Use of Haploid Model of to Uncover Mechanism of Action of a Novel Antifungal Agent.

Antifungal agents for the treatment of infections are limited. We recently discovered a novel antifungal small molecule, SM21, with promising activity. Herein, we employed the newly developed haploid toolbox to uncover the mechanism of action of SM21. Comprehensive RNA-Seq analyses of the haploid susceptible GZY803 strain revealed significant gene expression changes related to mitochondria when exposed to SM21. Mitochondrial structure visualization and measurement of ATP generation, reactive oxygen species (ROS) levels, and the antioxidant potential of SM21-treated and untreated GZY803, mitochondrial structure defective haploid mutant (Δ), and wild-type diploid SC5314 strains confirmed defects in mitochondria. Exploiting the advantage of haploids as a single ploidy model, we further exposed GZY803 to repetitive treatments of SM21 in order to generate resistant mutants. Three colonies designated S3, S5 and S6, which displayed resistance to SM21, were isolated. All resistant strains exhibited enhanced transcriptomic responses for peptide and protein metabolism and secreted aspartate proteases (SAPs) activity under SM21 treatment compared to the parent strain GZY803. Consistently, supplementing the resistant strains, GZY803, and SC5314 with peptone, a form of digested peptides, decreased susceptibility to SM21. The present study demonstrates the usefulness of haploid model in antifungal drug discovery. The findings will be invaluable to develop SM21 as a novel antifungal agent, which will benefit millions of patients suffering from infections.