Fks1 and Fks2 Are Functionally Redundant but Differentially Regulated in Candida glabrata: Implications for Echinocandin Resistance

The echinocandins caspofungin, micafungin, and anidulafungin, inhibitors of cell wall beta-1,3-glucan synthesis, were recently elevated to first-line agents for treating infections due to the azole-refractory yeast Candida glabrata. In Candida albicans, echinocandin resistance is strictly associated with mutations in Fks1, a large integral membrane protein and putative beta-1,3-glucan synthase, while mutations in both Fks1 and its paralog Fks2 (but not Fks3) have been associated with resistance in C. glabrata. To further explore their function, regulation, and role in resistance, C. glabrata fks genes were disrupted and subjected to mutational analysis, and their differential regulation was explored. An fks1 Delta fks2 Delta double disruptant was not able to be generated; otherwise, all three single and remaining two double disruptants displayed normal growth and echinocandin susceptibility, indicating Fks1-Fks2 redundancy. Selection on echinocandin-containing medium for resistant mutants was dependent on strain background: only fks1 Delta and fks1 Delta fks3 Delta strains consistently yielded mutants exhibiting high-level resistance, all with Fks2 hot spot 1 mutations. Thus, Fks1-Fks2 redundancy attenuates the rate of resistance; further analysis showed that it also attenuates the impact of resistance-conferring mutations. Growth of the fks1 Delta and, especially, fks1 Delta fks3 Delta strains was specifically susceptible to the calcineurin inhibitor FK506. Relatedly, FK506 addition or calcineurin gene CMP2 disruption specifically reversed Fks2-mediated resistance of laboratory mutants and clinical isolates. RNA analysis suggests that transcriptional control is not the sole mechanism by which calcineurin modulates Fks2 activity.