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: Paula Sampaio[Show abstract] [Hide abstract]
ABSTRACT: Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.PLoS ONE 01/2014; 9(1):e86270. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Digital image analysis of high time resolution video microscopy was used to investigate hyphal growth dynamics in different Candida albicans strains. The effects of the quorum sensing molecules tyrosol and farnesol, the deletion of the fungus specific protein phosphatase Z1 CaPPZ1), and the hypha-specific cyclin (HGC1) genes were analyzed by this method. Our system monitored cell growth in a CO2 incubator under near-physiological conditions and measured three major parameters under the following stringent conditions: (a) the time of yeast cell adherence, (b) the time of hyphal outgrowth, and (c) the rate of hyphal growth. This method showed that hyphal extension of wild-type SC5314 cells was accelerated by tyrosol and inhibited by farnesol. Hyphal growth rate was moderately lower in cappz1 and strongly reduced in hgc1 mutants. In addition, tyrosol treatment caused a firm adherence, while farnesol treatment and hgc1 mutation prevented the adherence of yeast cells to the surface of the culture flask. Transition from yeast-to-hyphal state was faster after tyrosol treatment, while it was reduced in farnesol-treated cells as well as in the cappz1 and hgc1 mutants. Our data confirm the notion that the attachment of yeast cells, the yeast-to-hyphal transition, and hyphal growth rate are closely related processes. Time-lapse video microscopy combined with image analysis offers a convenient and reliable method of testing chemicals, including potential drug candidates, and genetic manipulations on the dynamic morphological changes in C. albicans strains.Applied Microbiology and Biotechnology 04/2014; · 3.81 Impact Factor