Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease

Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight-threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines.Keywords: Neisseria gonorrhoeae, vaccine, proteomics, antibiotic resistance, surveillance, drugs, molecular targets, gonorrheaThis is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Frontiers Research Foundation. The published article can be found at: http://journal.frontiersin.org/journal/microbiolog

Proteomics, Bioinformatics and Structure-Function Antigen Mining For Gonorrhea Vaccines

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the serious health consequences combined with the prevalence and the dire possibility of untreatable gonorrhea. Reverse vaccinology, which includes genome and proteome mining, has proven successful in the discovery of vaccine candidates against many pathogenic bacteria. Here, we describe proteomic applications including comprehensive, quantitative proteomic platforms and immunoproteomics coupled with broad-ranging bioinformatics that have been applied for antigen mining to develop gonorrhea vaccine(s). We further focus on outlining the vaccine candidate decision tree, describe the structure-function of novel proteome-derived antigens as well as ways to gain insights into their roles in the cell envelope, and underscore new lessons learned about the fascinating biology of Neisseria gonorrhoeae

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.Keywords: Drug target, Mant guanine nucleotides, Neisseria gonorrhoeae, GTPase, Drug resistance, Obg protein

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

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.This is the publisher’s final pdf. The published article is copyrighted by The American Society for Biochemistry and Molecular Biology and can be found at: http://www.mcponline.org/content/15/7/233

Deciphering the Function of New Gonorrhea Vaccine Candidates in Cell Envelope Homeostasis

Neisseria gonorrhoeae is the sexually transmitted pathogen responsible for millions of cases of gonorrhea worldwide each year. Rapidly-spreading antibiotic resistance is diminishing the ability to effectively treat a disease with significant consequences to female and male reproductive health, as well as to neonatal well-being. A protective vaccine has the potential to substantially reduce the global morbidity of gonorrhea. Vaccine development has been challenged by the gonococcus’s tremendous modulation of its surface proteome, evasion of immune responses, and remarkable strain-to-strain variability. We performed high-throughput proteomic studies to identify proteins conserved across strains and expressed during exposure to host-relevant conditions that may represent promising targets for developing vaccines or antimicrobials against N. gonorrhoeae. In this work, we have characterized the functions of 8 proteome-derived vaccine candidates within the gonococcal cell envelope. In the first-ever Phenotype MicroArray examination of N. gonorrhoeae, we exposed knockout mutants of 7 candidate antigens, BamG (NGO1985), MlaA (NGO2121), NGO2054, NGO2111, NGO1205, NGO1344, and BamEGC (NGO1780), to over 1,000 conditions selected to probe bacterial resistance to cell envelope stress. This study suggested that BamG and MlaA were the most suitable proteins for inclusion in a vaccine due to their extensive chemical sensitivity phenomes. Additionally, knockout mutations of either protein resulted in similar chemical sensitivities in the extensively drug resistant strain WHO X. Further examination of phenotypes associated with MlaA deletion revealed that outer membrane integrity was altered, membrane vesicle secretion was increased, and bacterial colonies were smaller. To better understand the proposed role of MlaA in vesicle biogenesis, quantitative proteomics were performed and revealed that several adhesins and virulence factors were more abundant in the cell envelope and membrane vesicles of ∆mlaA mutant bacteria. Possibly as a result of these alterations, the MlaA knockout adhered to and invaded cervical cells more readily than wild type bacteria and was up to 16-fold more fit during competitive infections in the female mouse model of genital gonorrhea. MlaA was downregulated during iron deprivation and slightly upregulated under anaerobic conditions, but bacterial growth was not affected under either condition in the absence of MlaA. Additionally, polyclonal antiserum raised against MlaA cross-reacted with 37 heterogeneous laboratory strains and clinical isolates and recognized homologous proteins in N. meningitidis and N. lactamica, indicating that expression of MlaA is broadly conserved. Based on our observations, we suggest that N. gonorrhoeae fine-tunes its pathogenicity by modulating MlaA expression in response to the host environment. Finally, we evaluated the cupredoxin azurin (Laz), which is lipid-modified and surface-exposed in N. meningitidis, as a vaccine candidate. Our studies showed that in N. gonorrhoeae, the Laz cellular pool increased under anaerobiosis, although the ∆laz mutant was not attenuated during anaerobic growth. Laz expression was stable and broadly conserved across a diverse panel of geographically and temporally distinct gonococcal clinical isolates and during experimental infection of the murine genital tract. Although Laz appeared to influence expression of the nitrite reductase AniA, no interaction between the two proteins was revealed by in vivo cross-linking, suggesting that Laz is not an electron donor to AniA. Finally, our results demonstrated that Laz is not surface exposed in N. gonorrhoeae, eliminating its inclusion in a vaccine. Together, the studies performed here revealed insights into the physiological roles of eight proteome-derived vaccine candidate proteins in the cell envelope and evaluated their potential utility as gonorrhea vaccine candidates. Distinct phenotypes were associated with BamG and MlaA deletion while loss of NGO1344 and BamEGC resulted in similar phenotypes. These results provided valuable information for our vaccine candidate decision tree and suggested that vaccine-mediated inhibition of the function of any protein examined, especially BamG and MlaA, could be substantially detrimental to gonococcal viability in vivo. However, further evaluation of the suitability of MlaA and Laz as gonorrhea vaccine candidates revealed that the ∆mlaA mutant’s fitness was enhanced during infection and Laz was not surface exposed, implying that using either protein as a vaccine antigen may not be appropriate. In summary, our studies have broadened our understanding of N. gonorrhoeae physiology, illuminated a new MlaA-associated virulence pathway, and advanced gonorrhea vaccine research by evaluating 8 antigens

Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease

Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight- threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines