Suppressor screen to decipher a novel fungal virulence determinant
Cryptococcus neoformans is a pathogenic yeast and the most common cause of human fungal meningitis. Through systematic fitness screens in a murine pulmonary infection model, we identified a previously uncharacterized virulence protein, Rgh1, required for mammalian infectivity. Despite a strong requirement for RGH1 during in vivo infection, yeast lacking RGH1 displayed no growth defects in >100 in vitro growth environments except tissue culture (DMEM, pH 7.4, 37C, 5% CO2). To decipher the function of RGH1, we performed an ethyl methanesulfonate (EMS) mutagenesis screen to identify suppressors of the rgh1 phenotype. We isolated 3 mutants in the rgh1-null background capable of growth in tissue culture. We performed whole-genome nanopore sequencing and SNV calling to identify potentially causative mutations. We found that loss-of-function mutations in a putative cell wall alpha-amylase, AMY1, partially restored growth of rgh1-null mutants in tissue culture. This finding enables testing of mechanistic models for Rgh1's function during mammalian infection.