Mapping the Cryptococcus neoformans Hsp90 genetic interaction network identifies Ypk1 and Pkh202 as client proteins of the molecular chaperone

Hsp90 is a conserved molecular chaperone that orchestrates fungal stress responses by stabilizing proteins important for antifungal susceptibility and virulence. While targeting fungal-specific components within Hsp90 networks offers an attractive therapeutic strategy, the Hsp90 interaction network remains enigmatic in Cryptococcus neoformans , a leading cause of fatal meningitis in immunocompromised individuals. Here, we screened a C. neoformans mutant library for hypersensitivity to Hsp90 inhibitors. We identified 12 chemical-genetic interactors of Hsp90. Among these genes was YPK1 , a kinase that we showed physically associated with Hsp90 along with its upstream kinase Pkh202. While Hsp90 depletion destabilized Pkh202, it did not reduce Ypk1 protein abundance. Furthermore, we discovered that the Hsp90 genetic interactor CYC8 contributes to virulence in a mouse model of infection. Our findings define components of the Hsp90 genetic-interaction network and the first clients of Hsp90 in C. neoformans , revealing a relationship between Hsp90 genetic interactors and virulence in this fungal pathogen.