Calcineurin‐Dependent Stress Adaptation Enables Caspofungin Heteroresistance Leading to Stable Resistance in Candida Glabrata

ABSTRACT Antifungal heteroresistance has emerged as a clinical challenge across diverse species. In Candida glabrata , however, it has rarely been reported. Here, we characterized heteroresistance in a multicenter collection of 156 C. glabrata isolates, revealing a 25% prevalence with caspofungin specificity and 37°C dependent phenotypes. Transcriptomic profiling of the heteroresistant subpopulation under escalating drug pressure revealed an adaptive program centered on cell cycle and cell wall integrity, including several members of the calcineurin pathway. Mechanically, we identified that the phenotype was abolished by pharmacological inhibition or Δ cnb1 , Δ crz1 genetic deletion, confirming the regulatory role of calcineurin. Crucially, heteroresistance functioned as a reservoir for resistance, with in vitro descendant strains spanning a spectrum of MIC alterations. Mechanistically, we found the heteroresistance phenotype mostly independent of aneuploidy. To decipher the genetic basis, we applied an unbiased machine‐learning framework to genomic data, which not only identified the canonical FKS2_F659del mutation but also prioritized novel candidate PIR2_G149_I167del , demonstrating its power to uncover drivers of resistance from complex datasets. In summary, our study established a stepwise model of heteroresistance in C. glabrata , wherein calcineurin serves as a master regulator that promotes a resistance reservoir, revealing a potential vulnerability that could be exploited to prevent treatment failure.