The Crz1/Sp1 Transcription Factor of Cryptococcus neoformans Is Activated by Calcineurin and Regulates Cell Wall Integrity

Abstract

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δcna1, and a CRZ1/SP1 mutant (Δcrz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and Δcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30–37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…These observations are consistent with the finding in S. cerevisiae that the cyclin-dependent kinase complex Pho80-Pho85 and PKA negatively regulate the transcription factor Crz1, the major target of calcineurin (90,91). The Crz1 ortholog in C. neoformans influences growth at high temperature, the response to oxygen limitation, cell wall biosynthesis, fluconazole sensitivity, and biofilm formation in a calcium-dependent manner (92,93). In this context, it is interesting that recent work with Candida glabrata revealed that the aspartyl protease CgYps1 (yapsin 1) influences vacuolar and ion homeostasis, cell wall remodelling, calcineurin sensitivity, and polyphosphate levels (94).…”

Phospholipase C of Cryptococcus neoformans Regulates Homeostasis and Virulence by Providing Inositol Trisphosphate as a Substrate for Arg1 Kinase

“…Our previous findings established that, similar to other fungal species, C. neoformans calcineurin is activated by the addition of exogenous calcium to the growth medium (12). Furthermore, in filamentous fungi, addition of calcium restored some of the defects observed in the ⌬plc1 mutant, such as growth rate in Cryphonectria parasitica, and formation of infection structures (appressoria) in Magnaporthe oryzae (26,27).…”

“…After 4 h of incubation, the cells were treated with calcofluor white (CFW) (2.2 mg/ml) with or without FK506 (10 g/ml) for 1 h. The cells were collected and snap-frozen. RNA extraction and cDNA synthesis were performed as described previously (12). Real-time PCR was performed using the actin-encoding gene (ACT1) for normalization.…”

“…In S. cerevisiae, calcineurin conducts most of its transcriptional regulation via activation of the zinc finger transcription factor Crz1. In C. neoformans, the Crz1 ortholog is also activated by calcineurin and, during starvation, by protein kinase C, to regulate cell wall integrity (12,13).…”

Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach

“…To date, one of the well characterized substrates of calcineurin is the mammalian NFAT ortholog, Crz1, which mediates transcriptional response triggered by calcineurin activation in response to stress conditions in Candida species, C. neoformans 64,[95][96][97] and A. fumigatus. 98,99 As mentioned earlier, another important binding partner of calcineurin is the calcineurin binding protein, Cbp1, an element involved in fine tuning of calcineurin signaling that was also characterized from C. neoformans and A. fumigatus.…”