Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

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.

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.

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.

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.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

Jizanpeptins A-E (1-5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A-E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 µM) compared to chymotrypsin (IC50 = 1.4 to >10 µM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.

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.

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays.

The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344-in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.

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.

The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization.

OBJECTIVES: Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide. METHODS: The 2016 WHO reference strains (n = 14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n = 23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing. RESULTS: The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n = 14) were presented. CONCLUSIONS: The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.

Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC physiology, which may represent promising targets for designing new therapeutic interventions.

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.

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.

The type II secretion pathway in Vibrio cholerae is characterized by growth phase-dependent expression of exoprotein genes and is positively regulated by σE.

Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V. cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S. Subsequent screening of a V. cholerae genomic library suggested that σ(E) stress response, phosphate metabolism, and the second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ(E), we discovered that the upstream region of the T2S operon possesses both the consensus σ(E) and σ(70) signatures, and deletion of the σ(E) binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ(E) stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S complex belongs to the σ(E) regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches.

Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases.

The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961.

Antimicrobial rubrolides from a South African species of Synoicum tunicate.

The CH2Cl2-MeOH extract of a South African tunicate described as the new Synoicum globosum Parker-Nance sp. nov. (Ascidiacea, Aplousobranchia) was subjected to ¹H NMR-guided fractionation. This resulted in the identification of new 3″-bromorubrolide F (1), 3'-bromorubrolide E (2), 3'-bromorubrolide F (3), and 3',3″-dibromorubrolide E (4) and reisolation of known rubrolides E (5) and F (6), based on NMR spectroscopic and mass spectrometric data. Biological testing of both new and known members of this reported antimicrobial family of halogenated, aryl-substituted furanones indicated moderate antibacterial properties for 3'-bromorubrolide E (2), 3',3″-dibromorubrolide E (4), and rubrolide F (6) against methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis.

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.