The calcineurin pathway regulates extreme thermotolerance, cell membrane and wall integrity, antifungal resistance, and virulence in Candida auris

Abstract

Candida auris, an emerging fungal pathogen characterized by its multidrug resistance and high mortality rates, poses a significant public health challenge. Despite its importance, the signaling pathways governing virulence and antifungal resistance in C. auris remain poorly understood. This study investigates the calcineurin pathway in C. auris, critical for virulence and antifungal resistance in other fungal pathogens. Calcineurin, a calcium/calmodulin-dependent protein phosphatase, comprises a catalytic subunit (Cna1) and a regulatory subunit (Cnb1) in C. auris. Our findings reveal that deletion of CNA1 or CNB1 disrupts extreme thermotolerance and cell membrane and wall integrity, leading to increased susceptibility to azoles and echinocandins. Moreover, we identified a downstream transcription factor, Crz1, which plays a central role in this pathway in other fungal species. Deletion of CRZ1 resulted in similar membrane integrity defects observed in the cna1{Delta} and cnb1{Delta} mutants and increased susceptibility to azole drugs. Supporting it, fluconazole treatment induced Crz1 nuclear translocation in a Cna1-dependent manner. However, unlike cna1{Delta} and cnb1{Delta} mutants, the crz1{Delta} mutant displayed increased resistance to echinocandins, suggesting the opposing roles for Crz1 in regulating cell wall integrity. Nevertheless, echinocandins also promoted Crz1 nuclear translocation via Cna1, underscoring the complex regulatory mechanisms at play. Cna1 was found to be required for virulence in both the Drosophila systemic infection model and the murine skin infection model. However, in a systemic murine infection model, both calcineurin and Crz1 appeared dispensable for C. auris virulence. Our findings highlight that the evolutionarily conserved calcineurin pathway employs distinct regulatory mechanisms to perform divergent roles in regulating cell wall and membrane integrity, antifungal drug resistance, and virulence in C. auris. Author SummaryThe fungal pathogen Candida auris presents a global health threat due to its multidrug resistance and high mortality rates. Despite its clinical significance, the molecular mechanisms underlying its virulence and antifungal resistance remain poorly understood. This study investigates the complex role of the calcineurin signaling pathway in C. auris pathogenicity. Deletion of the calcineurin complex impairs extreme thermotolerance and compromises cell membrane and wall integrity, leading to increased susceptibility to azoles and echinocandins, antifungal agents targeting the cell membrane and wall, respectively. We also identified the Crz1 transcription factor as a downstream target of calcineurin signaling. Interestingly, unlike calcineurin mutants, Crz1 mutants are susceptible only to cell-membrane-targeting azoles but surprisingly exhibit increased resistance to cell-wall-targeting echinocandins, suggesting that the calcineurin pathway may regulate multiple transcription factors, including Crz1. Additionally, calcineurin was found to be essential for virulence in vivo, using two different animal infection models, Drosophila and mice. These findings highlight the essential role of the calcineurin pathway in C. auris virulence, offering novel insights into its role in antifungal resistance and virulence, and paving the way for targeted therapies.