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The high copper tolerance of Candida albicans is mediated by a P-type ATPase.

ABSTRACT: The pathogenic yeast Candida albicans has higher resistance than the baker's yeast Saccharomyces cerevisiae to elevated concentrations of copper. To understand the basis of this differential resistance, we performed a functional screen for C. albicans genes involved in copper detoxification. Here, we report the isolation of two such genes: a metallothionein, CaCUP1, and a copper-transporting P-type ATPase, CaCRP1. Both genes are induced by extracellular copper. Gene disruptions indicated that the copper extrusion pump is responsible for the unusual resistance of C. albicans to copper, whereas the metallothionein is responsible for the residual copper resistance of the Cacrp1Delta mutant. We show further that under acidic and anaerobic conditions, such as prevail in the natural niche of C. albicans, the digestive tract of animals, CaCRP1 function becomes essential for survival in the presence of even very low copper concentrations. These observations suggest that copper in the gastrointestinal tract may present a toxic challenge to which enteric organisms had to adapt.

SUBMITTER: Weissman Z

PROVIDER: S-EPMC16272 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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The high copper tolerance of Candida albicans is mediated by a P-type ATPase.

Weissman Z Z Berdicevsky I I Cavari B Z BZ Kornitzer D D

Proceedings of the National Academy of Sciences of the United States of America 20000301 7

The pathogenic yeast Candida albicans has higher resistance than the baker's yeast Saccharomyces cerevisiae to elevated concentrations of copper. To understand the basis of this differential resistance, we performed a functional screen for C. albicans genes involved in copper detoxification. Here, we report the isolation of two such genes: a metallothionein, CaCUP1, and a copper-transporting P-type ATPase, CaCRP1. Both genes are induced by extracellular copper. Gene disruptions indicated that th ...[more]

PMID: 10737803

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Project description:The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7? mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7? cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1? lsp1?), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7? in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161?, rvs167?, and arp2? arp3?). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2? mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7? cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

Project description:How cells exposed to one stress are later able to better survive other types of stress is not well understood. In eukaryotic organisms, physiological and pathological stresses can disturb endoplasmic reticulum (ER) function, resulting in "ER stress." Here, we found that exposure to tunicamycin, an inducer of ER stress, resulted in the acquisition of a specific aneuploidy, chromosome 2 trisomy (Chr2x3), in Candida albicans. Importantly, the resulting aneuploidy also conferred cross-tolerance to caspofungin, a first-line echinocandin antifungal, as well as to hydroxyurea, a common chemotherapeutic agent. Exposure to a range of tunicamycin concentrations induced similar ER stress responses. Extra copies of one Chr2 gene, MKK2, affected both tunicamycin and caspofungin tolerance, while at least 3 genes on chromosome 2 (ALG7, RTA2, and RTA3) affected only tunicamycin and not caspofungin responses. Other Chr2 genes (RNR1 and RNR21) affected hydroxyurea tolerance but neither tunicamycin nor caspofungin tolerance. Deletion of components of the protein kinase C (PKC) or calcineurin pathways affected tolerance to both tunicamycin and caspofungin, supporting the idea that the ER stress response and echinocandin tolerance are regulated by overlapping stress response pathways. Thus, antifungal drug tolerance can arise rapidly via ER stress-induced aneuploidy. IMPORTANCE Candida albicans is a prevalent human fungal commensal and also a pathogen that causes life-threatening systemic infections. Treatment failures are frequent because few therapeutic antifungal drug classes are available and because drug resistance and tolerance limit drug efficacy. We found that C. albicans rapidly overcomes the cellular stress induced by the drug tunicamycin by duplicating chromosome 2. Also, chromosome 2 duplication confers tolerance not only to tunicamycin but also to the following two unrelated drugs: caspofungin, an antifungal drug, and hydroxyurea, a chemotherapeutic. Cross tolerance to the three drugs involves different sets of genes, although some genetic pathways affect the tolerance to two of these three drugs. This work highlights a serious concern, namely, that changes in whole chromosome copy number can occur in response to one type of stress, and yet, they may facilitate the emergence of tolerance to multiple drugs, including the few antifungal drug classes available to treat Candida infections.

Project description:BackgroundThe emergence of tolerance to antifungal agents in Candida albicans complicates the treatment of fungal infections. Understanding the mechanisms underlying this tolerance is crucial for developing effective therapeutic strategies.ObjectiveThis study aims to elucidate the genetic and molecular basis of ketoconazole tolerance in C. albicans, focusing on the roles of chromosomal aneuploidy, Hsp90, and calcineurin.MethodsThe wild-type C. albicans strain SC5314 was exposed to increasing concentrations of ketoconazole (0.015-32 μg/mL) to select for tolerant adaptors. Disk diffusion and spot assays were used to assess tolerance. Whole-genome sequencing identified chromosomal changes in the adaptors. The roles of Hsp90 and calcineurin in maintaining and developing ketoconazole tolerance were investigated using specific inhibitors and knockout strains.ResultsAdaptors exhibited tolerance to ketoconazole concentrations up to 16 μg/mL, a significant increase from the parent strain's inhibition at 0.015 μg/mL. All tolerant adaptors showed amplification of chromosome R, with 29 adaptors having trisomy and one having tetrasomy. This aneuploidy was unstable, reverting to euploidy and losing tolerance in drug-free conditions. Both Hsp90 and calcineurin were essential for maintaining and developing ketoconazole tolerance. Inhibition of these proteins resulted in loss of tolerance. The efflux gene CDR1 was not required for the development of tolerance. Chromosome R trisomy and tetrasomy induce cross-tolerance to other azole antifungal agents, including clotrimazole and miconazole, but not to other antifungal classes, such as echinocandins and pyrimidines, exemplified by caspofungin and 5-flucytosine.ConclusionKetoconazole tolerance in C. albicans is mediated by chromosomal aneuploidy, specifically chromosome R amplification, and requires Hsp90 and calcineurin. These findings highlight potential targets for therapeutic intervention to combat antifungal tolerance and improve treatment outcomes.

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The high copper tolerance of Candida albicans is mediated by a P-type ATPase.

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The high copper tolerance of Candida albicans is mediated by a P-type ATPase.

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