Dissecting the Roles of the Calcineurin Pathway in Unisexual Reproduction, Stress Responses, and Virulence in Cryptococcus deneoformans

Abstract

The Ca/calmodulin-dependent protein phosphatase calcineurin orchestrates sexual reproduction, stress responses, and virulence via branched downstream pathways in the opportunistic human fungal pathogen The calcineurin-binding protein Cbp1, the calcineurin temperature suppressor Cts1, the calcineurin-responsive zinc finger transcription factor Crz1, and the calcineurin targets Pbp1, Tif3, and Puf4, all function downstream of calcineurin to orchestrate distinct cellular processes. To elucidate how the calcineurin pathway regulatory network governs unisexual reproduction, stress responses, and virulence, we have analyzed the self-filamentous strain, XL280α, and generated double mutants of these calcineurin downstream genes. We demonstrated that calcineurin governs unisexual reproduction at different sexual developmental stages, in which the initiation of the yeast-hyphal morphological transition is independent of Crz1, whereas the sporulation process is dependent on Crz1. Calcineurin-dependent unisexual reproduction is independent of the pheromone response pathway. Crz1 synergistically interacts with different calcineurin downstream targets in responding to ER, high-calcium, and cell wall stresses. We observed a widespread synergy suggesting that these proteins function in complex branched pathways downstream of calcineurin with some functional redundancy, which may allow efficient signaling network rewiring within the pathway for prompt adaptation to changing environments. Finally, we showed that deletion of or in the ∆ mutant background conferred a modest level of growth tolerance at 37°, but that the∆ ∆ and∆ ∆ double mutants were both avirulent, suggesting that calcineurin may control virulence via mechanisms beyond thermotolerance.

RNA sequencing reveals an additional Crz1-binding motif in promoters of its target genes in the human fungal pathogen Candida albicans

“…Similar to the homologs in S. cerevisiae, M. oryzae and another human fungal pathogen Aspergillus fumigatus [49], expression of PMC1 (C3_ 01250W_A) and RCT1 (C3_05710W) is positively controlled by CaCrz1, although expression of RCN1 (C6_ 01160W_A) is not regulated by CaCrz1 (SuppInfo 1 and 2; GEO Accession number: GSE123122). This is consistent with previous observations on Cryptococcus neoformans CBP1, the homolog of RCN1, that neither is regulated by nor interacts with Crz1 in this human fungal pathogen [54,55]. In contrast, expression of RCN1 is regulated by Crz1 in S. cerevisiae, M. oryzae and another human fungal pathogen Aspergillus fumigatus, which forms a feedback mechanism for the regulatory role of Rcn1 as an inhibitor of calcineurin [48,55,56].…”

Zinc Finger Proteins in the Human Fungal Pathogen Cryptococcus neoformans

“…Crz1(the calcineurin-responsive zinc finger transcription factor 1) is a downstream target of the calcineurin pathway and orchestrates distinct cellular processes in C. neoformans. Fu et al demonstrated that Crz1 is essential for the sporulation process although it is not required for yeast-hyphal morphological transition during unisexual reproduction of C. neoformans [68]. Furthermore, Jung et al showed that Crz1 plays roles in fungal virulence and stress survival in C. neoformans [69].…”

Ser/Thr protein phosphatases in fungi: structure, regulation and function

“…This same work identified a subset of calcineurin-regulated genes that, in response to a 24 to 37°C shift, were not dependent on the presence of Crz1, as well as a smaller number of genes dependent on Crz1 but not on calcineurin [313]. A recent study demonstrated that in Cryptococcus deneoformans the initiation of the yeast-hyphal morphological transition is independent of Crz1, whereas the sporulation process is dependent on the transcription factor [314].…”