of FHbp is low.

Figure 4.

Figure 4

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(A) The binding of standardized WC suspensions of strains MC58, MC58Lnt, MC58LntC and L91543 to anti‐FHbp antibody, JAR4, in an immuno‐dot blot assay to compare the level of cell surface expression of FHbp between these strains. The image is representative of multiple independent experiments (n = 6). (B) The reflective density was measured by a GS‐800™ calibrated densitometer. All columns represent mean ± SEM. *P ≤ 0.05, significantly different as indicated; NS, not significant. F achieved P < 0.05, no significant variance in homogeneity seen.

Disruption of Lnt causes major reduction in total cellular levels of FHbp

To test if the low level of FHbp at the cell surface in strain MC58Lnt is due to inefficiency in the sorting of the apo‐form of FHbp to the outer membrane, we initially tested FHbp expression in WC extracts of the mutant strain before proceeding to investigate its localization in the inner or outer membrane.

WC extracts of strains MC58, MC58Lnt and MC58LntC were fractionated by SDS‐PAGE and immunoblotted with JAR4 (Figure 5). Surprisingly MC58Lnt showed a significant 10‐fold reduction in band intensity and this was restored in strain MC58LntC. A consistent level of expression of the house‐keeping protein RecA (Loh et al., 2013) was observed between the 3 strains.

Figure 5.

Figure 5

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(A) Western immunblot of WC lysates from strains MC58, MC58Lnt and MC58LntC probed with anti‐FHbp antibody, JAR4. Equal protein loading was confirmed by the determination of RecA protein in each sample. The image is representative of multiple independent experiments (n = 8). (B) The expression of FHbp was determined in MC58, MC58Lnt and MC58LntC by densitometry and normalized to RecA protein. The reflective density was measured by a GS‐800™ calibrated densitometer. All columns represent mean ± SEM, *P ≤ 0.05, significantly different from strain MC58; NS, not significant. F achieved P < 0.05, no significant variance in homogeneity seen.

The reduction in ability of the diacylated FHbp to bind JAR4 is unlikely to be due to loss of the fatty acid affecting conformation of the epitope known to bind this antibody (previously characterized by Beernink and Granoff (2009)), since recombinant, non‐acylated FHbp (lacking the signal peptide) strongly binds this antibody (data not shown). Thus, we concluded that the mutation of Lnt in strain MC58 indeed results in a profound reduction in the cellular levels of FHbp.

Disruption of Lnt causes a reduction in fHbp transcription

To test if the marked reduction in FHbp in strain MC58Lnt could be attributed to down‐regulated transcription of fHbp, RNA was extracted from strains MC58Lnt, MC58 and MC58LntC and the level of transcription of fHbp compared between the strains and with the house‐keeping gene, recA. Strain MC58Lnt showed a significant 53% reduction in the level of fHbp transcript relative to the parent strain (Figure 6).

Figure 6.

Figure 6

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Determination of fHbp mRNA levels in bacterial strains MC58, MC58Lnt and MC58LntC by qRT‐PCR analysis. The data was obtained from six independent biological replicates (n = 6) with each including two technical replicates and normalized against the house‐keeping gene, recA. All columns represent mean ± SEM, *P ≤ 0.05, significantly different from strain MC58; NS, not significant. F achieved P < 0.05, no significant variance in homogeneity seen.

The Lnt mutant is more susceptibility to antibiotics

Since the disruption of Lnt likely affects a whole plethora of lipoproteins and lipoproteins are important for the integrity of the cell envelope, we predicted that the loss of Lnt would result in greater cell envelope permeability and therefore increased susceptibility of the mutant strain to antibiotics. The MICs of eight antibiotics were compared for strain MC58Lnt and the parent and complemented strains by the microbroth dilution method.

Strain MC58Lnt showed the following significant increases in antibiotic susceptibility; 64‐fold to rifampicin, 16‐fold to ciprofloxacin, 4‐fold to both globomycin and sulfanilamide and 2‐fold to ceftriaxone, penicillin G and chloramphenicol (Table 1). There was no change in MIC for tunicamycin.

Table 1.

Drug susceptibilities of MC58, MC58Lnt and MC58LntC

Drug Dose (μg·mL−1)
MC58 MC58Lnt MC58LntC
Tunicamycina 50 50 50
Ceftriaxone b 0.00097 0.00049 0.0097
Penicillin G b 0.025 0.0125 0.025
Chloramphenicol b 2.0 1.0 2.0
Sulfanilamide c 100 25 100
Globomycin c 6.25 1.5625 6.25
Ciprofloxacin d 0.00391 0.00024 0.00391
Rifampicin e 0.125 0.00495 0.125
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MICs determined using microbroth dilution assays. For each assay, every conditions was performed in triplicate and the assay repeated multiple times (n = 5).

a

No significant fold change observed between strains.

b,c,d,e

Strain MC58Lnt showed 2‐, 4‐, 16‐ and 64‐fold increased susceptibility respectively to the drugs tested.

Discussion

In this study, we set out to identify genes involved in meningococcal cell surface expression of the vaccine antigen FHbp. A random Tn library was constructed in strain MC58 and individual Tn mutants were screened by WC immuno‐dot blot using the anti‐FHbp antibody, JAR4, as a probe. A mutant strain was identified with markedly reduced binding to the antibody. The Tn was located, by Ion Torrent whole genome shot‐gun sequencing, in the lnt gene that encodes apolipoprotein N‐acyl transferase, Lnt.

The activity of Lnt is the third step of the biosynthetic pathway for post‐translational lipid modification of apolipoproteins that has been documented for E. coli (Kovacs‐Simon et al., 2011) (Figure 7). This processing begins with translation in the cytoplasm of the precursor or preprolipoprotein that carries a cleavable N‐terminal signal peptide possessing the conserved lipobox ([LVI][ASTVI][AGS][C]) at its C terminus. The preprolipoprotein is transported from the cytoplasm across the inner membrane usually by the general secretion pathway, which is mediated by the Sec translocon (Figure 7). In the periplasm, the thiol group of the invariant cysteine residue of the lipobox is diacylated by a preprolipoprotein diacylglyceryl transferase, Lgt, forming a prolipoprotein. Lipoprotein signal peptidase, Lsp, cleaves the signal peptide such that the di‐acylated cysteine residue becomes the new N terminal residue. Lnt then adds an additional amide‐linked fatty acid to this residue generating a triacylated protein. The mature lipoprotein can then be recognized by Lol, which transports it to the outer membrane, the default destination for lipoproteins. However, if lipoproteins contain a ‘Lol avoidance’ signal, (for example an aspartic acid at the +2 residue in E. coli or a lysine and a serine at positions +3 and +4 respectively, in Pseudomonas spp (Seydel et al., 1999, Narita and Tokuda, 2007), they are retained and anchored at the inner membrane (Hara et al., 2003). In E. coli , the Lol apparatus consists of LolCDE, an ABC transporter which releases mature lipoproteins from the inner membrane, LolA, a chaperone or carrier protein which shuttles lipoproteins from the inner membrane to the outer membrane, and LolB, an outer membrane lipoprotein receptor, which inserts the lipoprotein into the outer membrane (Narita and Tokuda, 2006) (Figure 7). Most outer membrane lipoproteins are thought to face into the periplasm however some are exposed on the outer surface to face extracellularly, such as FHbp.

Figure 7.

Figure 7

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Model for lipoprotein processing and sorting of tri‐ and diacylated lipoproteins in N. meningitidis. The signal peptide at the N terminus of the preprolipoprotein signals for its translocation across the inner membrane by the Sec apparatus. Once in the periplasm, Lgt adds a diacylglyceride to the conserved cysteine, the last amino acid of the lipobox at the C terminus of the signal peptide. LspA cleaves the signal peptide exposing the diacylated cysteine which becomes the +1 residue to which Lnt adds the third acyl chain. Both the fully mature lipoprotein and the apolipoprotein resulting from mutation of Lnt are recognized by the LolFD transporter complex (LoVullo et al., 2015). The LolA chaperone receives the lipoprotein or apolipoprotein and delivers it to the outer membrane anchored lipoprotein LolB, which inserts it into the outer membrane.

The generally accepted model, based largely on experiments performed in E. coli, is that complete triacylation of lipoproteins by Lnt is essential in Gram‐negative bacteria, as it permits the LolCDE‐dependent release of lipoproteins from the inner membrane for their sorting to the outer membrane (Fukuda et al., 2002, Robichon et al., 2005, Zuckert, 2014). Experiments using a conditionally lethal E. coli Lnt mutant showed that loss of Lnt led to incomplete maturation of lipoproteins and the retention in the inner membrane of those normally destined for the outer membrane, in particular, the retention of the highly abundant Braun's lipoprotein, Lpp, which subsequently cross‐linked with peptidoglycan (PGN) causing lethality (Yakushi et al., 1997, Robichon et al., 2005).

In our study, firstly we show that, in contrast to its function in E. coli, Lnt is not essential in N. meningitidis. This supports a recent study by LoVullo et al. (2015) who challenged the model outlined above. LoVullo and coworkers demonstrated Lnt was not essential in several Gram‐negative bacteria they investigated including Francisella novicida, Francisella tularensis and Neisseria gonorrhoeae (LoVullo et al., 2015).

We confirmed the role of Lnt in adding a fatty acid to FHbp, by adopting the approach of Kurokawa et al. (2012) and LoVullo et al. (2015) to differentiate diacylated from triacylated lipoproteins. A truncated version of FHbp consisting of the N‐terminal portion of FHbp (including its upstream signal peptide) fused to a His‐tag was expressed in the wild type and Lnt mutant strain. We could infer from the mobility differences observed upon SDS‐PAGE fractionation and Western immunoblotting that FHbp was diacylated in strain MC58Lnt (Figure 2).

We confirmed that the disruption of Lnt was directly responsible for the large reduction in binding of WCs to JAR4, shown by immuno‐dot blotting, by complementing strain MC58Lnt with an intact copy of the lnt gene. Immunofluorescence microscopy of meningococcal cells with JAR4 showed a low level of FHbp expression in the Lnt mutant and a restored level of expression in the complemented strain, like that of the wild type (Figure 3).

The level of expression of FHbp in strain MC58Lnt was assessed relative to strain L91543, which is known to express FHbp at the cell surface very poorly (Newcombe et al., 2014). Strain MC58Lnt showed a significantly higher level of reactivity to JAR4 compared with strain L91543 in WC immuno‐dot blot studies (Figure 4). This result suggests that N. meningitidis has the ability to sort diacylated lipoproteins to the outer membrane prior to export to the cell surface although the quantity of exported FHbp was low.

Together, our findings agree with those of LoVullo et al. (2015) who demonstrated that mutation of Lnt in F. tularensis resulted in lipoprotein Tul4 (LpnA) shifting from a triacylated form to a diacylated form and that this lipoprotein as well as others still sorted to the outer membrane.

LoVullo et al. (2015) showed that the Lol system of F. tularensis lacks a gene for LolE, which in E. coli forms a heterodimer with LolC to form the membrane component of the ABC transporter complex. They showed that the absence of lolE is not unique to Francisella but instead was represented by more than half of the bacterial genomes they analysed (LoVullo et al., 2015). Based on protein sequence analysis, LoVullo et al. (2015) concluded that the single LolC present in bacteria such as Francisella spp contains features found in both LolC and LolE proteins. This suggests that the single LolC is a hybrid protein, which they named LolF. LoVullo et al. (2015) proposed that a homodimer formed by LolF enables the LolFD transporter complex of Francisella to recognize diacylated as well as triacylated lipoproteins and to transfer either type of substrate to LolA for sorting to the outer membrane (Figure 7). To test if their hypothesis could be extended to another Gram‐negative bacterium, Neisseria gonorrhoeae, which has the same LolFD genomic organization as found in Francisella, LoVullo et al. (2015) demonstrated that viable Δlnt mutants could be constructed in this organism, with proper lipoprotein‐dependent functionality. LoVullo et al. (2015) proposed that the LolFD transporter may have a looser specificity for acyl chains and can recognize both with almost equal affinity in contrast to the LolCDE transporter of E. coli (Narita and Tokuda, 2011). Narita and Takuda (2011) demonstrated that LolCDE of E. coli does have some affinity for apolipoproteins but this is very low, and required over‐expression of LolCDE for apolipoproteins to be released from the inner membrane and sorted to the outer membrane in their Lnt null mutant strain (lacking Lpp or the transpeptidases that cross link Lpp to PGN).

N. meningitidis has the same LolFD genomic organization as that of N. gonorrhoeae and F. tularensis. However, given the low level of FHbp observed at the cell surface of strain MC58Lnt, we questioned if the LolFD transporter in this strain had low affinity for apolipoproteins resulting in inefficient sorting to the outer membrane and thus a low level of export to the cell surface. To test this, we investigated FHbp expression initially in WC extracts prior to assessing the compartmentalization of FHbp in the inner and outer membrane. Surprisingly, the total cellular level of FHbp expression of the mutant strain was reduced 10‐fold compared with that of the parent and complemented strain (Figure 5). This finding suggests that transcription or translation of the partly acylated FHbp was being affected.

We investigated fHbp transcription by qRT‐PCR in the Lnt mutant and showed that fHbp transcription was significantly down‐regulated by over 50% in strain MC58Lnt compared with the parent strain (Figure 6). However, given the 10‐fold decrease in FHbp levels in WC extracts, we predict that translation of FHbp is also down‐regulated or proteolysis of the apo‐form of FHbp is induced or both.

To combat the problem of envelope stress caused by altered proteins translocated from the cytoplasm to the inner membrane, Gram‐negative bacteria employ two major responses, one mediated by transcription factor σE and the other by Cpx to down‐regulate and degrade these proteins While the σE response senses and mediates adjustments to changes in the biogenesis of proteins in the periplasm, specifically outer membrane proteins (Ruiz and Silhavy, 2005), the Cpx response is activated by the over‐expression of proteins or misfolded proteins that aggregate at the inner membrane (MacRitchie et al., 2008). Both responses result in the activation of proteases to degrade these altered proteins and both can up‐regulate small RNAs (sRNA) to down‐regulate the synthesis of these proteins by interfering with their transcription or translation (Walsh et al., 2003, Zhang et al., 2003, Barchinger and Ades, 2013, Lima et al., 2013, Raivio et al., 2013). For example, σE regulated sRNA, MicL specifically targets Lpp of E. coli by decreasing lpp translation and accelerating the degradation of lpp mRNA (Guo et al., 2014). There is a growing body of evidence that σE regulated pathways are adopted by the meningococcus (Hopman et al., 2010, Huis in ‘t Veld et al., 2011, Fagnocchi et al., 2015).

Our study led us to propose the following model. The meningococcus can be grouped with F. tularensis and N. gonorrhoeae, and probably all other organisms that possess the LolFD transporter and can sort both di‐ and triacylated lipoproteins (LoVullo et al., 2015) negating the essentiality of Lnt; however, meningococcal LolFD has a higher affinity for triacylated lipoproteins than diacylated lipoproteins (Figure 7). We speculate that in strain MC58Lnt, the inefficient sorting of partially acylated lipoproteins such as the apo‐form of FHbp to the outer membrane (indicated from its strongly reduced surface expression) results in their accumulation in the periplasm inducing envelope stress responses to down‐regulate their expression and promote their degradation. Comparative quantification of total cellular apolipoprotein levels in Lnt mutants of other Gram‐negative bacteria (with that of their corresponding parent strains) will provide insight into whether apolipoproteins of these bacteria are also down‐regulated or proteolysed, due to the loss of Lnt, and whether our model therefore applies to other microorganisms. It is also important to note that the specific fate of apolipoproteins may vary if LolFD has different affinities for different apolipoproteins perhaps depending on their protein moiety, as has been suggested for LolCDE (Narita and Tokuda, 2011) or on their lipid moiety, or both.

Despite differences in the efficiency of sorting of diacylated lipoproteins in Gram‐negative bacteria possessing the LolFD apparatus, the important question we now ask is whether these bacteria have the ability to naturally generate both di‐ and triacylated lipoproteins by regulation of Lnt. Whilst the lipid moieties of bacterial lipoproteins play an important structural role by anchoring the lipoprotein to the cell wall, during infection lipoproteins can dissociate from the bacterium freeing the exposed lipid moieties to exert their immuno‐modulatory activities (Luo et al., 2016). Diacylated and triacylated lipoproteins induce cell signalling by interacting with two different Toll‐like receptor 2 (TLR2) heterodimers on antigen presenting cells. Specifically, diacylated lipoproteins signal via TLR2/TLR6 heterodimers (Kang et al., 2009) and triacylated lipoproteins signal via TLR2/TLR1 heterodimers (Jin et al., 2007) and the latter was recently demonstrated for tripalmitoylated FHbp of Trumenba (Luo et al., 2016). This difference in signalling affects the ability of lipoproteins or lipopeptides to activate macrophages and subsequently to activate B cells (Metzger et al., 1995, Mühlradt et al., 1997, 1998, Zeng et al., 2010). Thus, the ability to generate two lipid moieties with different immunomodulatory activities and potencies is likely to confer a fitness advantage to the bacterium in the host.

The intrinsic structure of the cell envelope of Gram‐negative bacteria presents a significant barrier for the penetration of antibiotics (Graef et al., 2016). However, in the meningococcal Lnt mutant strain, we anticipated that the inefficient sorting of FHbp to the outer membrane and the potential inefficient sorting of other apolipoproteins would result in a structurally weaker outer membrane that would be more permeable to antibiotics. The antibiotics commonly used as initial therapy, particularly in adolescents, to treat invasive meningococcal infection are penicillin G and third generation cephalosporins, cefotaxime and ceftriaxone, and chloramphenicol can be used in cases of penicillin allergy (Nadel, 2016). The antibiotics often used for follow up treatment are ceftrixone, ciprofloxacin or rifampicin (Nadel, 2016). We selected five of these antibiotics as well as tunicamycin, sulfanilamide and globomycin to compare the MICs for the Lnt mutant, parent and complemented strains.

The Lnt mutant was 64‐fold more susceptible to rifampicin, 16‐fold more susceptible to ciprofloxacin, four fold more susceptible to globomycin and sulfanilamide and two fold more susceptible to penicillin G, ceftriaxone and chloramphenicol. The results support our prediction that the cell envelope of the Lnt mutant is weaker and more permeable to antibiotics. This highlights the importance of developing antimicrobials that target enzymes involved in constituting envelope integrity that could be used in combination with antibiotics. An inhibitor of Lnt would target all meningococcal strains, as this protein is highly conserved between N. meningitidis strains as demonstrated by their 99 to 100% amino acid sequence identity across the entire protein (BLASTp analysis not shown). Importantly, this enzyme is absent in humans.

Interestingly LoVullo et al. (2015) reported only small increases in susceptibility of their ΔLnt mutant of N. gonorrhoeae to rifampicin, globomycin, polymixin B and vancomycin among the antibiotics and chemical compounds they tested. The greater increase in sensitivity of the meningococcal Lnt mutant to antibiotics, shown in this study, compared with the small increase observed for the gonococcal ΔLnt mutant (LoVullo et al., 2015) supports the difference in the two models proposed for these organisms. This model proposes that the LolFD of N. gonorrhoaea (as well as F. tularensis) sorts di‐ and triacylated lipoproteins efficiently maintaining the integrity of the outer membrane. In contrast, the LolFD of N. meningitidis appears to have a lower affinity for diacylated lipoproteins compared to triacylated lipoproteins and down‐regulates their expression resulting in a weaker outer membrane which is more permeable to antibiotics.

To conclude, from this study, we show that for the meningococcus Lnt is a promising drug target and for the gonococcus we highlight the importance of targeting enzymes that act earlier in the lipoprotein processing pathway, such as Lgt, to prevent any sorting of diacylated lipoproteins to the cell surface. Our study is very timely given the continuing rise in antibiotic resistance in both N. meningitidis and N. gonorrhoeae and the ever increasing need to develop new antimicrobials against these organisms (Zapun et al., 2016).

Author contributions

R.G. devised the project and supervised da Silva RAG to construct the Tn library of mutants and conduct the qRT‐PCR experiments, the MIC assays, generate the recombinant SFHbpHis strains and characterize these strains by Western analysis. R.G. complemented the mutant strain and performed the immuno‐dot blot assays and Western immunoblots with anti‐FHbp antibody and anti‐RecA antibody. R.G. wrote the manuscript. C.P.C. performed the immunofluorescence microscopy work. A.V.K. conducted the Ion Torrent Sequencing. O.E. assisted with the qRT‐PCR analysis and Western immunoblots. A.K.S. provided reagents and equipment. M.R.L. gave advice for the Western immunoblots. A.R. provided lab space, reagents, support and advice throughout this project.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1 PCR primer pairs used for PCR and qRT‐PCR. Restriction sites are underlined and the DNA uptake sequence element is shown in bold.

Supporting info item

Click here for additional data file. (106.8KB, jpg)

Acknowledgements

The authors would like to thank Johnjoe McFadden for kindly providing strain L91543, Lori Snyder for her suggestion to use piliated cells for transformation experiments, Mark Fielder and Sinead Holland for their advice regarding the MIC work, Amadou Jallow and Mamadou Jallow for their assistance with some of the qRT‐PCR analysis and Jonathan Cowan for his advice on statistics.

This work was funded by Kingston University and by the Brazilian foundation Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

da Silva, R. A. G. , Churchward, C. P. , Karlyshev, A. V. , Eleftheriadou, O. , Snabaitis, A. K. , Longman, M. R. , Ryan, A. , and Griffin, R. (2017) The role of apolipoprotein N‐acyl transferase, Lnt, in the lipidation of factor H binding protein of Neisseria meningitidis strain MC58 and its potential as a drug target. British Journal of Pharmacology, 174: 2247–2260. doi: 10.1111/bph.13660.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1 PCR primer pairs used for PCR and qRT‐PCR. Restriction sites are underlined and the DNA uptake sequence element is shown in bold.

Supporting info item

Click here for additional data file. (106.8KB, jpg)

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