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 PRA1 and PHR1, and repression of PHR2. We find that RIM101 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 in C. albicans through both RIM101-dependent and RIM101-independent pathways.

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes,ARG5 and ADE2, and two sequences newly identified through the Candida genome project,HRM101 and ENX3. HRM101 and ENX3are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways ofSaccharomyces cerevisiae and Aspergillus nidulans. We show that three independenthrm101/hrm101 mutants and two independentenx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 andENX3 sequences are indeed portions of genes and that the respective gene products have related functions.

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¯uconazole 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¯uconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506±¯uconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to¯u-conazole or membrane perturbation with SDS. FK506 was synergistic with¯uconazole 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. Keywords: calcineurin/Candida albicans/cyclosporin A/ uconazole/antifungal drugs IntroductionThe immunosuppressants cyclosporin A (CsA) and FK506 block rejection of transplanted organs by inhibiting signaling pathways required for T-cell activation (Schreiber and Crabtree, 1992). Both drugs are in widespread clinical use and have had a dramatic impact on transplant therapy. Interestingly, CsA and FK506 are natural products of soil-dwelling bacteria or fungi (reviewed in Cardenas et al., 1994). Both drugs are toxic to a variety of fungi, suggesting one natural role might be to inhibit competing microorganisms in the soil (Tropschug et al., 1989;Breuder et al., 1994;Odom et al., 1997a;Arndt et al., 1999). CsA and FK506 exert immunosuppressive and antifungal effects by inhibiting calcineurin (Liu et al., 1991;Foor et al., 1992;Nakamura et al., 1993;Breuder et al., 1994;Odom et al., 1997a;Fox et al., 2001), a conserved Ca 2+ -calmodulin activated protein phosphatase (reviewed in Klee et al., 1998;Hemenway and Heitman, 1999;Aramburu et al., 2000). Calcineurin is a heterodimer comprised of a catalytic A and a regulatory B subunit (Hubbard and Klee, 1989;Anglister et al., 1994;Watanabe et al., 1995). CsA and FK506 do not inhibit calcineurin on their own, but ®rst bind to small, abundant, conserved binding proteins (immunophilins). CsA associates with cyclophilin A, and FK506 with FKBP12, to form protein±drug complexes that inhibit calcineurin by binding to the interface between the A and B subunits (Haddy et al., 1992;Li and Handschumacher, 1993;Milan et al., 1994;Cardenas et al., 1995b;Grif®th et al., 1995;Kawamura and Su, 1995;Kissinger et al., 1995). CsA and FK506 target this unique region of calcineurin and do not inhibit other phosphatases.The mechanisms of action of CsA and FK506 are conserved from fungi to h...

SummaryThe 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.

The ability of Candida albicans to respond to diverse environments is critical for its success as a pathogen. The RIM101 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 the RIM101 pathway is necessary in vivo for pathogenesis. First, we show that rim101 ؊ /rim101 ؊ and rim8 ؊ /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, the rim101 ؊ /rim101 ؊ and rim8 ؊ /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.