Comparative proteomics reveal striking differences in biofilm formation and virulence potential of Gardnerella leopoldii, G. piotii, and G. vaginalis isolates

Abstract

ABSTRACT Bacterial vaginosis (BV) is a common, recurrent cause of abnormal vaginal discharge that is associated with an increased risk of sexually transmitted infections and adverse pregnancy outcomes. Despite its clinical significance, the pathogenesis of BV remains incompletely understood, limiting the effectiveness of current interventions. Although the Gardnerella genus has been proposed to play a pivotal role in BV pathogenesis via biofilm initiation, it was initially considered monotypic. This paradigm shifted with the recognition of species heterogeneity within the Gardnerella genus. Here, we examined in vitro biofilm growth and proteomes of a representative isolate from each of three Gardnerella species: G. vaginalis, G. leopoldii, and G. piotii. G. leopoldii and G. piotii formed more robust biofilms compared to G. vaginalis, with higher biomass, suggesting differences in extracellular matrix composition. Proteomic analysis identified significant differences in functional pathways and virulence proteins among the studied isolates. Cysteine synthase (CysK), implicated in cysteine synthesis and potentially possessing secondary functions, was enriched in the more robust G. leopoldii and G. piotii biofilms, suggesting a possible role in biofilm formation. Cytolysins were more abundant in the thinner G. vaginalis biofilms, whereas immune evasion proteins were more abundant in G. leopoldii biofilms. Adhesion and glycogen-degrading proteins varied in type and abundance across species. These findings reveal functional heterogeneity among the tested isolates and provide insight into how certain Gardnerella isolates establish biofilms. Targeting virulence factors identified in this study may offer novel strategies to disrupt Gardnerella biofilm formation to improve management of BV. IMPORTANCE Bacterial vaginosis (BV) is a leading cause of abnormal vaginal discharge, associated with high post-treatment recurrence. Gardnerella species play a central role in BV via initiation of polymicrobial biofilms, yet species-specific contributions remain poorly understood. This work demonstrates that tested isolates of G. leopoldii and G. piotii form more robust biofilms compared to G. vaginalis and differentially produce proteins associated with immune evasion, adhesion, and biofilm formation. Certain proteins were enriched in the more robust biofilm producers, suggestive of differences that may contribute to strain-specific biofilm formation. By demonstrating fundamental differences among the studied isolate biofilms, these findings highlight Gardnerella heterogeneity and provide a foundation for future studies aimed at targeting Gardnerella biofilm formation in BV. Bacterial vaginosis (BV) is a leading cause of abnormal vaginal discharge, associated with high post-treatment recurrence. Gardnerella species play a central role in BV via initiation of polymicrobial biofilms, yet species-specific contributions remain poorly understood. This work demonstrates that tested isolates of G. leopoldii and G. piotii form more robust biofilms compared to G. vaginalis and differentially produce proteins associated with immune evasion, adhesion, and biofilm formation. Certain proteins were enriched in the more robust biofilm producers, suggestive of differences that may contribute to strain-specific biofilm formation. By demonstrating fundamental differences among the studied isolate biofilms, these findings highlight Gardnerella heterogeneity and provide a foundation for future studies aimed at targeting Gardnerella biofilm formation in BV.