Dana A. Davis

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T‐cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1‐1/CNB1) abolished FK506–fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole‐resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.

RIM101-Dependent and -Independent Pathways Govern pH Responses in Candida albicans

ABSTRACT Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101,RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1and PHR1, and repression of PHR2. We find thatRIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation inC. albicans through both RIM101-dependent andRIM101-independent pathways.

Candida albicans RIM101 pH Response Pathway Is Required for Host-Pathogen Interactions

ABSTRACT The ability of Candida albicans to respond to diverse environments is critical for its success as a pathogen. TheRIM101 pathway controls gene expression and the yeast-to-hyphal transition in C. albicans in response to changes in environmental pH in vitro. In this study, we found that theRIM101 pathway is necessary in vivo for pathogenesis. First, we show thatrim101−/rim101− andrim8−/rim8− mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis. Second, these mutants show a marked reduction in kidney pathology. Third, therim101−/rim101− andrim8−/rim8− mutants show defects in the ability to damage endothelial cells in situ. Finally, we show that an activated allele of RIM101, RIM101-405, is a suppressor of the rim8− mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the rim8−/rim8−mutant. These results demonstrate that the RIM101 pathway is required for C. albicans virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.

A recyclable Candida albicans URA3 cassette for PCR product‐directed gene disruptions

For some time, gene disruptions in Candida albicans have been made with the hisG‐URA3‐hisG (‘Ura‐blaster’) cassette, which can be re‐used in successive transformations of a single strain after homologous excision of URA3. However, the hisG repeats are too large for efficient PCR amplification of the entire cassette, so it cannot be used for PCR product‐directed gene disruptions. We describe here a gene disruption cassette, URA3‐dpl200, with 200 bp flanking repeats that permit efficient PCR amplification. After transformation and integration to produce both arg5::URA3‐dpl200 and rim101::URA3‐dpl200 alleles, we find that arg5::dpl200 and rim101::dpl200 segregants, respectively, can be obtained. We have used the cassette to create rim101::dpl200/rim101::URA3‐dpl200 mutants exclusively through PCR product‐directed disruption. Copyright

Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p

The human pathogen Candida albicans grows and colonizes sites that can vary markedly in pH. The pH response in C. albicans is governed in part  by the Rim101p pathway. In Saccharomyces cerevisiae, Rim101p promotes alkaline responses by repressing expression of NRG1, itself a transcriptional repressor. Our studies reveal that in C. albicans, Rim101p‐mediated alkaline adaptation is not through repression of CaNRG1. Furthermore, our studies suggest that Rim101p and Nrg1p act in parallel pathways to regulate hyphal morphogenesis, an important contributor to virulence. To determine the wild‐type C. albicans transcriptional response to acidic and alkaline pH, we utilized microarrays and identified 514 pH‐responsive genes. Of these, several genes involved in iron acquisition were upregulated at pH 8, suggesting that alkaline pH induces iron starvation. Microarray analysis of rim101–/– cells indicated that Rim101p does not govern transcriptional responses at acidic pH, but does regulate a subset of transcriptional responses at alkaline pH, including the iron acquisition genes. We found that rim101–/– cells are sensitive to iron starvation, which suggests that one important aspect of the Rim101p‐dependent alkaline pH response is to adapt to iron starvation conditions.

Adaptation to environmental pH in Candida albicans and its relation to pathogenesis

Abstract. For microorganisms that grow over a wide range of extracellular pH, systems must have evolved to sense and respond appropriately. The human opportunistic pathogen Candida albicans colonizes and infects anatomical sites of diverse pH, including the oral and gastro-intestinal tracts and the vaginal cavity. The ability to sense and respond to neutral-alkaline environments is governed by signal transduction pathways, one of which culminates in the activation of the transcription factor, Rim101p. The RIM101/pacC pathway, which governs pH responses and differentiation, has been the focus of study in both Saccharomyces cerevisiae and Aspergillus nidulans. This pathway has been identified in C. albicans and governs pH responses, dimorphism, and pathogenesis. Although C. albicans and S. cerevisiae are related fungi, it is becoming apparent that there are unique aspects of the pH response and the role the RIM101 pathway plays in this response in C. albicans.

How human pathogenic fungi sense and adapt to pH: the link to virulence.

The ability of fungal pathogens to cause disease is dependent on the ability to grow within the human host environment. In general, the human host environment can be considered a slightly alkaline environment, and the ability of fungi to grow at this pH is essential for pathogenesis. The Rim101 signal transduction pathway is the primary pH sensing pathway described in the pathogenic fungi, and in Candida albicans, it is required for a variety of diseases. As more detailed analyses have been conducted studying pathogenesis at the molecular level, it has become clear that the Rim101 pathway, and pH responses in general, play an intimate role in pathogenesis beyond simply allowing the organism to grow. Here, several recent advances into Rim101-dependent functions implicated in disease progression are discussed.

Roles of Candida albicans Dfg5p and Dcw1p cell surface proteins in growth and hypha formation.

ABSTRACT The Candida albicans cell wall participates in both growth and morphological transitions between yeast and hyphae. Our studies here focus on Dfg5p and Dcw1p, two similar proteins with features of glycosylphosphatidylinositol-linked cell surface proteins. Mutants lacking Dfg5p are defective in alkaline pH-induced hypha formation; mutants lacking Dcw1p have no detected hypha formation defect. Both homozygote-triplication tests and conditional expression strategies indicate that dfg5 and dcw1 mutations are synthetically lethal. Therefore, Dfg5p and Dcw1p share a function required for growth. Epitope-tagged Dfg5p, created through an insertional mutagenesis strategy, is found in cell membrane and cell wall extract fractions, and endoglycosidase H digestion shows that Dfg5p undergoes N-linked mannosylation. Surprisingly, Dfg5p is required for expression of the hypha-specific gene HWP1 in alkaline media. Because Dfg5p is a cell surface protein, it is poised to generate or transmit an external signal required for the program of hypha-specific gene expression.

Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs.

ABSTRACT Candida albicans is an important commensal of mucosal surfaces that is also an opportunistic pathogen. This organism colonizes a wide range of host sites that differ in pH; thus, it must respond appropriately to this environmental stress to survive. The ability to respond to neutral-to-alkaline pHs is governed in part by the RIM101 signal transduction pathway. Here we describe the analysis of C. albicans Rim13p, a homolog of the Rim13p/PalB calpain-like protease member of the RIM101/pacC pathway from Saccharomyces cerevisiae and Aspergillus nidulans, respectively. RIM13, like other members of the RIM101 pathway, is required for alkaline pH-induced filamentation and growth under extreme alkaline conditions. Further, our studies suggest that the RIM101 pathway promotes pH-independent responses, including resistance to high concentrations of lithium and to the drug hygromycin B. RIM13 encodes a calpain-like protease, and we found that Rim101p undergoes a Rim13p-dependent C-terminal proteolytic processing event at neutral-to-alkaline pHs, similar to that reported for S. cerevisiae Rim101p and A. nidulans PacC. However, we present evidence that suggests that C. albicans Rim101p undergoes a novel processing event at acidic pHs that has not been reported in either S. cerevisiae or A. nidulans. Thus, our results provide a framework to understand how the C. albicans Rim101p processing pathway promotes alkaline pH-independent processes.

Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors

ABSTRACT Iron is an essential nutrient that is severely limited in the mammalian host. Candida albicans encodes a family of 15 putative ferric reductases, which are required for iron acquisition and utilization. Despite the central role of ferric reductases in iron acquisition and mobilization, relatively little is known about the regulatory networks that govern ferric reductase gene expression in C. albicans. Here we have demonstrated the differential regulation of two ferric reductases, FRE2 and FRP1, in response to distinct iron-limited environments. FRE2 and FRP1 are both induced in alkaline-pH environments directly by the Rim101 transcription factor. However, FRP1 but not FRE2 is also induced by iron chelation. We have identified a CCAAT motif as the critical regulatory sequence for chelator-mediated induction and have found that the CCAAT binding factor (CBF) is essential for FRP1 expression in iron-limited environments. We found that a hap5Δ/hap5Δ mutant, which disrupts the core DNA binding activity of CBF, is unable to grow under iron-limited conditions. C. albicans encodes three CBF-dependent transcription factors, and we identified the Hap43 protein as the CBF-dependent transcription factor required for iron-limited responses. These studies provide key insights into the regulation of ferric reductase gene expression in the fungal pathogen C. albicans.