Deletion of the fungus specific protein phosphatase Z1 exaggerates the consequences of oxidative stress elicited by tert-butyl-hydroperoxide in Candida albicans
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ABSTRACT:
ORGANISM(S): Candida albicans
PROVIDER: GSE134060 | GEO | 2019/11/04
REPOSITORIES: GEO
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Project description:Candida albicans is an opportunistic pathogen which is responsible for widespread nosocomial infections. It encompasses a fungus specific serine/threonine protein phosphatase gene CaPPZ1 that is involved in cation transport, cell wall integrity, oxidative stress response, morphological alterations, and virulence. We demonstrated that tert-butyl-hydoperoxide induced oxidative stress suppressed the growth rate and blocked the proliferation of the fungal cells without effecting their viability, both in the cappz1 KO mutant and in the genetically matching QMY23 control strains. To reveal the gene expression changes behind the above observation we carried out a global transcriptome analysis by DNA microarray hybridization as well as RNA sequencing, and confirmed our results by quantitative RT-PCR. The numbers of genes affected and the amplitudes of the transcript-level changes revealed that the deletion of CaPPZ1 enhances the effects of oxidative stress. Thus the roles of this fungus specific protein phosphatase in important physiological processes like membrane transport, cell surface interactions, oxidoreductase activity, cell signaling, translation, and RNA processing becomes more pronounced under oxidative conditions. We conclude that in the wild type C. albicans CaPPZ1 has a protective role against oxidative damage, and suggest that the specific inhibition of this phosphatase combined with mild oxidative treatment could be a feasible approach of topical antifungal therapy.
2019-11-04 | GSE133335 | GEO
Project description:Candida albicans is an opportunistic pathogen which is responsible for widespread nosocomial infections. It encompasses a fungus specific serine/threonine protein phosphatase gene CaPPZ1 that is involved in cation transport, cell wall integrity, oxidative stress response, morphological alterations, and virulence. We demonstrated that tert-butyl-hydoperoxide induced oxidative stress suppressed the growth rate and blocked the proliferation of the fungal cells without effecting their viability, both in the cappz1 KO mutant and in the genetically matching QMY23 control strains. To reveal the gene expression changes behind the above observation we carried out a global transcriptome analysis by DNA microarray hybridization as well as RNA sequencing, and confirmed our results by quantitative RT-PCR. The numbers of genes affected and the amplitudes of the transcript-level changes revealed that the deletion of CaPPZ1 enhances the effects of oxidative stress. Thus the roles of this fungus specific protein phosphatase in important physiological processes like membrane transport, cell surface interactions, oxidoreductase activity, cell signaling, translation, and RNA processing becomes more pronounced under oxidative conditions. We conclude that in the wild type C. albicans CaPPZ1 has a protective role against oxidative damage, and suggest that the specific inhibition of this phosphatase combined with mild oxidative treatment could be a feasible approach of topical antifungal therapy.
2019-11-04 | GSE133611 | GEO
Project description:The opportunistic pathogen Candida is one of the most common causes of nosocomial bloodstream infections. Because candidemia is associated with high mortality rates and because the incidences of multidrug-resistant Candida are increasing, efforts to identify novel targets for the development of potent antifungals are warranted. Here, we describe the structure and function of the first member of a family of protein phosphatases that is specific to fungi, protein phosphatase Z1 (PPZ1) from Candida albicans We show that PPZ1 not only is active but also is as susceptible to inhibition by the cyclic peptide inhibitor microcystin-LR as its most similar human homolog, protein phosphatase 1α (PP1α [GLC7 in the yeast Saccharomyces cerevisiae]). Unexpectedly, we also discovered that, despite its 66% sequence identity to PP1α, the catalytic domain of PPZ1 contains novel structural elements that are not present in PP1α. We then used activity and pulldown assays to show that these structural differences block a large subset of PP1/GLC7 regulatory proteins from effectively binding PPZ1, demonstrating that PPZ1 does not compete with GLC7 for its regulatory proteins. Equally important, these unique structural elements provide new pockets suitable for the development of PPZ1-specific inhibitors. Together, these studies not only reveal why PPZ1 does not negatively impact GLC7 activity in vivo but also demonstrate that the family of fungus-specific phosphatases-especially PPZ1 from C. albicans-are highly suitable targets for the development of novel drugs that specifically target C. albicans without cross-reacting with human phosphatases. Candida albicans is a medically important human pathogen that is the most common cause of fungal infections in humans. In particular, approximately 46,000 cases of health care-associated candidiasis occur each year in the United States. Because these infections are associated with high mortality rates and because multiple species of Candida are becoming increasingly resistant to antifungals, there are increasing efforts to identify novel targets that are essential for C. albicans virulence. Here we use structural and biochemical approaches to elucidate how a member of a fungus-specific family of enzymes, serine/threonine phosphatase PPZ1, functions in C. albicans We discovered multiple unique features of PPZ1 that explain why it does not cross-react with, and in turn compete for, PP1-specific regulators, a long-standing question in the field. Most importantly, however, these unique features identified PPZ1 as a potential target for the development of novel antifungal therapeutics that will provide new, safe, and potent treatments for candidiasis in humans.
| S-EPMC4999541 | biostudies-literature
Project description:Protein phosphatase Z is a "novel type" fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans.
| S-EPMC5570430 | biostudies-literature
Project description:BackgroundCandida albicans is an opportunistic pathogen which is responsible for widespread nosocomial infections. It encompasses a fungus specific serine/threonine protein phosphatase gene, CaPPZ1 that is involved in cation transport, cell wall integrity, oxidative stress response, morphological transition, and virulence according to the phenotypes of the cappz1 deletion mutant.ResultsWe demonstrated that a short-term treatment with a sublethal concentration of tert-butyl hydroperoxide suppressed the growth of the fungal cells without affecting their viability, both in the cappz1 mutant and in the genetically matching QMY23 control strains. To reveal the gene expression changes behind the above observations we carried out a global transcriptome analysis. We used a pilot DNA microarray hybridization together with extensive RNA sequencing, and confirmed our results by quantitative RT-PCR. Novel functions of the CaPpz1 enzyme and oxidative stress mechanisms have been unraveled. The numbers of genes affected as well as the amplitudes of the transcript level changes indicated that the deletion of the phosphatase sensitized the response of C. albicans to oxidative stress conditions in important physiological functions like membrane transport, cell surface interactions, oxidation-reduction processes, translation and RNA metabolism.ConclusionsWe conclude that in the wild type C. albicans CaPPZ1 has a protective role against oxidative damage. We suggest that the specific inhibition of this phosphatase combined with mild oxidative treatment could be a feasible approach to topical antifungal therapy.
| S-EPMC6862791 | biostudies-literature
Project description:Deletion of the fungus specific protein phosphatase Z1 exaggerates the consequences of oxidative stress elicited by tert-butyl-hydroperoxide in Candida albicans
| PRJNA553661 | ENA
Project description:Deletion of the fungus specific protein phosphatase Z1 exaggerates the consequences of oxidative stress elicited by tert-butyl-hydroperoxide in Candida albicans (Microarray expression)
| PRJNA551142 | ENA
Project description:Deletion of the fungus specific protein phosphatase Z1 exaggerates the consequences of oxidative stress elicited by tert-butyl-hydroperoxide in Candida albicans (RNAseq data set)
| PRJNA552040 | ENA
Project description:The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen, Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1-16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1-16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.
| S-EPMC6358084 | biostudies-literature
Project description:The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.
| S-EPMC8305657 | biostudies-literature