Microbial function and genital inflammation in young South African women at high risk of HIV infection

Abstract

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.

Lactic acid from vaginal microbiota enhances cervicovaginal epithelial barrier integrity by promoting tight junction protein expression

“…Samples were computational randomised and the eluted material subjected to metaproteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Q-Exactive quadrupole-Orbitrap MS (Thermo Fisher Scienti c, MA, USA) coupled with a Dionex UltiMate 3000 nano-UPLC system (120min per sample) [45]. Proteomic data were used to infer vaginal microbiome composition and host secretome as described previously [45].…”

“…Samples were computational randomised and the eluted material subjected to metaproteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Q-Exactive quadrupole-Orbitrap MS (Thermo Fisher Scienti c, MA, USA) coupled with a Dionex UltiMate 3000 nano-UPLC system (120min per sample) [45]. Proteomic data were used to infer vaginal microbiome composition and host secretome as described previously [45]. Brie y, proteins were identi ed using two different databases: (i) UniProt database restricted to human and microbial entries (73,910,451, release August-2017) and ltered using the MetaNovo pipeline [48]; (ii) human proteins combined with a vaginal metagenome-based database obtained from the study of A uni-Zadeh et al [49].…”

“…The taxonomic analysis of the proteins identi ed using both of the databases was similar; however, the rst database resulted in the identi cation of a greater number of proteins compared to the vaginal metagenome database. The rst database also showed a high degree of similarity compared to 16S rRNA gene sequence data from a subset of the same women and was thus used for downstream analysis [45]. As the majority of taxa identi ed had <2 proteins detected, a more stringent cut-off was applied to include only taxa with >3 detected proteins, or 2 proteins detected in multiple samples.…”

Protocol: evaluation of an optimised couples-focused intervention to increase testing for HIV in rural KwaZulu-Natal, South Africa, the Igugu Lethu (‘Our treasure’) cohort study

“…Proteins will be identified using a custom UniProt database including human and vaginal microbial entries. Taxonomy will be assigned using UniProt and relative abundance of each taxon will be determined by aggregating the intensity-based absolute quantification (iBAQ) values of all proteins identified for each taxon [41].…”

Determinants of Vaginal Microbiota Composition

“…We have a poor understanding of, and ability to accurately measure, metabolic products relevant to the optimal and dysbiotic VMB including short chain fatty acids and lactic acid (Aldunate et al, 2015 ). Metaproteomic analysis of mucosal inflammation and target cell activation in a cohort of African women has shown that minority microorganisms may have disproportionate metabolic activity and functional impact (Alisoltani et al, 2020 ). However, at this stage reference database limitations undermine the reliability of metaproteomics approaches.…”