Cryptococcus neoformans Ca ²⁺ homeostasis requires a chloride channel/antiporter Clc1 in JEC21, but not in H99

CLC-type chloride/proton antiporters are required for copper/iron homeostasis in fungi. A relationship between CLCs and Ca ²⁺ homeostasis has not been found before. Here we demonstrate the requirement of the antiporter CLC1 for Ca ²⁺ homeostasis/signaling in Cryptococcus neoformans. The deletion of CLC1 in JEC21 resulted in a mutant hypersensitive to cyclosporine A, an inhibitor of calcineurin. Intracellular Ca ²⁺ deficiency in the mutant Tx1 was confirmed with Fluo-3 staining epi-fluorescence microscopy. Tx1 failed to grow at elevated temperature and in SDS and displayed defects in cell wall integrity and cell separation. This defective phenotype is because of Ca ²⁺ deficiency that was restorable by exogenous Ca ²⁺. In contrast, H99 CLC1 was dispensable for Ca ²⁺ homeostasis and had no comparable defective consequences if deleted, suggesting divergent roles of CLCs in Ca ²⁺ homeostasis. Distinct Ca ²⁺ homeostasis mechanisms may contribute the virulence difference between the two strains. This work reveals a novel action of CLC antiporters in fungi and may provide information as to the evolution of pathogenicity among cryptococcal strains.