Role of a Candida albicans P1-Type ATPase in Resistance to Copper and Silver Ion Toxicity

Abstract

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C. albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1⌬ strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termed CRD1 (for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2. Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level. Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 M CuSO 4 . These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C. albicans as a nosocomial pathogen.

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae.

“…The E. coli homologue of this type of ATPase, copA, was shown to pump Cu(I) out of cells (Rensing et al, 2000). It was reported that overexpression of the CRD1 gene conferred copper-resistance on S. cerevisiae cells (Riggle and Kumamoto, 2000;Weissman et al, 2000). In S. cerevisiae, there is a structurally similar P-type ATPase encoded by PCA1 (Rad et al, 1994).…”

“…Different types of yeast have different mechanisms for developing copper-resistance. In Candida albicans, a P-type ATPase encoded by the CRD1 gene plays a major role in copper-resistance: excessive copper in the cytoplasm is exported from the cells by this enzyme (Riggle and Kumamoto, 2000;Weissman et al, 2000). The E. coli homologue of this type of ATPase, copA, was shown to pump Cu(I) out of cells (Rensing et al, 2000).…”

“…Copper-resistance can be mediated by several distinct mechanisms: a defect in the import of copper (Kampfenkel et al, 1995), hyper-production of metallothionein (Karin et al, 1984), or hyper-activity of a transporter such as the P-type-ATPase that pumps copper out of the C. albicans cells (Riggle and Kumamoto, 2000;Weissman et al, 2000). Which mechanism accounts for the copper-resistance of the las21 mutants?…”

Transcriptional Regulation of MDR1 , Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

“…C. albicans strains are listed in Table 1. Parent strains were CAI4 (⌬ura3/⌬ura3) (17) and CAPR306 (⌬ura3/⌬ura3 ⌬crd1/⌬crd1) (42). The crd1 deletion strain CAPR306 was used as a parent in these studies because the crd1 mutation provides a convenient genetic marker for experiments such as protoplast fusion.…”

Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis

“…A subtractive library enriched by T. rubrum genes expressed in the presence of cytotoxic components showed the upregulation of a gene encoding copper-resistanceassociated P-type ATPase after fluconazole contact (Paião et al, 2007). Copper is an essential metabolic element as a cofactor of many cellular enzymes, and copper-transporting P-type ATPases are found in eukaryotic organisms and require an iron transporter for adaptation in different environments and hosts (Riggle & Kumamoto, 2000). The absence of normal copper-transporter activity in Cryptococcus neoformans after VPH1 gene deletion was observed in an avirulent mutant interfering in laccase activity (Zhu & Williamson, 2003), a strong virulence factor in this opportunistic pathogen.…”