Biochemical Characterization of Recombinant EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against .

is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, , secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV-His on a large preparative scale, providing facile access to the C-terminal EntV construct. Under hyphal assay with specific inducers, we demonstrated that EntV-His exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV-His, we revealed that EntV-His enters 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 EntV-His does not affect yeast cell growth but potently exerts cytotoxicity against under hyphal-inducing conditions . The combination of EntV-His and GlcNAc displays rapid killing of , rendering it a promising antivirulent and antifungal agent.