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Background Increasing multidrug resistance rates in Neisseria gonorrhoeae have raised concerns and an urgent call for new antibiotics for treatment of gonorrhoea. Several decades of subdued drug development in this field and the recent failures of two new antibiotics to show non-inferiority compared with the current first-line antibiotics ceftriaxone plus azithromycin highlight the need for improved preclinical tools to predict clinical outcome of new drugs in the development process. Objectives To summarize current pharmacokinetic/pharmacodynamic (PK/PD) knowledge and dose-finding strategies for antibiotics against gonorrhoea. Sources Literature review of published papers and discussions by global experts at a special workshop on this topic. Content We review current knowledge of gonococcal specific PK/PD principles and provide an update on new in vitro and in vivo models to correlate drug exposure with clinical outcome, and identify challenges and gaps in gonococcal therapeutic research. Implications Identifying the ideal antimicrobial agent and dose for treating uncomplicated urogenital and pharyngeal gonococcal disease requires appropriate validated non-clinical PK/PD models. Recent advances in adapting in vitro and in vivo models for use in gonorrhoea are an important step for enabling the development of new drugs with reduced risk of failure in Phase 3 clinical development and diminish the risk of emergence of resistance.

Background Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea. Methods We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets. Results Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB. Conclusions Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Gonorrhea remains a major public health challenge, and current recommendations for gonorrhea treatment are threatened by evolving antimicrobial resistance and a diminished pipeline for new antibiotics. Evaluations of potential new treatments for gonorrhea currently make limited use of new understanding of the pharmacokinetic and pharmacodynamic contributors to effective therapy, the prevention of antimicrobial resistance, and newer designs for clinical trials. They are hampered by the requirement to utilize combination ceftriaxone/azithromycin therapy as the comparator regimen in noninferiority trials designed to seek an indication for gonorrhea therapy. Evolving gonococcal epidemiology and clinical trial design constraints hinder the enrollment of those populations at the greatest risk for gonorrhea (adolescents, women, and persons infected with antibiotic-resistant Neisseria gonorrhoeae). This article summarizes a recent meeting on the evaluation process for antimicrobials for urogenital gonorrhea treatment and encourages the consideration of new designs for the evaluation of gonorrhea therapy.

The bacterium Neisseria gonorrhoeae (Ngo) is the main cause of the sexually transmitted infection gonorrhea. The global incidence of 87 million new Ngo infections each year, rising infection rates, and the emergence of Ngo strains that are resistant to all clinically recommended antibiotics have raised the specter of untreatable infections (M. Unemo, H. S. Seifert, E. W. Hook, III, S. Hawkes, et al., Nat Rev Dis Primers 5:79, 2019, https://doi.org/10.1038/s41572-019-0128-6). Given their abundance in symptomatic disease, neutrophils are central to both Ngo infection and consequent damage to host tissues. This article highlights present knowledge and the main open questions about Ngo-neutrophil interactions in immunity versus disease pathogenesis.

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways., Antibiotic-resistant bacterial pathogens pose a substantial threat to human health. Here, aided by structural analyses, the authors describe the molecular mechanism behind the activity of a series of compounds that inhibit trans-translation and are effective in eradicating N. gonorrhoeae infection in mice.

Declining gonococcal susceptibility to ceftriaxone and azithromycin has raised the possibility of untreatable gonorrhea in the future and reignited interest in gonococcal vaccine development. Despite decades of research, previous gonococcal vaccine candidates have been ineffective. A growing body of data suggests that meningococcal group B outer-membrane vaccines may be cross-protective against Neisseria gonorrhoeae. Clinical trials of a licensed vaccine against Neisseria meningitidis serogroup B containing an outer-membrane vaccine component are underway to determine its efficacy against N. gonorrhoeae. Other experimental gonococcal vaccine candidates are in the preclinical phases. Population impact of future gonococcal vaccines with different levels of efficacy and duration of protection in various populations is being evaluated using modeling studies. Despite recent progress, gaps in gonococcal vaccine research remain. Research is needed to evaluate vaccine efficacy in preventing gonococcal infections acquired via various anatomic routes and among patients coinfected with other sexually transmitted infections. Studies that model the impact of a future vaccine on high-burden populations such as men who have sex with men and estimate both vaccine cost-effectiveness and the incremental cost-effectiveness ratio of vaccination to antimicrobial resistance and treatment costs are warranted. This narrative review examines the current state of gonococcal vaccine research, the possible impact of a gonococcal vaccine on gonorrhea rates based on modeling studies, gaps in the gonococcal vaccine literature, and public health implications of a future gonococcal vaccine on reducing the gonorrhea burden in the United States.

Renewed interest in developing vaccines against Neisseria gonorrhoeae has been sparked by the increasing threat of gonococcal antimicrobial resistance (AMR) and growing optimism that gonococcal vaccines are biologically feasible. Evidence suggests serogroup B Neisseria meningitidis vaccines might provide some cross-protection against N. gonorrhoeae, and new gonococcal vaccine candidates based on several approaches are currently in preclinical development. To further stimulate investment and accelerate development of gonococcal vaccines, greater understanding is needed regarding the overall value that gonococcal vaccines might have in addressing public health and societal goals in low-, middle-, and high-income country contexts and how future gonococcal vaccines might be accepted and used, if available. In January 2019, the World Health Organization (WHO) convened a multidisciplinary international group of experts to lay the groundwork for understanding the potential health, economic, and societal value of gonococcal vaccines and their likely acceptance and use, and for developing gonococcal vaccine preferred product characteristics (PPCs). WHO PPCs describe preferences for vaccine attributes that would help optimize vaccine value and use in meeting the global public health need. This paper describes the main discussion points and conclusions from the January 2019 meeting of experts. Participants emphasized the need for vaccines to control N. gonorrhoeae infections with the ultimate goals of preventing adverse sexual and reproductive health outcomes (e.g., infertility) and reducing the impact of gonococcal AMR. Meeting participants also discussed important PPC considerations (e.g., vaccine indications, target populations, and potential immunization strategies) and highlighted crucial research and data needs for guiding the value assessment and PPCs for gonococcal vaccines and advancing gonococcal vaccine development.

The concept of immunizing against gonorrhea has received renewed interest because of the recent emergence of strains of Neisseria gonorrhoeae that are resistant to most currently available antibiotics, an occurrence that threatens to render gonorrhea untreatable. However, despite efforts over many decades, no vaccine has yet been successfully developed for human use, leading to pessimism over whether this goal was actually attainable. Several factors have contributed to this situation, including extensive variation of the expression and specificity of many of the gonococcal surface antigens, and the ability of N. gonorrhoeae to resist destruction by complement and other innate immune defense mechanisms. The natural host restriction of N. gonorrhoeae for humans, coupled with the absence of any definable state of immunity arising from an episode of gonorrhea, have also complicated efforts to study gonococcal pathogenesis and the host's immune responses. However, recent findings have elucidated how the gonococcus exploits and manipulates the host's immune system for its own benefit, utilizing human-specific receptors for attachment to and invasion of tissues, and subverting adaptive immune responses that might otherwise be capable of eliminating it. While no single experimental model is capable of providing all the answers, experiments utilizing human cells and tissues in vitro, various in vivo animal models, including genetically modified strains of mice, and both experimental and observational human clinical studies, have combined to yield important new insight into the immuno-pathogenesis of gonococcal infection. In turn, these have now led to novel approaches for the development of a gonococcal vaccine. Ongoing investigations utilizing all available tools are now poised to make the development of an effective human vaccine against gonorrhea an achievable goal within a foreseeable time-frame.

Mouse models of infection are important tools in the study of infectious disease or host the development of products to prevent or treat infections. The estradiol-treated mouse model of Neisseria gonorrhoeae genital tract infection has proved to be a valuable system for determining the importance of gonococcal factors that mediate evasion of host innate effectors in vivo or host gonococcal adaptation to hormonally driven host factors in females. Examination of mechanisms that Neisseria gonorrhoeae uses to subvert the host immune response also has been greatly aided by this whole model system, as have studies on the consequence of antibiotic resistance mutations on gonococcal fitness in vivo and the search for new antibiotics to treat antibiotic-resistant infections. The strict human specificity of N. gonorrhoeae limits the ability of experimental murine infection to mimic human infection. However, in recent years, the development of transgenic mice and protocols for supplementing mice with human factors has improved animal modeling of gonorrhea. To date, however, because the mouse estrous cycle is much shorter than the human reproductive cycle, all reported gonorrhea mouse models require treatment with estradiol and antibiotics to maintain an estrus-like state and suppress the overgrowth of inhibitory commensal flora that occurs under the influence of estrogen to allow sustained N. gonorrhoeae infection. In this chapter, we detail the methods used to (1) prepare the mice for experimental infection with N. gonorrhoeae, (2) inoculate mice and quantitatively culture vaginal swabs for noncompetitive and competitive infection experiments, and (3) monitor the host innate immune response to infection.

Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea.

There is an urgent need for the development of an antigonococcal vaccine due to the increasing drug resistance found in this pathogen. The U.S. Centers for Disease Control (CDC) have identified multidrug-resistant gonococci (GC) as among 3 “urgent” hazard-level threats to the U.S. population. In light of this, on 29 to 30 June 2015, the National Institute for Allergy and Infectious Diseases (NIAID) sponsored a workshop entitled “Gonorrhea Vaccines: the Way Forward.” The goal of the workshop was to gather leaders in the field to discuss several key questions on the current status of gonorrhea vaccine research and the path forward to a licensed gonorrhea vaccine. Representatives from academia, industry, U.S. Government agencies, and a state health department were in attendance. This review summarizes each of the 4 scientific sessions and a series of 4 breakout sessions that occurred during the one and a half days of the workshop. Topics raised as high priority for future development included (i) reinvigoration of basic research to understand gonococcal infection and immunity to allow intervention in processes essential for infection; (ii) clinical infection studies to establish parallels and distinctions between in vitro and animal infection models versus natural human genital and pharyngeal infection and to inform in silico modeling of vaccine impact; and (iii) development of an integrated pipeline for preclinical and early clinical evaluation and direct comparisons of potential vaccine antigens and adjuvants and routes of delivery.

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.

In 2014, the World Health Organization, the US National Institutes of Health, and global technical partners published a comprehensive roadmap for development of new vaccines against sexually transmitted infections (STIs). Since its publication, progress has been made in several roadmap activities: obtaining better epidemiologic data to establish the public health rationale for STI vaccines, modeling the theoretical impact of future vaccines, advancing basic science research, defining preferred product characteristics for first-generation vaccines, and encouraging investment in STI vaccine development. This article reviews these overarching roadmap activities, provides updates on research and development of individual vaccines against herpes simplex virus, Chlamydia trachomatis, Neisseria gonorrhoeae, and Treponema pallidum, and discusses important next steps to advance the global roadmap for STI vaccine development.

There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development., Author summary Eighty-seven million Neisseria gonorrhoeae (Ng) infections occur globally each year and control of gonorrhea through vaccination is challenged by a lack of strong evidence that immunity to gonorrhea is possible. This contention was recently challenged by epidemiological evidence suggesting that an outer membrane vesicle (OMV) vaccine from the related species Neisseria meningitidis (Nm) protected humans against gonorrhea. Here we provide experimental evidence in support of this hypothesis by demonstrating that a licensed, modified version of this Nm OMV-based vaccine accelerates clearance of Ng in a mouse infection model. These results confirm the possibility cross-species protection and are important in that they support the biological feasibility of vaccine-induced immunity against gonorrhea. We also showed that several Ng outer membrane proteins that may be protective targets of the vaccine are recognized by antiserum from vaccinated mice, at least two of which (PilQ and NHBA) are also detected with sera from 4CMenB-immunized human subjects. Our demonstration that a vaccine that may reduce the risk of gonorrhea in humans protects mice against Ng, a highly host-restricted pathogen, also validates the mouse model as a useful tool for guiding the development of other candidate gonorrhea vaccines.

Introduction Sexually transmitted infections (STIs) continue to plague militaries and defense forces. While the historical recognition of the impact of STIs on operations is evident, contemporary surveillance and research activities are limited. As Neisseria gonorrhoeae and other sexually transmitted pathogens become increasingly resistant to antibiotics, the role of the Department of Defense (DoD) in disease surveillance and clinical research is essential to military Force Health Protection. Methods The Infectious Disease Clinical Research Program (IDCRP) of the Uniformed Services University of the Health Sciences partnered with the DoD Global Emerging Infections Surveillance (GEIS) program to monitor the distribution of gonorrhea antimicrobial resistance (AMR) both domestically and abroad. The DoD gonococcal reference laboratory and repository was established in 2011 as a resource for confirmatory testing and advanced characterization of isolates collected from sites across the continental United States (CONUS) and GEIS-funded sites outside the continental United States (OCONUS). The IDCRP is currently implementing surveillance efforts at CONUS military clinics, including Madigan Army Medical Center, Naval Medical Center Camp Lejeune, Naval Medical Center Portsmouth, Naval Medical Center San Diego, and San Antonio Military Medical Center (efforts were also previously at Womack Army Medical Center). The reference laboratory and repository receives specimens from OCONUS collaborators, including Armed Forces Research Institute of Medical Sciences (AFRIMS; Bangkok, Thailand), Naval Medical Research Unit No. 3 (NAMRU-3), Ghana Detachment (Accra, Ghana), Naval Medical Research Unit No. 6 (NAMRU-6; Lima, Peru), U.S. Army Medical Research Unit – Georgia (USAMRD-G; Tbilisi, Republic of Georgia), and U.S. Army Medical Research Directorate – Kenya (USAMRD-K; Nairobi, Kenya). The gonococcal surveillance program, to include findings, as well as associated clinical research efforts are described. Results Among N. gonorrhoeae isolates tested within the United States, 8% were resistant to tetracycline, 2% were resistant to penicillin, and 30% were resistant to ciprofloxacin. To date, only one of the 61 isolates has demonstrated some resistance (MIC=1 μg/ml) to azithromycin. No resistance to cephalosporins has been detected; however, reduced susceptibility (MIC=0.06–0.125 μg/ml) has been observed in 13% of isolates. Resistance is commonly observed in N. gonorrhoeae isolates submitted from OCONUS clinical sites, particularly with respect to tetracycline, penicillin, and ciprofloxacin. While no azithromycin-resistant isolates have been identified from OCONUS sites, reduced susceptibility (MIC=0.125–0.5 μg/ml) to azithromycin was observed in 23% of isolates. Conclusion Continued monitoring of circulating resistance patterns on a global scale is critical for ensuring appropriate treatments are prescribed for service members that may be infected in the U.S. or while deployed. Domestic surveillance for gonococcal AMR within the Military Health System has indicated that resistance patterns, while variable, are not dramatically different from what is seen in U.S. civilian data. Global patterns of gonococcal AMR have been described through the establishment of a central DoD gonococcal reference laboratory and repository. This repository of global isolates provides a platform for further research and development into biomedical countermeasures against gonococcal infections.

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., Author summary The Gram-negative outer membrane is a formidable barrier, primarily because of its asymmetric composition. A layer of lipopolysaccharide is exposed to the external environment and phospholipids are on the internal face of the outer membrane. MlaA is part of a bacterial system that prevents phospholipid accumulation within the lipopolysaccharide layer. If MlaA is removed, membrane asymmetry is disrupted and bacteria become more vulnerable to certain antimicrobials. Neisseria gonorrhoeae causes millions of infections worldwide annually. A growing number are resistant to available antibiotics. Improving our understanding of gonococcal pathogenicity and basic biological processes is required to facilitate the discovery of new weapons against gonorrhea. We investigated the role of MlaA in N. gonorrhoeae and found that when MlaA was absent, bacteria were more sensitive to antibiotics and human defensins. However, the mutant bacteria produced more membrane vesicles–packages of proteins wrapped in membrane material. Mutant vesicles and cell envelopes were enriched in proteins that contribute to disease. These alterations significantly increased mutant fitness during experimental infection of the female mouse genital tract. Our results provide new insights into the processes N. gonorrhoeae uses to fine-tune its ability to stay fit in the hostile environment of the genital tract.

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., Author summary Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is a clinically important pathogen due to the emergence of multi-drug resistance and the lack of a vaccine(s). During host colonization, pathogenic and commensal Neisseria inevitably encounter lysozyme, a major host innate defense factor that is abundantly present in epithelial secretions and phagocytic cells. Although Neisseria spp produce a c-type lysozyme inhibitor, the Adhesin Complex Protein, the significance of lysozyme inhibition for host colonization has not been addressed. Here we demonstrate the existence of a new c-type lysozyme inhibitor in Neisseria. We show that it is a surface-displayed lipoprotein in N. gonorrhoeae and, through its lysozyme-blocking function, plays a critical role in colonization of genital tract mucosae during infection in the female gonorrhea mouse model. We named the protein SliC as a surface-exposed lysozyme inhibitor of c-type lysozyme. Understanding the mechanisms underlying anti-lysozyme strategies may facilitate antimicrobial development.

Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae ; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.

Highlights • 78 million new infections annually; greatest impact on women and neonates in LMIC. • Current control measures are inadequate and challenged by antibiotic resistance. • Conserved candidate vaccine antigens and adjuvant strategies are being developed. • There is a need for human studies to investigate correlates of immunity. • A meningococcal outer membrane vesicle vaccine may protect against gonorrhea., There is a growing public health interest in controlling sexually transmitted infections (STIs) through vaccination due to increasing recognition of the global disease burden of STIs and the role of STIs in women’s reproductive health, adverse pregnancy outcomes, and the health and well-being of neonates. Neisseria gonorrhoeae has historically challenged vaccine development through the expression of phase and antigenically variable surface molecules and its capacity to cause repeated infections without inducing protective immunity. An estimated 78 million new N. gonorrhoeae infections occur annually and the greatest disease burden is carried by low- and middle-income countries (LMIC). Current control measures are clearly inadequate and threatened by the rapid emergence of antibiotic resistance. The gonococcus now holds the status of “super-bug” as there is currently no single reliable monotherapy for empirical treatment of gonorrhea. The problem of antibiotic resistance has elevated treatment costs and necessitated the establishment of large surveillance programs to track the spread of resistant strains. Here we review the need for a gonorrhea vaccine with respect to global disease burden and related socioeconomic and treatment costs, with an emphasis on the impact of gonorrhea on women and newborns. We also highlight the challenge of estimating the impact of a gonorrhea vaccine due to the need for more data on the burden of gonococcal pelvic inflammatory disease and related sequelae and of gonorrhea-associated adverse pregnancy outcomes and the problem of empirical diagnosis and treatment of STIs in LMIC. There is also a lack of clinical and basic science research in the area of gonococcal/chlamydia coinfection, which occurs in a high percentage of individuals with gonorrhea and should be considered when testing the efficacy of gonorrhea vaccines. Finally, we review recent research that suggests a gonorrhea vaccine is feasible and discuss challenges and research gaps in gonorrhea vaccine development.

The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type N. gonorrhoeae strain FA1090 has a survival advantage relative to a PEA transferase A ( lptA ) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an lptA mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokines were not elevated in female mice infected with the isogenic lptA mutant, in contrast to mice infected with the wild-type and complemented lptA mutant bacteria. We also demonstrated that lptA mutant bacteria were more susceptible to human and murine cathelicidins due to increased binding by these peptides and that the differential induction of NF-κB by wild-type and unmodified lipid A was more pronounced in the presence of CAMPs. This work demonstrates that PEA decoration of lipid A plays both protective and immunostimulatory roles and that host-derived CAMPs may further reduce the capacity of PEA-deficient lipid A to interact with TLR4 during infection.

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

Neisseria gonorrhoeae is a common bacterial sexually transmitted infection. Like all Gram-negative bacteria, the outer membrane of the gonococcus is rich in endotoxin, a known ligand for Toll-like receptor (TLR)4. However, the role of endotoxin and that of its cognate receptor TLR4 in the mucosal response to acute gonococcal infection in the genital tract of women is unclear. To test this, we examined the course of infection after vaginal inoculation of N. gonorrhoeae in mice carrying the Lps(d) mutation in Tlr4, which renders them unresponsive to endotoxin. Although there was no difference in the duration of colonization, Lps(d) mice had a significantly higher peak bacterial burden which coincided with a massive polymorphonuclear cell influx and concomitant upregulation of a subset of inflammatory cytokine and chemokine markers. Notably, infected Lps(d) mice showed a decrease in interleukin-17, suggesting that Th17 responses are more dependent on TLR4 signaling in vivo. Defective polymorphonuclear cell-mediated and complement-independent serum killing of gonococci in Lps(d) mice was also observed and may account for the increased bacterial burden. This is the first in vivo evidence that TLR4-regulated factors modulate early inflammatory responses to gonococcal infection in the female reproductive tract and control bacterial replication.

Efforts to develop vaccines against Neisseria gonorrhoeae have become increasingly important, given the rising threat of gonococcal antimicrobial resistance (AMR). Recent data suggest vaccines for gonorrhoea are biologically feasible; in particular, epidemiological evidence shows that vaccines against a closely related pathogen, serogroup B Neisseria meningitidis outer membrane vesicle (OMV) vaccines, may reduce gonorrhoea incidence. Vaccine candidates using several approaches are currently in preclinical development, including meningococcal and gonococcal OMV vaccines, a lipooligosaccharide epitope and purified protein subunit vaccines. The Global STI Vaccine Roadmap provides action steps to build on this technical momentum and advance gonococcal vaccine development. Better quantifying the magnitude of gonorrhoea-associated disease burden, for outcomes like infertility, and modelling the predicted role of gonococcal vaccines in addressing AMR will be essential for building a full public health value proposition, which can justify investment and help with decision making about future vaccine policy and programs. Efforts are underway to gain consensus on gonorrhoea vaccine target populations, implementation strategies and other preferred product characteristics that would make these vaccines suitable for use in low- and middle-income, as well as high-income, contexts. Addressing these epidemiological, programmatic and policy considerations in parallel to advancing research and development, including direct assessment of the ability of meningococcal B OMV vaccines to prevent gonorrhoea, can help bring about the development of viable gonococcal vaccines.

Host immune responses, including the characteristic influx of neutrophils, against Neisseria gonorrhoeae are poorly understood; adaptive immunity is minimal and non-protective. We hypothesize that N. gonorrhoeae selectively elicits Th17-dependent responses, which trigger innate defense mechanisms, including neutrophils and antimicrobial proteins, that it can resist. We found that N. gonorrhoeae induced the production of interleukin-17 (IL-17) in mouse T-cells and Th17-inducing cytokines in mouse and human APCs in vitro. IL-17 was induced in the iliac lymph nodes in vivo in a female mouse model of genital tract gonococcal infection. Antibody blockade of IL-17 or deletion of the major IL-17 receptor (IL-17R) in IL-17RA(KO) mice led to prolonged infection and diminished neutrophil influx. Genital tract tissue from IL-17RA(KO) mice showed reduced production of neutrophil-attractant chemokines in response to culture with N. gonorrhoeae. These results imply a crucial role for IL-17 and Th17 cells in the immune response to N. gonorrhoeae.

The neisserial opacity (Opa) proteins are a family of antigenically distinct outer membrane proteins that undergo phase-variable expression. Opa + variants of Neisseria gonorrhoeae strain FA1090 are selected in a cyclical pattern from the lower genital tract of estradiol-treated mice. Here we show that cyclical recovery of Opa + gonococci does not occur in ovariectomized mice; therefore, the reproductive cycle plays a role in the selection kinetics in vivo . As predicted by the selection pattern shown by wild-type gonococci, we demonstrated that a constitutive Opa-expressing strain was more fit than an Opa-deficient mutant in the early and late phases of infection. We found no evidence that Opa-mediated colonization selects for Opa + variants during murine infection based on adherence assays with cultured murine epithelial cells. We also tested the hypothesis that complement selects for Opa protein expression during infection. Although some Opa + variants of a serum-sensitive derivative of strain FA1090 were more resistant to the bactericidal activity of normal human serum, selection for Opa expression was not abrogated in C3-depleted mice. Finally, as previously reported, Opa + gonococci were more sensitive to serine proteases. Thus, proteases or protease inhibitors may contribute to the observed in vivo selection pattern. We concluded that Opa proteins promote persistence of N. gonorrhoeae in the female genital tract and that opa gene phase variation allows gonococci to evade or capitalize upon unidentified host factors of the mammalian reproductive cycle. This work revealed an intimate interaction between pathogen and host and provides evidence that hormonally related factors shape bacterial adaptation.

Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind complement clown-regulatory proteins, C4b-binding protein (C4BP) and factor H (M), to evade killing by human complement. In addition, sialylation of gonococcal lipooligosaccharide (LOS) also enables N. gonorrhoeae to bind fH. Strains of N. gonorrhoeae that resist killing by human serum complement are killed by serum from rodent, lagomorph and primate species, which cannot be readily infected experimentally with this organism and whose C4BP and/or fH molecules do not bind to N. gonorrhoeae. Serum resistance of gonococci is restored in these sera by human C4BP and/or fH Direct binding specificity of human and chimpanzee C4BP and human fH to gonococci may explain, in part, species-specific restriction of natural gonococcal infection and address why Por1B, but not Por1A containing gonococcal strains, have been successful in experimental chimpanzee infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates. (C) 2008 Elsevier Ltd. All rights reserved.

Neisseria gonorrhoeae produces several antioxidant defenses, including high levels of catalase, which may facilitate the persistence during an inflammatory response via neutralization of H 2 O 2 produced by phagocytes. In vivo testing of the role of catalase in gonococcal survival is critical since several physiological factors impact interactions between N. gonorrhoeae and polymorphonuclear leukocytes (PMNs). Here we assessed the importance of gonococcal catalase in a surrogate model of female genital tract infection. Female BALB/c mice were treated with 17-β estradiol to promote susceptibility to N. gonorrhoeae and inoculated intravaginally with wild-type gonococci or a catalase ( kat ) deletion mutant. A localized PMN influx occurred in an average of 43 and 81% of mice infected with wild-type or kat mutant gonococci, respectively, and PMNs associated with numerous wild-type or catalase-deficient bacteria were observed in vaginal smears. The combined results of six experiments showed a significant difference in the number of days wild-type bacteria were recovered compared to the catalase-deficient gonococci. However, there was much variability between experiments, and we found no correlation between PMN influx, colonization load, and clearance of wild-type or kat mutant bacteria. Estradiol treatment did not impair bacterial uptake, the luminol-dependent chemiluminescence response, or the killing capacity of isolated murine PMNs against N. gonorrhoeae or Staphylococcus aureus . Our data suggest N. gonorrhoeae is not significantly challenged by H 2 O 2 produced by PMNs in the murine lower genital tract; alternatively, redundant defense mechanisms may protect the gonococcus from reactive oxygen species during infection.

COL Jose L. Sanchez, MC USA (Ret.)*†; Brian K. Agan, MD‡; Alice Y. Tsai, MPH*; Grace E. Macalino, PhD‡; LTC Eyako Wurapa, MC USA§; Margaret Mbuchi, PhD§; Erica Dueger, DVM, PhD∥; Katherine C. Horton, MPH∥; Silvia M. Montano-Torres, MD, MPH¶; LCDR Drake H. Tilley, MC USN¶; Karen E. Saylors, PhD**; Naiki Puplampu, MPhil, PhD††; LCDR Christopher C. Duplessis, MC USN††; LCDR Dustin J. Harrison, MSC USN‡‡; CDR Shannon D. Putnam, MSC USN (Ret.)§§; MAJ Eric C. Garges, MC USA∥∥; LCDR Benjamin J. Espinosa, MSC USN¶¶; LCDR Jamal Dejli, MSC USN***; COL Mitchell Meyers, MC USA†††; LTC Samuel L. Yingst, VC USA†††; Ann E. Jerse, PhD‡‡‡; Hala H. Maktabi, PhD, MPH*; MAJ Ronald L. Burke, VC USA*; Nikki N. Jordan, MPH§§§; Gosia Nowak, MSc, MPH∥∥∥; Katherine Hsu, MD, MPH¶¶¶; Olusegun O. Soge, PhD****††††; King K. Holmes, MD, PhD****††††; R. Scott McClelland, MD, MPH****††††; Michael R. MacDonald, MS‡‡‡‡; COL Julie A. Pavlin, MC USA (Ret.)†; COL Joel C. Gaydos, MC USA (Ret.)*†; COL Edmund C. Tramont, MC USA (Ret.)§§§§

Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments. IMPORTANCE Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A’s structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea., IMPORTANCE Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A’s structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.

Gonorrhea occurs at high incidence throughout the world and significantly impacts reproductive health and the spread of human immunodeficiency virus. Current control measures are inadequate and seriously threatened by the rapid emergence of antibiotic resistance. Progress on gonorrhea vaccines has been slow; however, recent advances justify significant effort in this area. Conserved vaccine antigens have been identified that elicit bactericidal antibodies and, or play key roles in pathogenesis that could be targeted by a vaccine-induced response. A murine genital tract infection model is available for systematic testing of antigens, immunization routes and adjuvants, and transgenic mice exist to relieve some host restrictions. Furthermore, mechanisms by which Neisseria gonorrhoeae avoids inducing a protective adaptive response are being elucidated using human cells and the mouse model. Induction of a Th1 response in mice clears infection and induces a memory response, which suggests Th1-inducing adjuvants may be key in vaccine-induced protection. Continued research in this area should include human testing and clinical studies to confirm or negate findings from experimental systems and to define protective host factors.

Gonococcal pilin variation is thought to allow immune evasion and change the adherence properties of the pilus. We have examined the process of pilin antigenic variation in human volunteers inoculated with strain FA1090. Our data show that pilin variation occurred throughout the process of infection, that at each time sampled after inoculation multiple pilin variants were present, and that later pilin variants appear to be recombinants between previously expressed genes and the silent storage pilin copies. Thus, during infection a large repertoire of proteins are available to the population to help avoid immune responses, to provide pili with varying functions, and to transmit to a new host.

The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of outer membrane proteins demonstrating phase and antigenic variation. N. gonorrhoeae strain FA0190 has 11 opa loci that encode at least 8 antigenically distinct Opa proteins. To determine if expression of one Opa protein or a subset of them is favored during gonococcal infection, we inoculated Opa-negative variants of strain FA1090 intraurethrally into male volunteers. The Opa phenotype of gonococci isolated from urine and urethral swab cultures from nine infected subjects was determined. Opa proteins were expressed in a large proportion of the reisolates from the infected subjects. Gonococci cultured from urine or urethral swab samples from six of the subjects were uniformly Opa positive, with the predominant Opa variants differing among subjects. Three different Opa proteins were represented as the predominant type in at least one subject each. In three subjects, there was more heterogeneity in Opa phenotype of the reisolates, including the presence of Opa-negative variants. An increase in the proportion of isolates expressing multiple Opa proteins occurred over time in most subjects. Passage of the inoculum in vitro did not result in similar changes in Opa expression. There was no detectable difference in infectivity of an Opa-negative variant and one expressing an Opa protein (OpaF) that was highly represented in reisolates from the original nine subjects. Reisolates from three infected volunteers inoculated with the OpaF variant showed continued expression of OpaF alone or in conjunction with other Opa proteins. These results demonstrate that there is strong selection for expression of one or more Opa proteins by strain FA1090 in vivo, but that no single protein is preferentially expressed during early infection in the male urethra.

Epidemiological studies suggest H 2 O 2 -producing lactobacilli protect women against gonorrhea. Here we demonstrate that Lactobacillus crispatus and Lactobacillus jensenii , the most common lactobacilli in the female genital tract, inhibit gonococci in both acidic and neutral pH conditions. Inhibition was neutralized by bovine catalase, suggesting that H 2 O 2 is the primary mediator of inhibition.

The MtrC-MtrD-MtrE multidrug efflux pump of Neisseria gonorrhoeae confers resistance to a diverse array of antimicrobial agents by transporting these toxic compounds out of the gonococcus. Frequently in gonococcal strains, the expression of the mtrCDE operon is differentially regulated by both a repressor, MtrR, and an activator, MtrA. The mtrR gene lies 250 bp upstream of and is transcribed divergently from the mtrCDE operon. Previous research has shown that mutations in the mtrR coding region and in the mtrR-mtrCDE intergenic region increase levels of gonococcal antibiotic resistance and in vivo fitness. Recently, a C-to-T transition mutation 120 bp upstream of the mtrC start codon, termed mtr120, was identified in strain MS11 and shown to be sufficient to confer high levels of antimicrobial resistance when introduced into strain FA19. Here we report that this mutation results in a consensus −10 element and that its presence generates a novel promoter for mtrCDE transcription. This newly generated promoter was found to be stronger than the wild-type promoter and does not appear to be subject to MtrR repression or MtrA activation. Although rare, the mtr120 mutation was identified in an additional clinical isolate during sequence analysis of antibiotic-resistant strains cultured from patients with gonococcal infections. We propose that cis-acting mutations can develop in gonococci that significantly alter the regulation of the mtrCDE operon and result in increased resistance to antimicrobials., IMPORTANCE Gonorrhea is the second most prevalent sexually transmitted bacterial infection and a worldwide public health concern. As there is currently no vaccine against Neisseria gonorrhoeae, appropriate diagnostics and subsequent antibiotic therapy remain the primary means of infection control. However, the effectiveness of antibiotic treatment is constantly challenged by the emergence of resistant strains, mandating a thorough understanding of resistance mechanisms to aid in the development of new antimicrobial therapies and genetic methods for antimicrobial resistance testing. This study was undertaken to characterize a novel mechanism of antibiotic resistance regulation in N. gonorrhoeae. Here we show that a single base pair mutation generates a second, stronger promoter for mtrCDE transcription that acts independently of the known efflux system regulators and results in high-level antimicrobial resistance.

Genital tract infections caused by Neisseria gonorrhoeae and Chlamydia trachomatis serovars D to K occur at high incidence in many areas of the world. Despite high rates of coinfection with these pathogens, investigations of host-parasite interactions have focused on each pathogen individually. We describe here a coinfection model in which female BALB/c mice were first infected with the mouse Chlamydia species C. muridarum and then inoculated with N. gonorrhoeae following treatment with water-soluble 17β-estradiol to promote long-term gonococcal infection. Viable gonococci and chlamydiae were recovered for an average of 8 to 10 days, and diplococci and chlamydial inclusions were observed in lower genital tract tissue by immunohistochemical staining. Estradiol treatment reduced proinflammatory cytokine and chemokine levels in chlamydia-infected mice; however, coinfected mice had a higher percentage of vaginal neutrophils compared to mice infected with either pathogen alone. We detected no difference in pathogen-specific antibody levels due to coinfection. Interestingly, significantly more gonococci were recovered from coinfected mice compared to mice infected with N. gonorrhoeae alone. We found no evidence that C. muridarum increases gonococcal adherence to, or invasion of, immortalized murine epithelial cells. However, increased vaginal concentrations of inflammatory mediators macrophage inflammatory protein 2 and tumor necrosis factor alpha were detected in C. muridarum -infected mice prior to inoculation with N. gonorrhoeae concurrently with the downregulation of cathelicidin-related antimicrobial peptide and secretory leukocyte peptidase inhibitor genes. We conclude that female mice can be successfully infected with both C. muridarum and N. gonorrhoeae and that chlamydia-induced alterations in host innate responses may enhance gonococcal infection.

BACKGROUND:The development of a gonorrhea vaccine is challenged by the lack of correlates of protection. The antigenically variable neisserial opacity (Opa) proteins are expressed during infection and have a semivariable (SV) and highly conserved (4L) loop that could be targeted in a vaccine. Here we compared antibodies to linear (Ab(linear)) and cyclic (Ab(cyclic)) peptides that correspond to the SV and 4L loops and selected hypervariable (HV(2)) loops for surface-binding and protective activity in vitro and in vivo. METHODS/FINDINGS:Ab(SV cyclic) bound a greater number of different Opa variants than Ab(SV linear), including variants that differed by seven amino acids. Antibodies to the 4L peptide did not bind Opa-expressing bacteria. Ab(SV) (cyclic) and Ab(HV2) (cyclic), but not Ab(SV) (linear) or Ab(HV2 linear) agglutinated homologous Opa variants, and Ab(HV2BD) (cyclic) but not Ab(HV2BD) (linear) blocked the association of OpaB variants with human endocervical cells. Only Ab(HV2BD) (linear) were bactericidal against the serum resistant parent strain. Consistent with host restrictions in the complement cascade, the bactericidal activity of Ab(HV2BD) (linear) was increased 8-fold when rabbit complement was used. None of the antibodies was protective when administered vaginally to mice. Antibody duration in the vagina was short-lived, however, with

Commensal lactobacilli that produce hydrogen peroxide (H(2)O(2)) inhibit Neisseria gonorrhoeae in vitro, and clinical data suggest that they are associated with a reduced risk of gonorrhea. We precolonized mice with Lactobacillus crispatus and then challenged them with N. gonorrhoeae, to measure the effects of H(2)O(2)-producing lactobacilli on gonococcal infection. We found no difference in the duration of infection or the number of gonococci recovered from untreated mice and mice colonized with L. crispatus. A gonococcal catalase mutant and a catalase, cytochrome C peroxidase mutant exhibited greater susceptibility to L. crispatus in vitro than did wild-type bacteria; however, recovery of these mutants from mice was not affected by L. crispatus. We also found no evidence that utilization of lactobacillus-produced lactate by N. gonorrhoeae balances the detrimental effects of H(2)O(2) during infection. We conclude that the association between lactobacilli and gonococci is complex and may be subject to factors that have not been reproduced in vitro.

Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, β-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered.

The 17beta-estradiol-treated mouse model is the only small animal model of gonococcal genital tract infection. Here we show gonococci localized within vaginal and cervical tissue, including the lamina propria, and high numbers of neutrophils and macrophages in genital tissue from infected mice. Infection did not induce a substantial or sustained increase in total or gonococcal-specific antibodies. Mice could be reinfected with the same strain and repeat infection did not boost the antibody response. However, intravaginal immunization of estradiol-treated mice induced gonococcal-specific primary and secondary serum antibody responses. We conclude that similar to human infection, experimental murine infection induces local inflammation but not an acquired immune response or immunological memory.

Enteropathogenic Escherichia coli (EPEC) causes a characteristic histopathology in intestinal epithelial cells called the attaching and effacing lesion. Although the histopathological lesion is well described the bacterial factors responsible for it are poorly characterized. We have identified four EPEC chromosomal genes whose predicted protein sequences are similar to components of a recently described secretory pathway (type III) responsible for exporting proteins lacking a typical signal sequence. We have designated the genes sepA, sepB, sepC, and sepD (sep, for secretion of E. coli proteins). The predicted Sep polypeptides are similar to the Lcr (low calcium response) and Ysc (yersinia secretion) proteins of Yersinia species and the Mxi (membrane expression of invasion plasmid antigens) and Spa (surface presentation of antigens) regions of Shigella flexneri. Culture supernatants of EPEC strain E2348/69 contain several polypeptides ranging in size from 110 kDa to 19 kDa. Proteins of comparable size were recognized by human convalescent serum from a volunteer experimentally infected with strain E2348/69. A sepB mutant of EPEC secreted only the 110-kDa polypeptide and was defective in the formation of attaching and effacing lesions and protein-tyrosine phosphorylation in tissue culture cells. These phenotypes were restored upon complementation with a plasmid carrying an intact sepB gene. These data suggest that the EPEC Sep proteins are components of a type III secretory apparatus necessary for the export of virulence determinants.

Background and Objectives: The spread of sexually transmitted diseases (STDs), including gonorrhea, is affected by the duration of infection. Oral antibiotic therapy for gonococcal infection has been shown to be as effective as conventional intramuscular injection with ceftriaxone. Rapid cure would be expected to limit further spread of gonorrhea. However, the speed with which Neisseria gonorrhoeae is eliminated from the urogenital tract has not been evaluated. Goal of this Study: To determine the time required for elimination of Neisseria gonorrhoeae from the urine, mucosa, and semen in male subjects after treatment with ceftriaxone (250 mg intramuscularly), ciprofloxacin (500 mg by mouth, single dose) or cefixime (400 mg by mouth, single dose). Results: In 14 subjects, gonococci were eliminated from the urine within 4 hours of therapy and the mucosa within 24 hours after therapy. In 9 additional subjects, gonococci were eliminated from the semen by 24 hours after therapy. Conclusion: These results support the efficacy of single-dose oral therapy for gonorrhea and suggest that earlier follow-up for proof of cure in clinical trials of new antibiotics for gonorrhea may be acceptable. Rapid elimination of gonorrhea reduces the risk for continued transmission of the organism

Neisseria gonorrhoeae infection is limited to the human host. Experimental urethral infection in male volunteers was used to study different aspects of the infection. Urethral installation of a variety of gonococcal variants (10(4)-10(6)) led to infection in 27 subjects, who developed pyuria and shed bacteria in urine before urethritis developed 1-6 days after gonococcal inoculation. The incubation period was affected by the inoculation procedure and size of the inoculum. Subjects were treated with intramuscular ceftriaxone (250 mg) if urethritis developed or at 6 days after inoculation. Urine cultures became negative within several hours of therapy, and symptoms resolved within 1 day of therapy. Infected patients suffered no major complications. Experimental male urethral gonococcal infection provides a unique opportunity to understand the biology and immunology of gonococcal infection and is an efficient method to test gonococcal vaccine candidates.

The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% of E. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produce attaching and effacing intestinal lesions, such as RDEC-1 (an EPEC of weanling rabbits) and enterohemorrhagic E. coli. The predicted amino acid sequence derived from the nucleotide sequence of eae shows significant homology to that of the invasin of Yersinia pseudotuberculosis.