Fig. 5

Relative abundance of gene ontologies in independent samples and in inflammatory versus non-inflammatory Lactobacillus isolates. a The top 14 microbial biological process (BP) and cellular component (CC) gene ontology (GO) terms that distinguished women with low versus high inflammation in the full cohort (n = 113) were validated in an independent sample of ten women from Cape Town, South Africa. Liquid chromatography-tandem mass spectrometry was used to evaluate the metaproteome in lateral vaginal wall swabs from these women. Inflammation groups were defined based on hierarchical followed by K-means clustering of these ten women according to the concentrations of nine pro-inflammatory cytokines [interleukin (IL)-1α, IL-1β, IL-6, IL-12p40, IL-12p70, tumor necrosis factor (TNF)-α, TNF-β, TNF-related apoptosis-inducing ligand (TRAIL), interferon (IFN)-γ]. GO relative abundance was determined by aggregation of microbial protein intensity-based absolute quantification (iBAQ) values assigned to a same GO term. The relative abundance of the top BPs and CCs (except ATP-binding cassette transporter complex which was not detected in these samples) is shown as bar graphs, with blue indicating women with low inflammation (n = 5) and red indicating women with high inflammation (n = 5). Welch’s t test was used for comparisons. *p < 0.05. b–i Twenty-two Lactobacillus isolates that induced relatively high inflammatory responses and 22 isolates that induced lower inflammatory responses were adjusted to 4.18 × 10⁶ CFU/ml in bacterial culture medium and incubated for 24 h under anaerobic conditions. Following incubation, protein was extracted and digested and liquid chromatography-tandem mass spectrometry was conducted. Raw files were processed with MaxQuant against a database including the Lactobacillus genus and common contaminants. The iBAQ values for proteins with the same gene ontologies were aggregated, log₁₀-transformed, and compared using the Mann-Whitney U test. Box-and-whisker plots show log₁₀-transformed iBAQ values, with lines indicating medians and whiskers extending to 1.5 times the interquartile range from the box. A false discovery rate step-down procedure was used to adjust for multiple comparisons, and adjusted p values < 0.05 were considered statistically significant