Biochemical Characterization of Recombinant <i>Enterococcus faecalis</i> EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against <i>Candida albicans</i>

<i>Candida albicans</i> is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit <i>C. albicans</i> hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, <i>Enterococcus faecalis</i>, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against <i>C. albicans</i> in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on <i>C. albicans</i> has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV⁸⁸-His₆ on a large preparative scale, providing facile access to the C-terminal EntV construct. Under <i>in vitro</i> <i>C. albicans</i> hyphal assay with specific inducers, we demonstrated that EntV⁸⁸-His₆ exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV⁸⁸-His₆, we revealed that EntV⁸⁸-His₆ enters <i>C. albicans</i> via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV⁸⁸-His₆ does not affect <i>C. albicans</i> yeast cell growth but potently exerts cytotoxicity against <i>C. albicans</i> under hyphal-inducing conditions <i>in vitro</i>. The combination of EntV⁸⁸-His₆ and GlcNAc displays rapid killing of <i>C. albicans</i>, rendering it a promising antivirulent and antifungal agent.