The antifungal mechanism of EntV-derived peptides is associated with a reduction in extracellular vesicle release.

Candida albicans, an opportunistic fungal pathogen, causes systemic and superficial infections, especially in immunocompromised patients. Treatment of fungal infections is complicated by limited antifungal options and the development of drug resistance. Previous work from our group demonstrated the efficacy of the anti-virulence peptide EntV and shorter variants against C. albicans infection in various animal models, including mouse models of oropharyngeal candidiasis and disseminated infection and a rat venous catheter model. However, the mechanism of action, which abrogates fungal virulence without fungicidal or fungistatic activity, has remained unknown. We used a combination of cell biological, biochemical, genomic, and genetic approaches to identify this mechanism. We demonstrate that EntV-based peptides bind to the fungal cell envelope in a punctate and dynamic manner, co-localizing with extracellular vesicles (EVs), which play a critical role in fungal biofilm formation and virulence. Transcriptomic and genetic analyses further indicate that this activity is linked to the intracellular vesicular trafficking machinery, especially the ESCRT pathway, as mutations in this pathway alter sensitivity to EntV peptides and regulate virulence. We also show that EntV treatment significantly reduces EV secretion in C. albicans, supporting a novel mechanism of antifungal action through inhibition of EV-mediated virulence. These findings further develop EntV as a promising anti-virulence agent with potential for therapeutic development against drug-resistant fungal pathogens.