Cch1 Mediates Calcium Entry in Cryptococcus neoformans and Is Essential in Low-Calcium Environments

Abstract

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37°C to 39°C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca 2؉ /calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low (ϳ50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise (ϳ1 to 10 M) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca 2؉ -permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca 2؉ ] extracellular , ϳ100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca 2؉ ] extracellular (ϳ100 nM) became more severe with increasing temperature (25°C to 38.5°), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca 2؉ ] extracellular , ϳ0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.

Components of the Calcium-Calcineurin Signaling Pathway in Fungal Cells and Their Potential as Antifungal Targets

“…Moreover, hypha formation and maintenance defects, as well as sensitivity to oxidant agents, were identified in the mutant strains, which demonstrates that CCH1 and MID1 play important roles in morphogenesis, the oxidative stress response, and virulence in Candida albicans (86,87). CCH1 also plays a role in mediating the virulence of C. neoformans and is required for the growth of C. neoformans at low extracellular Ca 2ϩ concentrations, especially at mammalian body temperatures (45). In aspergilli, the homologs of CCH1 and MID1, cchA and midA, not only have the functional benefits of fast growth but also play important roles in calcium homeostasis and virulence (50,51).…”

“…The major calcium influx system components CCH1 and MID1 have been identified as important factors in the survival of many fungi (45,48,50,87). Previous reports have demonstrated that CCH1 and MID1 are responsible for the resistance of Candida albicans to azoles, as the deletion of the CCH1 and MID1 genes attenuated the strain's resistance to fluconazole and itraconazole, and the cch1⌬/⌬ or mid1⌬/⌬ mouse models displayed attenuated virulence (85).…”

“…The HACS consists of two putative proteins, Cch1p and Mid1p, which are expressed and colocalize to the plasma membrane in a variety of fungi, such as the saprophytes Schizosaccharomyces pombe and Neurospora crassa (41,42), the animal-pathogenic fungi Candida albicans and Cryptococcus neoformans (43)(44)(45), and the plantpathogenic fungi Gibberella zeae, Claviceps purpurea, and Uromyces appendiculatus (46)(47)(48)(49). Notably, Aspergillus species (50,51) express the putative Ca 2ϩ channel homologs of CCH1 and MID1, namely, cchA and midA, whose topology is similar to the overall topology of Ca 2ϩ voltage-gated channels in higher eukaryotes.…”

Eca1, a Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase, Is Involved in Stress Tolerance and Virulence inCryptococcus neoformans

“…Another component of calcium homeostasis that has been recently identified is the Cch1 voltage-gated Ca 2ϩ channel that is required for calcium uptake in C. neoformans and predicted to be in the plasma membrane (44). Strains with cch1 mutations show sensitivity to calcium chelation, like eca1 mutants, but no temperature sensitivity.…”

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

“…All these data show that the Ca 2ϩ -permeable channels seem to be more involved in fungal development than in the interaction with host plants, with one marked exception: deletion of mid1 in the phytopathogenic, biotrophic ascomycete Claviceps purpurea resulted in a complete loss of virulence (28), demonstrating the different roles Mid1 may play in some fungi. However, all mid1 and cch1 deletion mutants generated so far have one phenotype in common: the inability to grow under low-calcium (about 100 nM) conditions, indicating that they are part of the high-affinity calcium influx system (HACS) (29,30). Besides HACS, there also exists a low-affinity calcium influx system (LACS), with the Ca 2ϩ channel Fig1 as the main component.…”