Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Eukaryotic Cell (Impact Factor: 3.18). 06/2011; 10(6):803-19. DOI: 10.1128/EC.00310-10
Source: PubMed


Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic
fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast
to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly
demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity,
hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin
is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination
therapy with azoles or echinocandins against emerging C. dubliniensis infections.

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    • "For example in S. cerevisiae, calcineurin/Crz1 regulates expression of genes encoding ion pumps required for distinct aspects of ion homeostasis: PMC1 encodes a Ca2+-ATPase responsible for calcium sequestration in vacuoles, and PMR1 similarly transports Ca2+ and Mn2+ into the Golgi; ENA1, ENA2 and ENA5 encode plasma membrane Na+-ATPase exporters [9]. Orthologs of these genes are also regulated by calcineurin and Crz1 in C. albicans [21], C. dubliniensis [29], M. oryzae [14] and A. fumigatus [13]. Genes encoding chitin synthases (CHS), which are essential for maintaining cell wall integrity, are regulated by calcineurin and Crz1 in C. albicans and S. cerevisiae, and in the filamentous fungi M. oryzae and A. nidulans [9], [14], [15], [31], [32]. "
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    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.
    PLoS ONE 12/2012; 7(12):e51403. DOI:10.1371/journal.pone.0051403 · 3.23 Impact Factor
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    • "In Saccharomyces cerevisiae [10], [17]–[19] and Schizosaccharomyces pombe [20], the importance of CN has been established in various stress responses including high temperature, high concentrations of ions, cell wall stress and exposure to mating pheromone. In the human pathogens Candida albicans, Candida dubliniensis and Aspergillus fumigatus, the CN-dependent cascade controls cell wall integrity, stress resistance and response, morphogenesis, serum survival and virulence [4], [21]–[26]. For C. albicans it has also been shown that CN is a key mediator of Hsp90-dependent azole resistance [6]. "
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    ABSTRACT: In the gray mold fungus Botrytis cinerea the Gα subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/calmodulin-dependent phosphatase calcineurin. In this study we focused on the functional characterization of the catalytic subunit of calcineurin (BcCnA) and its putative regulator calcipressin (BcRcn1). We deleted the genes encoding both proteins to examine their role concerning growth, differentiation and virulence. The ΔbccnA mutant shows a severe growth defect, does not produce conidia and is avirulent, while the loss of BcRcn1 caused retardation of hyphal growth and delayed infection of host plants, but had no impact on conidiation and sclerotia formation. Expression of several calcineurin-dependent genes and bccnA itself is positively affected by BcRcn1. Complementation of the Δbcrcn1 mutant with a GFP-BcRcn1 fusion construct revealed that BcRcn1 is localized in the cytoplasm and accumulates around the nuclei. Furthermore, we showed that BcCnA physically interacts with BcRcn1 and the regulatory subunit of calcineurin, BcCnB. We investigated the impact of several protein domains characteristic for modulation and activation of BcCnA via BcRcn1, such as the phosphorylation sites and the calcineurin-docking site, by physical interaction studies between BcCnA and wild-type and mutated copies of BcRcn1. Based on the observed phenotypes we conclude that BcRcn1 acts as a positive modulator of BcCnA and the Ca(2+)/calcineurin-mediated signal transduction in B. cinerea, and that both proteins regulate fungal development and virulence.
    PLoS ONE 07/2012; 7(7):e41761. DOI:10.1371/journal.pone.0041761 · 3.23 Impact Factor
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    • "This result suggests that inhibition of Ca 2+ tolerance by calcineurin is conserved in S. cerevisiae and C. glabrata. In contrast, C. albicans and C. dubliniensis calcineurin mutants are hypersensitive to Ca 2+ (Chen et al. 2011; Sanglard et al. 2003), suggesting a divergent role of calcineurin in Ca 2+ tolerance between C. albicans/C. dubliniensis and C. glabrata/S. "
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    ABSTRACT: Candida glabrata is an emerging human fungal pathogen that is frequently drug tolerant, resulting in difficulties in treatment and a higher mortality in immunocompromised patients. The calcium-activated protein phosphatase calcineurin plays critical roles in controlling drug tolerance, hyphal growth, and virulence in diverse fungal pathogens via distinct mechanisms involving survival in serum or growth at host temperature (37° and higher). Here, we comprehensively studied the calcineurin signaling cascade in C. glabrata and found novel and uncharacterized functions of calcineurin and its downstream target Crz1 in governing thermotolerance, intracellular architecture, and pathogenesis in murine ocular, urinary tract, and systemic infections. This represents a second independent origin of a role for calcineurin in thermotolerant growth of a major human fungal pathogen, distinct from that which arose independently in Cryptococcus neoformans. Calcineurin also promotes survival of C. glabrata in serum via mechanisms distinct from C. albicans and thereby enables establishment of tissue colonization in a murine systemic infection model. To understand calcineurin signaling in detail, we performed global transcript profiling analysis and identified calcineurin- and Crz1-dependent genes in C. glabrata involved in cell wall biosynthesis, heat shock responses, and calcineurin function. Regulators of calcineurin (RCN) are a novel family of calcineurin modifiers, and two members of this family were identified in C. glabrata: Rcn1 and Rcn2. Our studies demonstrate that Rcn2 expression is controlled by calcineurin and Crz1 to function as a feedback inhibitor of calcineurin in a circuit required for calcium tolerance in C. glabrata. In contrast, the calcineurin regulator Rcn1 activates calcineurin signaling. Interestingly, neither Rcn1 nor Rcn2 is required for virulence in a murine systemic infection model. Taken together, our findings show that calcineurin signaling plays critical roles in thermotolerance and virulence, and that Rcn1 and Rcn2 have opposing functions in controlling calcineurin signaling in C. glabrata.
    G3-Genes Genomes Genetics 06/2012; 2(6):675-91. DOI:10.1534/g3.112.002279 · 3.20 Impact Factor
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