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

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Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV 88 –His 6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV 88 –His 6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV 88 –His 6, we revealed that EntV 88 –His 6 enters C. albicans 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 88 –His 6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro . The combination of EntV 88 –His 6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

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