Whole genome sequencing of C. neoformans H99 suppressor strains of the RAM pathway downstream kinase Cbk1 knockout mutant

Through natural evolution, we isolated multiple suppressor strains of the conserved RAM pathway downstream kinase Cbk1 knockout mutant. By whole genome sequencing and comparative genomics, we found multiple distinct mutations in the RNA-binding protein encoding gene SSD1 among the suppressor strains. Indeed, deletion of SSD1 in cbk1D was able to partially restore CO2 tolerance, thermotolerance, and morphological defects in vitro. Deletion of SSD1 also partially restored cbk1D's defect in virulence in both inhalation and intravenous infection mouse models of cryptococcosis. The findings of this study suggest that CO2 tolerance is co-regulated with other virulence factors such as morphology and thermotolerance, and highlights the essential role of the RAM pathway in adaptation to the host environment in Cryptococcus.