Abstract
While pathogenic fungi can acquire resistance to the current arsenal of antifungals through genetic mutations, heteroresistance has emerged as an important new cause of therapeutic failures. Heteroresistance is generally thought to arise in small subpopulations that display phenotypic resistance to antifungals without genetic mutations. This study blurs that line by showing gain-of-resistance mutations in FKS2 , which encodes a target of echinocandins and fungerps, cause large amounts of heteroresistance in Candida glabrata through heterogeneous expression of the gene even in clonal cell populations. Heteroresistance decreased when stress-responsive transcription factors (Crz1, Rlm1) were eliminated and was nearly abolished when the upstream regulators (calcineurin, Slt2) were mutated or inhibited. Identical gain-of-resistance mutations in FKS1 , a paralog of FKS2 , showed much less heteroresistance due to its constitutive expression coupled with variable levels of antagonism by wild-type FKS2 . A genome-wide screen using Tn-seq revealed additional regulators of heteroresistance and resistance including IRA1 , an inhibitor of the Ras1-PKA signaling pathway that senses glucose availability. IRA1 increased expression of FKS2 and decreased expression of FKS1 , which increased heteroresistance and decreased resistance, respectively, when these genes carried resistance mutations. Similar principles may govern heteroresistance in other fungal pathogens such as Candida parapsilosis , which naturally carries resistance mutations in FKS1 and frequently exhibits heteroresistance to echinocandins, and Candidozyma auris , which easily acquires such mutations.