Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans

Abstract

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

Cryptococcal dissemination to the central nervous system requires the vacuolar calcium transporter Pmc1

“…Recently, Chow et al () demonstrated that the calcineurin signalling pathway positively regulates the transcription of VCX1 and PMC1 . In agreement with this finding, our data showed that VCX1 and PMC1 were indeed regulated by calcineurin and also that the EcaI transporter was positively regulated by this pathway.…”

“…Recently, Chow et al (2017) demonstrated that the calcineurin signalling pathway positively regulates the transcription of VCX1 and PMC1. In agreement with this finding, our data showed that VCX1…”

“…Nuclear Crz1 then stimulates the expression of calcineurin-responsive genes, the products of which regulate Ca 2+ homeostasis and stress tolerance (Lev, Desmarini, Chayakulkeeree, Sorrell, & Djordjevic, 2012;Park et al, 2016;Stathopoulos-Gerontides, Guo, & Cyert, 1999;Yoshimoto et al, 2002). However, a recent study showed that Crz1 can activate some of its downstream targets independently of calcineurin activation (Chow et al, 2017). Furthermore, calcineurin can function at the post-transcriptional level through activation of RNA-binding proteins such as Pbp1, a poly(A)-binding protein that localises in P-bodies and regulates sexual reproduction in C. neoformans upon calcineurin activation (Chow et al, 2017).…”

Roles for stress response and cell wall biosynthesis pathways in caspofungin tolerance inCryptococcus neoformans

“…The Crz1 transcription factor is activated by the calcineurin protein, and 289 Crz1 mediates many of the known effects of this calcineurin signaling. Accordingly, we assessed 290 whether an mCherry-tagged Crz1 fusion protein (Crz1-mCherry) localizes to the nucleus after 291 treatment with caspofungin (Chow et al 2017). We assessed Crz1 nuclear localization by 292 examining Crz1-mCherry co-localization with the GFP-Nop1 nucleolar marker as well as with 293 DAPI staining.…”

Leveraging Fungal Calcineurin-Inhibitor Structures, Biophysics and Dynamics to Design Selective and Non-Immunosuppressive FK506 Analogs

“…The immunosuppressants first bind to their respective immunophilins, FKBP12 (12-kDa FK506 binding protein) and CypA (cyclophilin A), which subsequently bind to calcineurin in a groove between the CnA and CnB subunits. The immunophilin-immunosuppressant complex inhibits calcineurin serine-threonine phosphatase activity blocking the dephosphorylation of downstream targets, such as the human nuclear transcription factor NFAT involved in T-cell activation and interleukin-2 transcription, and the fungal transcription factor Crz1 (NFAT ortholog), implicated in virulence, stress response, and thermotolerance [6][7][8][9][10] . In humans, this leads to potent immunosuppression and is critical in preventing graft rejection, but also precludes FK506 and CsA usage as antifungals in already immunocompromised patients.…”