Calcium binding protein Ncs1 is calcineurin-regulated inCryptococcus neoformansand essential for cell division and virulence

Abstract

AbstractIntracellular calcium (Ca2+) is crucial for signal transduction inCryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signalling 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 non-stress conditions, as thencs1Δmutant is sensitive to a high Ca2+concentration and has an elevated basal Ca2+level that correlates with increased expression of the Ca2+transporter genes,CCH1andMID1. Furthermore,NCS1expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast toSaccharomyces cerevisiae,NCS1expression inC. neoformansis regulated by the calcineurin pathway via the transcription factor Crz1, asNCS1expression is reduced by FK506 treatment andCRZ1deletion. Moreover, thencs1Δmutant shares a high temperature and high Ca2+sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ andcam1Δ) and theNCS1promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1-deficency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data shows that Ncs1 plays distinct roles in Ca2+sensing inC. neoformansdespite widespread functional conservation of Ncs1 and other regulators of Ca2+homeostasis.ImportanceCryptococcus neoformansis the major cause of fungal meningitis in HIV infected patients. Several studies have highlighted the important contribution of Ca2+signalling and homeostasis to the virulence ofC. 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 signalling cascade. Our work provides evidence of a link between Ca2+homeostasis and cell cycle progression inC. neoformans.