Calcineurin Is Essential for Virulence in Candida albicans

ABSTRACT Calcineurin is a conserved Ca2+-calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37°C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans Δcmp1 mutants displayed hypersensitivity to elevated Na+, Li+, and Mn2+ concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca2+ concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl2 after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the Δcmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.

[1]  Gerald R. Fink,et al.  Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: Regulation by starvation and RAS , 1992, Cell.

[2]  G. Reboux,et al.  Fluconazole-resistant recurrent oral candidiasis in human immunodeficiency virus-positive patients: persistence of Candida albicans strains with the same genotype , 1994, Journal of clinical microbiology.

[3]  J. O'connor,et al.  Isolation of a Candida albicans gene, tightly linked to URA3, coding for a putative transcription factor that suppresses a Saccharomyces cerevisiae aft1 mutation , 2001, Yeast.

[4]  D. Irwin,et al.  Isogenic strain construction and gene mapping in Candida albicans. , 1993, Genetics.

[5]  F. Rubio,et al.  The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. , 1994, The Journal of biological chemistry.

[6]  N. Morin,et al.  Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase , 1995, Molecular and cellular biology.

[7]  J. Heitman,et al.  Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12–FK506 in Cryptococcus neoformans , 2001, Molecular microbiology.

[8]  H. Prokisch,et al.  Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient , 1997, Molecular and General Genetics MGG.

[9]  M. Cyert,et al.  Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone , 1992, Molecular and cellular biology.

[10]  L Timmermann,et al.  The mechanism of action of cyclosporin A and FK506. , 1996, Clinical immunology and immunopathology.

[11]  D. Dignard,et al.  Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans , 2001, Molecular microbiology.

[12]  Ramón Serrano,et al.  Yeast putative transcription factors involved in salt tolerance , 1998, FEBS letters.

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

[14]  J. Heitman,et al.  Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Sanglard,et al.  Potent Synergism of the Combination of Fluconazole and Cyclosporine in Candida albicans , 2000, Antimicrobial Agents and Chemotherapy.

[16]  A. Gillum,et al.  Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations , 2004, Molecular and General Genetics MGG.

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

[18]  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.

[19]  N. Gow,et al.  Growth and guidance of the fungal hypha. , 1994, Microbiology.

[20]  J. Staab,et al.  Reevaluation of the Role of HWP1 in Systemic Candidiasis by Use of Candida albicans Strains with Selectable Marker URA3 Targeted to the ENO1 Locus , 2002, Infection and Immunity.

[21]  S. Rupp,et al.  The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans , 2000, Molecular microbiology.

[22]  D. Kirsch,et al.  Pathogenicity of Candida albicans auxotrophic mutants in experimental infections , 1991, Infection and immunity.

[23]  J. Ariño,et al.  The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium‐mediated signalling , 2002, Molecular microbiology.

[24]  K. L. Lee,et al.  An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida Albicans. , 1975, Sabouraudia.

[25]  W. Bowen,et al.  Avirulence of Candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis. , 1995, FEMS microbiology letters.

[26]  J. Heitman,et al.  Comparison of the Roles of Calcineurin in Physiology and Virulence in Serotype D and Serotype A Strains ofCryptococcus neoformans , 2000, Infection and Immunity.

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

[28]  D. Sanglard,et al.  Fluconazole plus Cyclosporine: a Fungicidal Combination Effective against Experimental Endocarditis Due to Candida albicans , 2000, Antimicrobial Agents and Chemotherapy.

[29]  J. Hacker,et al.  Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation , 1998, Molecular and General Genetics MGG.

[30]  M. Cyert,et al.  Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[31]  N. Gow,et al.  Ura-status-dependent adhesion of Candida albicans mutants. , 2001, FEMS microbiology letters.

[32]  G. Fink,et al.  Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. , 1994, Science.

[33]  Y. Liu,et al.  Protein phosphatase type 2B (calcineurin)‐mediated, FK506‐sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. , 1993, The EMBO journal.

[34]  T. Davis,et al.  Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase , 1996, Molecular and cellular biology.

[35]  T. C. White,et al.  Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid , 1997, Journal of bacteriology.

[36]  J. Becker,et al.  Altered Expression of Selectable Marker URA3 in Gene-Disrupted Candida albicans Strains Complicates Interpretation of Virulence Studies , 1998, Infection and Immunity.

[37]  G. Fink,et al.  Nonfilamentous C. albicans Mutants Are Avirulent , 1997, Cell.

[38]  K. Cunningham,et al.  Essential role of calcineurin in response to endoplasmic reticulum stress , 2002, The EMBO journal.

[39]  P. Staib,et al.  Sequential gene disruption in Candida albicans by FLP‐mediated site‐specific recombination , 1999, Molecular microbiology.

[40]  J. Ernst.  Transcription factors in Candida albicans - environmental control of morphogenesis. , 2000, Microbiology.

[41]  R. Akada,et al.  TheSaccharomyces cerevisiae genes (CMP1 andCMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B , 1991, Molecular and General Genetics MGG.

[42]  C. Nombela,et al.  Virulence genes in the pathogenic yeast Candida albicans. , 2001, FEMS microbiology reviews.

[43]  T. Kuno,et al.  cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. , 1991, Biochemical and biophysical research communications.

[44]  S. Katiyar,et al.  Antifungal activity in Saccharomyces cerevisiae is modulated by calcium signalling , 2002, Molecular microbiology.

[45]  D. Matheos,et al.  Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. , 1997, Genes & development.

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

[47]  Joseph Heitman,et al.  Calcineurin is essential for survival during membrane stress in Candida albicans , 2002, The EMBO journal.

[48]  D. Hirata,et al.  Protein phosphatase 2B of Saccharomyces cerevisiae is required for tolerance to manganese, in blocking the entry of ions into the cells. , 1995, European journal of biochemistry.

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

[50]  G. Fink,et al.  Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases , 1994, The Journal of cell biology.