Development of a Tag/Catcher-mediated capsid virus-like particle vaccine presenting the conserved Neisseria gonorrhoeae SliC antigen that blocks human lysozyme.

Virus-like particles (VLPs) are promising nanotools for the development of subunit vaccines due to high immunogenicity and safety. Herein, we explored the versatile and effective Tag/Catcher-AP205 capsid VLP (cVLP) vaccine platform to address the urgent need for the development of an effective and safe vaccine against gonorrhea. The benefits of this clinically validated cVLP platform include its ability to facilitate unidirectional, high-density display of complex/full-length antigens through an effective split-protein Tag/Catcher conjugation system. To assess this modular approach for making cVLP vaccines, we used a conserved surface lipoprotein, SliC, that contributes to the Neisseria gonorrhoeae defense against human lysozyme, as a model antigen. This protein was genetically fused at the N- or C-terminus to the small peptide Tag enabling their conjugation to AP205 cVLP, displaying the complementary Catcher. We determined that SliC with the N-terminal SpyTag, N-SliC, retained lysozyme-blocking activity and could be displayed at high density on cVLPs without causing aggregation. In mice, the N-SliC-VLP vaccines, adjuvanted with AddaVax or CpG, induced significantly higher antibody titers compared to controls. In contrast, similar vaccine formulations containing monomeric SliC were non-immunogenic. Accordingly, sera from N-SliC-VLP-immunized mice also had significantly higher human complement-dependent serum bactericidal activity. Furthermore, the N-SliC-VLP vaccines administered subcutaneously with an intranasal boost elicited systemic and vaginal IgG and IgA, whereas subcutaneous delivery alone failed to induce vaginal IgA. The N-SliC-VLP with CpG (10 µg/dose) induced the most significant increase in total serum IgG and IgG3 titers, vaginal IgG and IgA, and bactericidal antibodies.

Comprehensive Bioinformatic Assessments of the Variability of Neisseria gonorrhoeae Vaccine Candidates.

A protective vaccine is the only viable way to stop the spread of gonorrhea in the face of rising antibiotic resistance. However, the notorious phase and antigenic variation of Neisseria gonorrhoeae surface proteins remains one of the challenges in vaccine development. To facilitate vaccine advancement efforts, we carried out comprehensive bioinformatic analyses of sequence variation by comparing 34 gonorrhea antigen candidates among >5,000 clinical N. gonorrhoeae isolates deposited in the Neisseria PubMLST database. Eight protein antigens showed exceptional conservation by having a single allele variant distributed in >80% of isolates. An additional 18 vaccine candidates were represented by ≤3 alleles in >50% of N. gonorrhoeae isolates globally. Phylogenetic analyses highlighted closely related antigen variants and additionally showed that AniA and FetB were the closest between N. gonorrhoeae and N. meningitidis Up to 44% of N. meningitidis alleles for both antigens have premature stop codons, suggesting differential expression. Mapping polymorphisms to the available three-dimensional structures of 12 antigens revealed low-frequency surface polymorphisms. PorB and TbpB possessed numerous high-prevalence polymorphic sites. While TbpA was also highly variable, conserved loops were nonetheless identified. A high degree of sequence conservation, the distribution of a single antigen variant among N. gonorrhoeae strains globally, or low-frequency sequence polymorphisms in surface loops make ACP, AniA, BamA, BamE, MtrE, NspA, NGO0778, NGO1251, NGO1985, OpcA, PldA, Slam2, and ZnuD promising candidates for a gonorrhea vaccine. Finally, the commonly used N. gonorrhoeae FA1090 strain emerges as a vaccine prototype, as it carries antigen sequence types identical to the most broadly distributed antigen variants.IMPORTANCENeisseria gonorrhoeae, the Gram-negative bacterium responsible for the sexually transmitted infection gonorrhea, is categorized as a high-priority pathogen for research and development efforts. N. gonorrhoeae's "superbug" status, its high morbidity, and the serious health impact associated with gonorrhea highlight the importance of vaccine development. One of the longstanding barriers to developing an effective vaccine against N. gonorrhoeae is the remarkable variability of surface-exposed antigens. In this report, we addressed this roadblock by applying extensive bioinformatic analyses to 34 gonorrhea antigen candidates among >5,000 clinical N. gonorrhoeae isolates. Our studies are important, as they reveal promising, conserved gonorrhea vaccine candidates and aid structural vaccinology. Moreover, these approaches are broadly applicable to other infectious diseases where surface antigen variability impedes successful vaccine design.

A novel gonorrhea vaccine composed of MetQ lipoprotein formulated with CpG shortens experimental murine infection.

Bacterial surface lipoproteins are emerging as attractive vaccine candidates due to their biological importance and the feasibility of their large-scale production for vaccine manufacturing. The global prevalence of gonorrhea, resistance to antibiotics, and serious consequences to reproductive and neonatal health necessitate development of effective vaccines. Reverse vaccinology identified the surface-displayed L-methionine binding lipoprotein MetQ (NGO2139) and its homolog GNA1946 (NMB1946) as gonococcal and meningococcal vaccine candidates, respectively. Here, we assessed the suitability of MetQ for inclusion in a gonorrhea vaccine by examining MetQ conservation, its function inNeisseria gonorrhoeae (Ng) pathogenesis, and its ability to induce protective immune responses using a female murine model of lower genital tract infection. In-depth bioinformatics, phylogenetics and mapping the most prevalent Ng polymorphic amino acids to the GNA1946 crystal structure revealed remarkable MetQ conservation: ~97% Ng isolates worldwide possess a single MetQ variant. Mice immunized with rMetQ-CpG (n = 40), a vaccine containing a tag-free version of MetQ formulated with CpG, exhibited robust, antigen-specific antibody responses in serum and at the vaginal mucosae including IgA. Consistent with the activity of CpG as a Th1-stimulating adjuvant, the serum IgG1/IgG2a ratio of 0.38 suggested a Th1 bias. Combined data from two independent challenge experiments demonstrated that rMetQ-CpG immunized mice cleared infection faster than control animals (vehicle, p < 0.0001; CpG, p = 0.002) and had lower Ng burden (vehicle, p = 0.03; CpG, p < 0.0001). We conclude rMetQ-CpG induces a protective immune response that accelerates bacterial clearance from the murine lower genital tract and represents an attractive component of a gonorrhea subunit vaccine.

Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production.

The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA-which recodes proteins through mRNA editing-as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence.

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants.

The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally because of its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n = 15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics data bank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.

PubMLST for Antigen Allele Mining to Inform Development of Gonorrhea Protein-Based Vaccines.

Neisseria gonorrhoeae (Ng) is a human-specific pathogen and the etiological agent of gonorrhea, a sexually transmitted infection with a significant global health burden. While often asymptomatic, untreated gonorrhea can lead to pelvic inflammatory disease, ectopic pregnancy, infertility, and increased transmission/acquisition of HIV. A protective gonorrhea vaccine may be the only way to control disease transmission in the future due to the inexorable development of antibiotic resistance. Subunit antigens are proven candidates for vaccine development due to their safety, cost-effectiveness, and rapid preparation. To inform protein-based gonorrhea vaccine design by including different antigen variants, herein we present bioinformatics mining of alleles and single nucleotide/amino acid polymorphisms using DNA/protein sequences of all Ng isolates deposited into the PubMLST database and MtrE and BamA as model antigens. We also present phylogenetic analyses that can be performed using sequence data to gain insights into the evolutionary relationships between the polymorphisms found among the population of isolates using a convenient tool: Molecular Evolutionary Genetics Analysis (MEGA) software. Finally, we perform antigen polymorphism mapping onto the MtrE and BamA structures. This methodology can be applied for rational vaccine design to increase vaccine coverage and cross-protection by heteroligand presentation achieved via inclusion of diverse antigen variants and is relevant to over 100 different species and genera deposited into the PubMLST family of databases.

Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA.

Lipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigations focused on discovering new vaccine and therapeutic targets for this increasingly difficult to treat pathogen. Its surface exposure in N. meningitidis suggested Laz could be a vaccine candidate for N. gonorrhoeae. Here we characterized the localization, expression, and role of Laz within the gonococcal cell envelope and challenged the hypothesis that Laz and AniA interact. While we demonstrate that Laz indeed shows some good features of a vaccine antigen, such as stable expression, high conservation, and ability to elicit antibodies that cross-react with a diverse panel of Neisseria, it is not a surface-displayed lipoprotein in the gonococcus. This discovery eliminates Laz as a gonorrhea vaccine candidate, further highlighting the necessity of examining homologous protein localization between closely related species. Absence of Laz slightly altered cell envelope integrity but was not associated with growth defects in vitro, including during anoxia, implicating the presence of other electron pathways to AniA. To further dissect the implied AniA-Laz interaction, we utilized biolayer interferometry and optimized and executed chemical cross-linking coupled with immunoblotting to covalently link interacting protein partners in living gonococci. This method, applied for the first time in N. gonorrhoeae research to interrogate protein complexes, was validated by the appearance of the trimer form of AniA, as well as by increased formation of the ß-barrel assembly machinery complex, in the presence of cross-linker. We conclude that Laz is not an electron donor to AniA based on their distinct subcellular localization, discordant expression during infection of the female mouse lower genital tract, and lack of interaction in vivo and in vitro.

Transkingdom network reveals bacterial players associated with cervical cancer gene expression program.

Cervical cancer is the fourth most common cancer in women worldwide with human papillomavirus (HPV) being the main cause the disease. Chromosomal amplifications have been identified as a source of upregulation for cervical cancer driver genes but cannot fully explain increased expression of immune genes in invasive carcinoma. Insight into additional factors that may tip the balance from immune tolerance of HPV to the elimination of the virus may lead to better diagnosis markers. We investigated whether microbiota affect molecular pathways in cervical carcinogenesis by performing microbiome analysis via sequencing 16S rRNA in tumor biopsies from 121 patients. While we detected a large number of intra-tumor taxa (289 operational taxonomic units (OTUs)), we focused on the 38 most abundantly represented microbes. To search for microbes and host genes potentially involved in the interaction, we reconstructed a transkingdom network by integrating a previously discovered cervical cancer gene expression network with our bacterial co-abundance network and employed bipartite betweenness centrality. The top ranked microbes were represented by the families Bacillaceae, Halobacteriaceae, and Prevotellaceae. While we could not define the first two families to the species level, Prevotellaceae was assigned to Prevotella bivia. By co-culturing a cervical cancer cell line with P. bivia, we confirmed that three out of the ten top predicted genes in the transkingdom network (lysosomal associated membrane protein 3 (LAMP3), STAT1, TAP1), all regulators of immunological pathways, were upregulated by this microorganism. Therefore, we propose that intra-tumor microbiota may contribute to cervical carcinogenesis through the induction of immune response drivers, including the well-known cancer gene LAMP3.

SliC is a surface-displayed lipoprotein that is required for the anti-lysozyme strategy during Neisseria gonorrhoeae infection.

Lysozymes are nearly omnipresent as the first line of immune defense against microbes in animals. They exert bactericidal action through antimicrobial peptide activity and peptidoglycan hydrolysis. Gram-negative bacteria developed several weapons to battle lysozymes, including inhibitors of c-type lysozymes in the MliC/PliC family and the Neisseria adhesin complex protein (ACP). Until the recent discovery of ACP, no proteinaceous lysozyme inhibitors were reported for the genus Neisseria, including the important human pathogen N. gonorrhoeae. Here, we describe a previously unrecognized gonococcal virulence mechanism involving a protein encoded by the open reading frame ngo1063 that acts to counteract c-type Iysozyme and provides a competitive advantage in the murine model of gonorrhea. We named this protein SliC as a surface-exposed lysozyme inhibitor of c-type lysozyme. SliC displays low overall primary sequence similarity to the MliC/PliC inhibitors, but we demonstrate that it has a parallel inhibitory mechanism. Our studies provide the first evidence that bacterial proteinaceous lysozyme inhibitors protect against host lysozyme during infection based on lack of attenuation of the ΔsliC mutant in lysozyme knock-out mice, and that the conserved residues involved in lysozyme inhibition, S83 and K103, are functionally indispensable during infection in wild type mice. Recombinant SliC completely abrogated the lytic activity of human and chicken c-type lysozymes, showing a preference towards human lysozyme with an IC50 of 1.85 µM and calculated KD value of 9.2 ± 1.9 µM. In contrast, mutated SliC bearing S83A and K103A substitutions failed to protect fluorescein-labeled cell-wall from lysozyme-mediated hydrolysis. Further, we present data revealing that SliC is a surface-displayed lipoprotein released in membrane vesicles that is expressed throughout all phases of growth, in conditions relevant to different niches of the human host, and during experimental infection of the murine genital tract. SliC is also highly conserved and expressed by diverse gonococcal isolates as well as N. meningitidis, N. lactamica, and N. weaveri. This study is the first to highlight the importance of an anti-lysozyme strategy to escape the innate immune response during N. gonorrhoeae infection.

Proteomics of Vibrio cholerae.

Combining high-throughput mass spectrometry with isobaric tags for relative and absolute quantification (iTRAQ) allows for the identification and relative quantification of proteins from multiple samples. Furthermore, low-abundance proteins that are usually not detected can be enriched by using only the relevant fraction of the proteome, e.g., cytoplasmic, membrane proteins, or secreted proteins. Described here is a workflow for isolation and enrichment of secreted and membrane proteins that is compatible with mass spectrometry. Isolated proteins are reduced, alkylated, and digested with trypsin, and obtained peptides are labeled with iTRAQ reagent and separated by strong cation exchange to reduce the complexity. Finally, the peptides are separated by reverse-phase chromatography, spotted on a MALDI target plate, and analyzed by MALDI TOF-TOF.

Structural and functional insights into the role of BamD and BamE within the ß-barrel assembly machinery in Neisseria gonorrhoeae.

The ß-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.

Detection of bacterial-reactive natural IgM antibodies in desert bighorn sheep populations.

Ecoimmunology is a burgeoning field of ecology which studies immune responses in wildlife by utilizing general immune assays such as the detection of natural antibody. Unlike adaptive antibodies, natural antibodies are important in innate immune responses and often recognized conserved epitopes present in pathogens. Here, we describe a procedure for measuring natural antibodies reactive to bacterial antigens that may be applicable to a variety of organisms. IgM from desert bighorn sheep plasma samples was tested for reactivity to outer membrane proteins from Vibrio coralliilyticus, a marine bacterium to which sheep would have not been exposed. Immunoblotting demonstrated bighorn sheep IgM could bind to a variety of bacterial cell envelope proteins while ELISA analysis allowed for rapid determination of natural antibody levels in hundreds of individual animals. Natural antibody levels were correlated with the ability of plasma to kill laboratory strains of E. coli bacteria. Finally, we demonstrate that natural antibody levels varied in two distinct populations of desert bighorn sheep. These data demonstrate a novel and specific measure of natural antibody function and show that this varies in ecologically relevant ways.

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA.

Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.

Functional and structural studies on the Neisseria gonorrhoeae GmhA, the first enzyme in the glycero-manno-heptose biosynthesis pathways, demonstrate a critical role in lipooligosaccharide synthesis and gonococcal viability.

Sedoheptulose-7-phosphate isomerase, GmhA, is the first enzyme in the biosynthesis of nucleotide-activated-glycero-manno-heptoses and an attractive, yet underexploited, target for development of broad-spectrum antibiotics. We demonstrated that GmhA homologs in Neisseria gonorrhoeae and N. meningitidis (hereafter called GmhAGC and GmhANM , respectively) were interchangeable proteins essential for lipooligosaccharide (LOS) synthesis, and their depletion had adverse effects on neisserial viability. In contrast, the Escherichia coli ortholog failed to complement GmhAGC depletion. Furthermore, we showed that GmhAGC is a cytoplasmic enzyme with induced expression at mid-logarithmic phase, upon iron deprivation and anaerobiosis, and conserved in contemporary gonococcal clinical isolates including the 2016 WHO reference strains. The untagged GmhAGC crystallized as a tetramer in the closed conformation with four zinc ions in the active site, supporting that this is most likely the catalytically active conformation of the enzyme. Finally, site-directed mutagenesis studies showed that the active site residues E65 and H183 were important for LOS synthesis but not for GmhAGC function in bacterial viability. Our studies bring insights into the importance and mechanism of action of GmhA and may ultimately facilitate targeting the enzyme with small molecule inhibitors.

Akkermansia muciniphila mediates negative effects of IFNγ on glucose metabolism.

Cross-talk between the gut microbiota and the host immune system regulates host metabolism, and its dysregulation can cause metabolic disease. Here, we show that the gut microbe Akkermansia muciniphila can mediate negative effects of IFNγ on glucose tolerance. In IFNγ-deficient mice, A. muciniphila is significantly increased and restoration of IFNγ levels reduces A. muciniphila abundance. We further show that IFNγ-knockout mice whose microbiota does not contain A. muciniphila do not show improvement in glucose tolerance and adding back A. muciniphila promoted enhanced glucose tolerance. We go on to identify Irgm1 as an IFNγ-regulated gene in the mouse ileum that controls gut A. muciniphila levels. A. muciniphila is also linked to IFNγ-regulated gene expression in the intestine and glucose parameters in humans, suggesting that this trialogue between IFNγ, A. muciniphila and glucose tolerance might be an evolutionally conserved mechanism regulating metabolic health in mice and humans.

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea.

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform-isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry-to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.

Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics.

A promising new drug target for the development of novel broad-spectrum antibiotics is the highly conserved small GTPase Obg (YhbZ, CgtA), a protein essential for the survival of all bacteria including Neisseria gonorrhoeae (GC). GC is the agent of gonorrhea, a prevalent sexually transmitted disease resulting in serious consequences on reproductive and neonatal health. A preventive anti-gonorrhea vaccine does not exist, and options for effective antibiotic treatments are increasingly limited. To address the dire need for alternative antimicrobial strategies, we have designed and optimized a 384-well GTPase assay to identify inhibitors of Obg using as a model Obg protein from GC, ObgGC. The assay was validated with a pilot screen of 40,000 compounds and achieved an average Z' value of 0.58 ± 0.02, which suggests a robust assay amenable to high-throughput screening. We developed secondary assessments for identified lead compounds that utilize the interaction between ObgGC and fluorescent guanine nucleotide analogs, mant-GTP and mant-GDP, and an ObgGC variant with multiple alterations in the G-domains that prevent nucleotide binding. To evaluate the broad-spectrum potential of ObgGC inhibitors, Obg proteins of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were assessed using the colorimetric and fluorescence-based activity assays. These approaches can be useful in identifying broad-spectrum Obg inhibitors and advancing the therapeutic battle against multidrug resistant bacteria.

The Neisseria gonorrhoeae Obg protein is an essential ribosome-associated GTPase and a potential drug target.

BACKGROUND: Neisseria gonorrhoeae (GC) is a Gram-negative pathogen that most commonly infects mucosal surfaces, causing sexually transmitted urethritis in men and endocervicitis in women. Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility. The incidence of gonorrhea cases remains high globally while antibiotic treatment options, the sole counter measures against gonorrhea, are declining due to the remarkable ability of GC to acquire resistance. Evaluating of potential drug targets is essential to provide opportunities for developing antimicrobials with new mechanisms of action. We propose the GC Obg protein, belonging to the Obg/CgtA GTPase subfamily, as a potential target for the development of therapeutic interventions against gonorrhea, and in this study perform its initial functional and biochemical characterization. RESULTS: We report that NGO1990 encodes Obg protein, which is an essential factor for GC viability, associates predominantly with the large 50S ribosomal subunit, and is stably expressed under conditions relevant to infection of the human host. The anti-Obg antisera cross-reacts with a panel of contemporary GC clinical isolates, demonstrating the ubiquitous nature of Obg. The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth. The in vitro binding and hydrolysis of the fluorescent guanine nucleotide analogs mant-GTP and mant-GDP by recombinant wild type and T192AT193A mutated variants of Obg are also assessed. CONCLUSIONS: Characterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.

A metalloprotease secreted by the type II secretion system links Vibrio cholerae with collagen.

Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.

Quantitative proteomic analysis of the cell envelopes and native membrane vesicles derived from gram-negative bacteria.

Proteins localized to the cell envelope and naturally released membrane vesicles (MVs) play diverse functions in physiology and pathogenesis of Gram-negative bacteria. Study of these proteome fractions is essential for better understanding the basic physiological processes, development of vaccines, and identification of potential drug targets. This unit presents gel-free quantitative proteomic methods for comprehensive proteomic profiling of the cell envelopes and MVs. The procedure starts with the precipitation of the isolated proteome fractions to remove any potential compounds that may interfere with downstream experimental steps. Subsequently, the proteins are reduced, alkylated, and subjected to trypsin digestion. The trypsinized peptides are labeled using isobaric tagging for relative and absolute quantification (iTRAQ), and analyzed samples are pooled and subjected to rigorous prefractionations by strong cation exchange (SCX) and reversed-phase (RP) liquid chromatography (LC). Finally, the tandem mass spectrometry (MS/MS) fragmentation enables peptides identification and quantification.