Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans.
ABSTRACT The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.
- SourceAvailable from: Joaquín Ariño[show abstract] [hide abstract]
ABSTRACT: The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.Yeast 02/2001; 18(2):115-24. · 1.96 Impact Factor
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ABSTRACT: Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li+, the intracellular content of the cation was markedly decreased in ppz1 delta ppz2 delta mutants when compared to wild type cells. No significant differences were observed between both strains when the Li+ influx was measured, but ppz1 delta ppz2 delta mutants eliminated Li+ more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 delta ppz2 delta cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na+ and Li+ of a ena1 delta strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.Journal of Biological Chemistry 07/1995; 270(22):13036-41. · 4.65 Impact Factor
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ABSTRACT: Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels-which are susceptible to chromosome position effects-can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Delta/leu2Delta, his1Delta/his1Delta; arg4Delta/arg4Delta, his1Delta/his1Delta; and arg4Delta/arg4Delta, leu2Delta/leu2Delta, his1Delta/his1Delta) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.Eukaryotic Cell 03/2005; 4(2):298-309. · 3.59 Impact Factor