Unlabelled: Enterococcus faecalis sortase-assembled endocarditis and biofilm-associated pili (Ebp) are virulence factors implicated in enterococcal biofilm-associated infections and gastrointestinal colonization. We previously showed that E. faecalis biofilm metabolism is influenced by extracellular electron transfer (EET) under iron-rich conditions, raising the question of whether Ebp pili also play a role in EET. Here, we report a novel role of Ebp pili in E. faecalis adhesion to the iron oxides magnetite, goethite, and hematite, where the EbpA tip adhesin contributes to this interaction. Adhesion by Ebp pili is conditionally important for EET to iron oxides, as pilus mutants are attenuated in EET under non-static growth conditions. In alignment with the established role of EET in redox homeostasis, we find that EET to ferricyanide supports E. faecalis anaerobic growth on glycerol. Furthermore, in an antibiotic-treated mouse gastrointestinal colonization model, we show that E. faecalis mutants deficient in EET poorly colonize the intestinal niche. Taken together, our findings suggest that Ebp pili can influence E. faecalis metabolic fitness by promoting EET to iron oxides, raising new questions about how Ebp pili shape E. faecalis interactions with environmental ecosystems. Additionally, the important role of EET in E. faecalis colonization of the dysbiotic gastrointestinal environment highlights the need for further inquiry into how EET contributes to E. faecalis microbial pathogenesis.

Importance: In this study, we explored the interplay between extracellular electron transfer (EET) and an Enterococcus faecalis biofilm factor, the endocarditis and biofilm-associated pili (Ebp). We demonstrate that Ebp pili have a novel role in adhesion to iron oxides, which consequently promotes EET to iron oxides under non-static conditions. Along with our findings that E. faecalis EET can be coupled to anaerobic cell growth, our results point to a potential ecological role of Ebp pili in natural environments, outside of its established function in adhesion to host ligands. We provide the first evidence of the contribution of EET to E. faecalis colonization of the antibiotic-treated murine intestinal niche, which adds to the limited experimental evidence linking EET and microbial pathogenesis, as well as highlights the need for further studies of EET in bacterial pathogens.