Characterization of the mechanism of action of the Enterococcus faecalis bacteriocin EntV on Candida albicans

Candida albicans shares communal niches with multiple bacterial species. Previous work from our group demonstrated that the Gram-positive bacterium Enterococcus faecalis, a normal constituent of the oral and gut microbiome that is often co-isolated with C. albicans, antagonizes hyphal morphogenesis, biofilm formation, and virulence in C. albicans. These effects are mediated by EntV, a bacteriocin and antimicrobial peptide produced by E. faecalis. The main aim of this work is to unveil the molecular mechanism behind the activity of EntV on C. albicans. Using fluorescence microscopy, we determined that EntV binds to the cell walls of several Candida species, including both yeast and hyphae of C. albicans. Contrary to other antimicrobial peptides, it does not cause cell lysis and does not synergize with cell wall damaging agents. Moreover, we screened a library of C. albicans mutants for strains with altered susceptibility to the peptide; most of the positive hits had functions related to cell wall maintenance and were further screened to ascertain changes in the staining patterns. Furthermore, to identify the target layer on the cell wall, pull-down assays were performed. Mannan was identified as the major wall component able to bind the peptide. Finally, live imaging of macrophages incubated with Candida was carried out in order to assess any change in the phagocytic behaviour in presence of the peptide. Identifying the molecular target of EntV in regard to the anti-virulence mechanisms of C. albicans is an important step in its further development as a therapeutic addition to the classical antifungal agents.