Calcium Binding Protein Ncs1 Is Calcineurin Regulated in Cryptococcus neoformans and Essential for Cell Division and Virulence

Abstract

Intracellular calcium (Ca²⁺) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca²⁺-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca²⁺ binding mediated by the EF-hand domains of the Ca²⁺ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca²⁺ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca²⁺ concentration and has an elevated basal Ca²⁺ level. Furthermore, NCS1 expression is induced by Ca²⁺, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca²⁺ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca²⁺ sensor in C. neoformans, working with calcineurin to regulate Ca²⁺ homeostasis and, consequently, promote fungal growth and virulence.IMPORTANCECryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca²⁺ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca²⁺ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca²⁺ homeostasis and cell cycle progression in C. neoformans.

Keywords: Cryptococcus neoformans; calcium binding protein; calcium sensor; calcium signaling.