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

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.

[1]  J. Heitman,et al.  The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling Circuit during High-Temperature Stress Responses in Cryptococcus neoformans , 2011, Eukaryotic Cell.

[2]  J. Heitman,et al.  Association of Calcineurin with the COPI Protein Sec28 and the COPII Protein Sec13 Revealed by Quantitative Proteomics , 2011, PloS one.

[3]  J. Heitman,et al.  Calcineurin Colocalizes with P-Bodies and Stress Granules during Thermal Stress in Cryptococcus neoformans , 2011, Eukaryotic Cell.

[4]  J. Heitman,et al.  Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis , 2011, Eukaryotic Cell.

[5]  T. Myers,et al.  A Novel Specificity Protein 1 (SP1)-like Gene Regulating Protein Kinase C-1 (Pkc1)-dependent Cell Wall Integrity and Virulence Factors in Cryptococcus neoformans* , 2011, The Journal of Biological Chemistry.

[6]  D. Nilsson,et al.  Cellular stress induces cytoplasmic RNA granules in fission yeast. , 2011, RNA.

[7]  Chung-Yu Lan,et al.  Candida albicans Hap43 Is a Repressor Induced under Low-Iron Conditions and Is Essential for Iron-Responsive Transcriptional Regulation and Virulence , 2010, Eukaryotic Cell.

[8]  R. Dean,et al.  Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus , 2010, PLoS pathogens.

[9]  Yuanchao Wang,et al.  MgCRZ1, a transcription factor of Magnaporthe grisea, controls growth, development and is involved in full virulence. , 2009, FEMS microbiology letters.

[10]  Jongsun Park,et al.  MoCRZ1, a gene encoding a calcineurin-responsive transcription factor, regulates fungal growth and pathogenicity of Magnaporthe oryzae. , 2009, Fungal genetics and biology : FG & B.

[11]  Roy Parker,et al.  P bodies promote stress granule assembly in Saccharomyces cerevisiae , 2008, The Journal of cell biology.

[12]  U. Stahl,et al.  Two zinc finger transcription factors, CrzA and SltA, are involved in cation homoeostasis and detoxification in Aspergillus nidulans. , 2008, The Biochemical journal.

[13]  Jean-Michel Claverie,et al.  Phylogeny.fr: robust phylogenetic analysis for the non-specialist , 2008, Nucleic Acids Res..

[14]  G. Goldman,et al.  Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA , 2008, Molecular microbiology.

[15]  J. Schumacher,et al.  Calcineurin-Responsive Zinc Finger Transcription Factor CRZ1 of Botrytis cinerea Is Required for Growth, Development, and Full Virulence on Bean Plants , 2008, Eukaryotic Cell.

[16]  J. Heitman,et al.  Calcineurin Inhibitor Agents Interact Synergistically with Antifungal Agents In Vitro against Cryptococcus neoformans Isolates: Correlation with Outcome in Solid Organ Transplant Recipients with Cryptococcosis , 2007, Antimicrobial Agents and Chemotherapy.

[17]  S. Harrison,et al.  Structure of calcineurin in complex with PVIVIT peptide: portrait of a low-affinity signalling interaction. , 2007, Journal of molecular biology.

[18]  G. Wagner,et al.  Structure of the calcineurin-NFAT complex: defining a T cell activation switch using solution NMR and crystal coordinates. , 2007, Structure.

[19]  Alistair J. P. Brown,et al.  The PKC, HOG and Ca2+ signalling pathways co‐ordinately regulate chitin synthesis in Candida albicans , 2007, Molecular microbiology.

[20]  J. Heitman,et al.  Cryptococcus neoformans in organ transplant recipients: impact of calcineurin-inhibitor agents on mortality. , 2007, The Journal of infectious diseases.

[21]  T. Kuno,et al.  Real-time monitoring of calcineurin activity in living cells: evidence for two distinct Ca2+-dependent pathways in fission yeast. , 2006, Molecular biology of the cell.

[22]  D. Sanglard,et al.  CRZ1, a target of the calcineurin pathway in Candida albicans , 2006, Molecular microbiology.

[23]  I. F. Larrinoa,et al.  Functional characterization of the Candida albicans CRZ1 gene encoding a calcineurin-regulated transcription factor , 2005, Current Genetics.

[24]  P. R. Kraus,et al.  Identification of Cryptococcus neoformans Temperature-Regulated Genes with a Genomic-DNA Microarray , 2004, Eukaryotic Cell.

[25]  平山 園子.  Zinc finger protein Prz1 regulates Ca[2+] but not Cl[-] homeostasis in fission yeast : Identification of distinct branches of calcineurin signaling pathway in fission yeast , 2003 .

[26]  D. Botstein,et al.  Genome-wide Analysis of Gene Expression Regulated by the Calcineurin/Crz1p Signaling Pathway in Saccharomyces cerevisiae * , 2002, The Journal of Biological Chemistry.

[27]  A. Bruce-Keller,et al.  Polyglutamine Expansion, Protein Aggregation, Proteasome Activity, and Neural Survival* , 2002, The Journal of Biological Chemistry.

[28]  M. Cyert,et al.  Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site. , 2002, Genes & development.

[29]  K. Williams,et al.  The eukaryotic response regulator Skn7p regulates calcineurin signaling through stabilization of Crz1p , 2001, The EMBO journal.

[30]  J. Heitman,et al.  Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans , 2001, The EMBO journal.

[31]  Joseph Heitman,et al.  Synergistic Antifungal Activities of Bafilomycin A1, Fluconazole, and the Pneumocandin MK-0991/Caspofungin Acetate (L-743,873) with Calcineurin Inhibitors FK506 and L-685,818 against Cryptococcus neoformans , 2000, Antimicrobial Agents and Chemotherapy.

[32]  J. Heitman,et al.  Immunosuppressive and Nonimmunosuppressive Cyclosporine Analogs Are Toxic to the Opportunistic Fungal Pathogen Cryptococcus neoformans via Cyclophilin-Dependent Inhibition of Calcineurin , 2000, Antimicrobial Agents and Chemotherapy.

[33]  M. Cyert,et al.  Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. , 1999, Genes & development.

[34]  H. Okamura,et al.  Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT. , 1998, Molecular cell.

[35]  M. Cyert,et al.  Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. , 1997, Genes & development.

[36]  J. Heitman,et al.  Calcineurin is required for virulence of Cryptococcus neoformans , 1997, The EMBO journal.

[37]  J. Perfect,et al.  Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA , 1993, Journal of bacteriology.

[38]  Stuart L. Schreiber,et al.  Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes , 1991, Cell.

[39]  R. Sentandreu,et al.  Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. , 1983, Journal of general microbiology.