Gonorrhea, an obligate human infection, is on the rise worldwide and gonococcal strains resistant to many antibiotics are emerging. Appropriate antimicrobial treatment and prevention, including effective vaccines, are urgently needed. To guide investigation, an experimental model of genital tract infection has been developed in female mice to study mechanisms by which Neisseria gonorrhoeae evades host-derived antimicrobial factors and to identify protective and immunosuppressive pathways. Refinements of the animal model have also improved its use as a surrogate host of human infection and accelerated the testing of novel therapeutic and prophylactic compounds against gonococcal infection. Reviewed herein are the (a) history of antibiotic usage and resistance against gonorrhea and the consequences of resistance mechanisms that may increase gonococcal fitness and therefore the potential for spread, (b) use of gonococcal infection in the animal model system to study mechanisms of pathogenesis and host defenses, and (c) current status of 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 inNeisseria 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 BNeisseria meningitidis(Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis thatNmOMVs induce cross-protection against gonorrhea in a well-characterized female mouse model ofNggenital tract infection. We found that immunization with the licensedNmOMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced theNgbacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted withNgOMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized severalNgsurface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognizedNgPilQ 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 bactericidal₅₀titers 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 thatNmOMVs confer cross-species protection against gonorrhea, and implicate severalNgsurface 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.

Resistance to ceftriaxone in Neisseria gonorrhoeae is mainly conferred by mosaic penA alleles that encode penicillin-binding protein 2 (PBP2) variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mosaic penA alleles on gonococcal fitness, we introduced the mosaic penA alleles from two ceftriaxone-resistant (Cro ^r ) clinical isolates (H041 and F89) into a Cro ^s strain (FA19) by allelic exchange and showed that the resultant Cro ^r mutants were significantly outcompeted by the Cro ^s parent strain in vitro and in a murine infection model. Four Cro ^r compensatory mutants of FA19 penA41 were isolated independently from mice that outcompeted the parent strain both in vitro and in vivo . One of these compensatory mutants (LV41C) displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D) in the acnB gene encoding the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduction of the acnB _G348D allele into FA19 penA41 conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of additional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq) analysis of FA19 penA41 acnB _G348D revealed a large set of upregulated genes involved in carbon and energy metabolism. We conclude that compensatory mutations can be selected in Cro ^r gonococcal strains that increase metabolism to ameliorate their fitness deficit. IMPORTANCE The emergence of ceftriaxone-resistant (Cro ^r ) Neisseria gonorrhoeae has led to the looming threat of untreatable gonorrhea. Whether Cro resistance is likely to spread can be predicted from studies that compare the relative fitnesses of susceptible and resistant strains that differ only in the penA gene that confers Cro resistance. We showed that mosaic penA alleles found in Cro ^r clinical isolates are outcompeted by the Cro ^s parent strain in vitro and in vivo but that compensatory mutations that allow ceftriaxone resistance to be maintained by increasing bacterial fitness are selected during mouse infection. One compensatory mutant that was studied in more detail had a mutation in acnB , which encodes the aconitase that functions in the tricarboxylic acid (TCA) cycle. This study illustrates that compensatory mutations can be selected during infection, which we hypothesize may allow the spread of Cro resistance in nature. This study also provides novel insights into gonococcal metabolism and physiology.