Multi-omics uncover interactions in the vaginal microbiome and a type II secretion/Tad pilus system in Gardnerella species

Abstract

Background: The vaginal microbiome is a key determinant of women's health, with Lactobacillus-dominant communities protecting against bacterial vaginosis (BV) and related adverse outcomes. Dysbiosis involves a decline in lactobacilli and overgrowth of anaerobes, with Gardnerella species central to initiating polymicrobial aggregates. However, the genetic determinants of community assembly, biofilm formation, and interspecies interactions remain poorly understood. This study investigated the genomic and functional basis of these traits using an integrated multi-omics approach. Results: Long-read whole-genome sequencing of clinical Gardnerella isolates yielded closed genomes and revealed a pangenomic split correlating with high- versus low-biofilm phenotypes, linked to clade-specific genomic islands and allelic variants. Functional gene modules within these regions distinguished G. vaginalis from G. piotii, contributing to nutrient acquisition, cell wall metabolism, and colonization. Integrated 16S rRNA gene sequencing, and proteomics of all pairwise dual-species co-cultures of five key vaginal bacteria showed that G. vaginalis and L. iners acted as dominant ecological drivers, outcompeting neighbors and triggering asymmetric proteomic remodeling. Differential expression of a gene within a putative operon encoding a conserved type II secretion/Tad pilus system, revealed by integrating the pangenome with the proteome, implicated this system in interspecies interactions. AI-based structural predictions of the complex suggested isoform-specific differences in protein folding between G. vaginalis and G. piotii. In parallel, metagenomic analysis of 93 women showed the operon was more abundant in BV samples than in healthy controls, linking it to dysbiosis. Integrated metabolomics further identified a methyl-{beta}-carboline compound elevated in Prevotella bivia-containing co-cultures that selectively inhibited G. vaginalis and P. bivia while sparing L. crispatus. Several dipeptides influenced growth and biofilm formation of vaginal microbes, and methyl-{beta}-carboline and sphingosine additionally inhibited multidrug-resistant pathogens, suggesting new strategies for managing polymicrobial infections. Conclusions: This work links Gardnerella genomic features to virulence-associated phenotypes and demonstrates how interspecies interactions shape community dynamics and antimicrobial metabolite production. The findings support a model in which biofilm capacity is genetically encoded within an accessory genome, deployed through tailored proteomic responses, and modulated by an interaction-specific metabolic landscape, providing a foundation for identifying bioactive molecules and developing diagnostics and targeted therapeutics to restore and maintain vaginal health.