Natural compounds from freshwater mussels disrupt fungal virulence and overcome fluconazole resistance in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen responsible for fatal infections, especially in patients with a depressed immune system. Overexposure to antifungal drugs due to prolonged treatment regimens and structure-similar applications in agriculture have weakened the efficacy of current antifungals in the clinic. The rapid evolution of antifungal resistance urges the discovery of new compounds that inhibit fungal virulence factors, rather than directly killing the pathogen as alternative strategies to overcome disease and reduce selective pressure towards resistance. Recent studies, including our own, have highlighted the antimicrobial properties of natural sources, such as invertebrates, against human fungal pathogens, including C. neoformans, through virulence factor impairment. Here, we evaluated the efficacy of freshwater mussel extracts (crude and clarified) against virulence factor production (i.e., thermotolerance, melanin, capsule, and biofilm) in C. neoformans. Similarly, we demonstrated the critical potential of these extracts to increase the susceptibility of the pathogen to fluconazole across resistant strains, overcoming a globally devastating problem. Additionally, we measured the inhibitory activity of the extracts against peptidases related to fungal virulence and drug resistance. Furthermore, we integrated these phenotypic findings with quantitative proteomics profiling to define distinct signatures of each treatment and validated a new mechanism of anti-virulence action for a selected extract. By understanding the mechanisms driving the antifungal activity of mussels, we may develop innovative treatments for fungal infections lacking susceptibility to conventional drugs.