Fig. 3. BV-associated bacteria (BVAB) induce distinct metabolomic profiles from mock-infected controls and immunomodulatory signatures of lipids and amino acids.

a Principal component analysis (PCA) demonstrates that F. nucleatum and F. gonidiaformans induce similar and partially overlapping metabolic profiles that cluster separately from mock-infected controls. PC1 and PC2 scores were analyzed by unpaired two-tailed Student’s t-tests. ***p < 0.001; ****p < 0.0001. b Hierarchical cluster analysis (HCA) of metabolite Z-scores from 3-D cervical cells infected with BVAB and mock-infected controls. HCA was performed using Euclidean distance and average linkage clustering on both rows (metabolites) and columns (treatments). c Venn diagrams indicating the number of unique or overlapping significantly (p < 0.05) accumulated or depleted metabolites (infection vs. mock-infected controls). d Pie charts represent the percent of significantly different (p < 0.05) metabolites (infection vs. mock-infected controls) relative to all metabolites in the superpathway (right). *p < 0.05; Chi-squared (χ²) analysis. Infection with BVAB induces global changes in metabolites corresponding to amino acid and lipid superpathways. All bacteria were processed in PBS and adjusted to an optical density at 600 nm (OD₆₀₀) of 0.5. Human 3-D cervical cells were infected with bacterial suspensions (20 μl per 1 × 10⁵ epithelial cells) for 24 h under anaerobic conditions. A minimum of n = 3 independent replicates were performed and measured for each condition. Cell culture supernatants were used for global metabolomics analysis.