Affiliation:
1. Departments of Molecular Genetics and Microbiology
2. Medicine
3. Pharmacology and Cancer Biology
4. and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710
Abstract
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
/
cam1
Δ 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 Ca
2+
-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 Ca
2+
- and calcineurin-independent and -dependent roles in controlling
C. neoformans
morphogenesis and high-temperature growth.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
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