Calcium- and Calcineurin-Independent Roles for Calmodulin in Cryptococcus neoformans Morphogenesis and High-Temperature Growth

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Abstract
The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37°C. One insertion mutant, cam1-ts, that displayed a growth defect at 37°C and hypersensitivity to the calcineurin inhibitor FK506 at 25°C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3′ untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37°C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Δ diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37°C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37°C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.