Calcium signaling components in the human pathogen Cryptococcus neoformans

Abstract

Calcium signaling through calmodulin and the phosphatase calcineurin are required for key events of the biology of the human pathogen Cryptococcus neoformans, including mating, morphogenesis, growth at 37°C and virulence. In a recent work we described the functional characterization of a new component of this calcium signaling network: the vacuolar calcium exchanger Vcx1. This transporter is involved in calcium tolerance and virulence in C. neoformans. Two other uncharacterized calcium transporters which are putative orthologs of Saccharomyces cerevisiae PMC1 (a vacuolar calcium ATPase) and PMR1 (a Golgi calcium ATPase) are also functional in C. neoformans. No ortholog of CRZ1, the target of calcineurin in other fungi, has been identified in C. neoformans, indicating a high complexity in cryptococcal calcium-related pathways. Future studies are necessary for the complete understanding of calcium signaling regulation in C. neoformans.

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The calcium-calcineurin signaling pathway in Cryptococcus neoformans is fundamental for sensing and adaptation to the human host milieu.1 In our recent work, a new component of the C. neoformans Ca²⁺ signaling network was characterized: the vacuolar calcium exchanger Vcx1. Mutant cells lacking Vcx1 expression had altered calcineurin-dependent Ca²⁺ tolerance and a reduced ability to kill mice. The loss of VCX1 gene activity did not influence cell wall integrity or capsule size, but resulted in decreased secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) to culture supernatants. The Vcx1 knockout strain was also more susceptible to killing by murine macrophages.2 The cellular and molecular connections that could explain the relationship of calcium transport with polysaccharide secretion remain to be elucidated.

The phosphatase calcineurin, the major component of the calcium signaling pathway, is required for several crucial events of the C. neoformans biology, such as mating, morphogenesis, growth at 37°C and virulence.3–7 C. neoformans calmodulin, which senses cytosolic calcium and activates calcineurin, is essential for viability and acts in response to high temperature.1 Besides Vcx1, two important calcium transporters, Cch1 and EcaI, were described in reference 8 and 9. The plasma membrane calcium channel Cch1 mediates calcium entry in C. neoformans cells and is required for calcium uptake in low-calcium environments.8 The sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase EcaI participates in stress tolerance.9 All these components, Vcx1, Cch1 and EcaI, are involved in C. neoformans virulence, making clear the importance of calcium transport in fungal virulence.2,8,9 In fact, calcium-related pathways have been associated to key pathogenic steps in important fungal pathogens, including Candida albicans and Aspergillus fumigatus.10,11 Searches in the C. neoformans genome database (www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) revealed the presence of two putative orthologs of well-characterized calcium transporters of Saccharomyces cerevisiae, PMC1 (a vacuolar calcium ATPase) and PMR1 (a Golgi calcium ATPase).12,13 In C. neoformans, we demonstrated that the PMC1 ortholog is upregulated in the Vcx1 knockout strain, probably due to a compensatory effect. We speculate that this observation is related to the fact that PMC1 also transports calcium into vacuoles, generating functional redundancy. This finding indicates the existence of a complex system that regulates calcium transport, including the participation of uncharacterized C. neoformans calcium transporters.

The transcriptional regulation of the calcium-calcineurin signaling pathway has been extensively studied in filamentous fungi and yeasts.14–20 The zinc finger transcription factor Crz1 is the molecular target of calcineurin, that mediates nuclear translocation of Crz1 after dephosphorylation.19 Orthologs of S. cerevisiae Crz1 are involved in virulence and pathogenesis in several plant and human fungal pathogens, such as Magnaporthe oryzae, Botrytis cinerea, A. fumigatus and Candida glabrata.17,18,21–23 Up to now, no ortholog of the CRZ1 gene has been identified in C. neoformans. Probably, C. neoformans contains diverse or more than one transcription factor responsive to calcineurin.1 This scenario suggests that the characterization of potential Crz1 orthologs and/or novel calcium transporters is necessary to a broader understanding of the complex regulation of calcium signaling in the human pathogen C. neoformans.

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