Differentiated extracts from freshwater and terrestrial mollusks inhibit virulence factor production in Cryptococcus neoformans

The human fungal pathogen, Cryptococcus neoformans, is responsible for deadly infections among immunocompromised individuals with the evolution of antifungal resistance driving the solution to discover new compounds that inhibit fungal virulence factors rather than kill the pathogen. Recently, exploration into natural sources (e.g., plants, invertebrates, microbes) of antifungal agents has garnered attention by integrating a One Health approach for new compound discovery. Here, we explore extracts from three mollusk species (freshwater and terrestrial) and evaluate effects against the growth and virulence factor production (i.e., thermotolerance, melanin, capsule, and biofilm) in C. neoformans. We demonstrate that clarified extracts of Planorbella pilsbryi have a fungicidal effect on cryptococcal cells comparable to fluconazole. Similarly, all extracts of Cipangopaludina chinensis affect cryptococcal thermotolerance and impair biofilm and capsule production, with clarified extracts of Cepaea nemoralis also conveying the latter effect. Next, inhibitory activity of extracts against peptidases related to specific virulence factors, combined with stress assays and quantitative proteomics, defined distinct proteome signatures and proposed proteins driving the observed anti-virulence properties. Overall, this work highlights the potential of compounds derived from natural sources to inhibit virulence factor production in a clinically important fungal pathogen.