Global Analysis of the Evolution and Mechanism of Echinocandin Resistance

Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host. We report on a series of C. glabrata isolates that evolved echinocandin resistance in a patient undergoing treatment with the echinocandin caspofungin for recurring bloodstream C. glabrata candidemia over a 10-month period. Whole genome sequencing revealed that a mutation occurred in the drug target, Fks2, accompanying a major increase in resistance, as well as 8 other non-synonymous mutations in 8 genes not previously implicated in echinocandin resistance. The FKS2 mutation was sufficient to confer echinocandin resistance in a sensitive laboratory strain, however, the mutant allele also imparted a growth defect in clinically relevant conditions. Analysis of 10 independent C. glabrata clinical isolates recovered at different time points from separate patients in distinct geographical locations revealed an association between Fks2-mediated resistance and mutations in MOH1 and CDC6. To uncover mechanisms that abrogate echinocandin resistance we turned to Hsp90 and found that pharmacological inhibition of Hsp90 both reduced basal tolerance of C. glabrata and abrogated resistance of clinical isolates. Compromising calcineurin function either pharmacologically or genetically phenocopied compromising Hsp90 function. We propose a model in which Hsp90 and calcineurin mediate echinocandin resistance in C. glabrata by regulating expression of the resistance determinant FKS2, consistent with our findings that caspofungin induced both calcineurin and FKS2 expression and that increased FKS2 expression was dependent upon calcineurin. Furthermore, we report that one of the clinical isolates in the series is a petite mutant due to its morphology and the inability to respire; although the petite phenotype was not intrinsically involved in echinocandin resistance, it imparted resistance to the combination of echinocandins and Hsp90 inhibitors. Thus, our results provide the first global view of mutations that accompany the evolution of fungal drug resistance in a human host, establish novel resistance mutations, and reveal a new molecular mechanism of echinocandin resistance, with broad therapeutic potential.