Background Female genital tract (FGT) inflammation is an important risk factor for HIV acquisition. The FGT microbiome is closely associated with inflammatory profile; however, the relative importance of microbial activities has not been established. Since proteins are key elements representing actual microbial functions, this study utilized metaproteomics to evaluate the relationship between FGT microbial function and inflammation in 113 young and adolescent South African women at high risk of HIV infection. Women were grouped as having low, medium, or high FGT inflammation by K-means clustering according to pro-inflammatory cytokine concentrations. Results A total of 3186 microbial and human proteins were identified in lateral vaginal wall swabs using liquid chromatography-tandem mass spectrometry, while 94 microbial taxa were included in the taxonomic analysis. Both metaproteomics and 16S rRNA gene sequencing analyses showed increased non-optimal bacteria and decreased lactobacilli in women with FGT inflammatory profiles. However, differences in the predicted relative abundance of most bacteria were observed between 16S rRNA gene sequencing and metaproteomics analyses. Bacterial protein functional annotations (gene ontology) predicted inflammatory cytokine profiles more accurately than bacterial relative abundance determined by 16S rRNA gene sequence analysis, as well as functional predictions based on 16S rRNA gene sequence data (p < 0.0001). The majority of microbial biological processes were underrepresented in women with high inflammation compared to those with low inflammation, including a Lactobacillus-associated signature of reduced cell wall organization and peptidoglycan biosynthesis. This signature remained associated with high FGT inflammation in a subset of 74 women 9 weeks later, was upheld after adjusting for Lactobacillus relative abundance, and was associated with in vitro inflammatory cytokine responses to Lactobacillus isolates from the same women. Reduced cell wall organization and peptidoglycan biosynthesis were also associated with high FGT inflammation in an independent sample of ten women. Conclusions Both the presence of specific microbial taxa in the FGT and their properties and activities are critical determinants of FGT inflammation. Our findings support those of previous studies suggesting that peptidoglycan is directly immunosuppressive, and identify a possible avenue for biotherapeutic development to reduce inflammation in the FGT. To facilitate further investigations of microbial activities, we have developed the FGT-DB application that is available at http://fgtdb.org/.

Female genital tract (FGT) inflammation is an important risk factor for HIV acquisition. Previous studies have identified the FGT microbiota as a major determinant of altered inflammatory cytokine profiles, however, the relative importance of microbial activities and functions is unknown. Liquid chromatography-tandem mass spectrometry was used to evaluate the FGT metaproteomic profiles of 113 young South African women. FGT inflammation was defined by inflammatory cytokine concentration profile. A total of 3,186 microbial and human proteins were identified and both were analyzed for the functional and the former also for taxonomic distributions. The functional annotations of bacterial proteins predicted genital inflammation more accurately than bacterial relative abundance determined by 16S rRNA analysis (p<0.0001). The majority of microbial biological pathways were underrepresented in women with inflammation, including a signature of reduced cell wall organization and peptidoglycan biosynthesis. This signature remained associated with FGT inflammatory profiles after adjusting for Lactobacillus relative abundance and potential confounders. These biological pathways were also associated with inflammatory cytokine responses to Lactobacillus isolates from the same women in a vaginal epithelial cell model. Comparison of metaproteomics taxonomic assignment with 16S rRNA gene survey analysis revealed differences in bacterial relative abundance between the methods, however both approaches showed increased non-optimal bacteria and decreased lactobacilli in women with FGT inflammation. These findings suggest that microbial function is a critical determinant of FGT inflammation, and that specific microbial properties may be harnessed for biotherapeutic development.

Problem Data on the effects of contraceptives on female genital tract (FGT) immune mediators are inconsistent, possibly in part due to pre‐existing conditions that influence immune mediator changes in response to contraceptive initiation. Methods This study included 161 South African women randomised to injectable depot medroxyprogesterone acetate (DMPA‐IM), copper intrauterine device (IUD), or levonorgestrel (LNG) implant in the Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial. We measured thirteen cytokines and antimicrobial peptides previously associated with HIV acquisition in vaginal swabs using Luminex and ELISA, before, and at 1 and 3 months after contraceptive initiation. Women were grouped according to an overall baseline inflammatory profile. We evaluated modification of the relationships between contraceptives and immune mediators by baseline inflammation, demographic, and clinical factors. Results Overall, LNG implant and copper IUD initiation were associated with increases in inflammatory cytokines, while no changes were observed following DMPA‐IM initiation. However, when stratifying by baseline inflammatory profile, women with low baseline inflammation in all groups experienced significant increases in inflammatory cytokines, while those with a high baseline inflammatory profile experienced no change or decreases in inflammatory cytokines. Conclusion We conclude that pre‐contraceptive initiation immune profile modifies the effect of contraceptives on the FGT innate immune response.

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