- 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.