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Cell Wall Integrity (cell + wall_integrity)
Selected AbstractsThe Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin functionMOLECULAR MICROBIOLOGY, Issue 5 2003Peter R. Kraus Summary Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37°C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a ,-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall. [source] Role of the Slt2 mitogen-activated protein kinase pathway in cell wall integrity and virulence in Candida glabrataFEMS YEAST RESEARCH, Issue 3 2010Taiga Miyazaki Abstract The Slt2 mitogen-activated protein kinase pathway plays a major role in maintaining fungal cell wall integrity. In this study, we investigated the effects of SLT2 deletion and overexpression on drug susceptibility and virulence in the opportunistic fungal pathogen Candida glabrata. While the ,slt2 strain showed decreased tolerance to elevated temperature and cell wall-damaging agents, the SLT2 -overexpressing strain exhibited increased tolerance to these stresses. A mutant lacking Rlm1, a transcription factor downstream of Slt2, displayed a cell wall-associated phenotype intermediate to that of the ,slt2 strain. When RLM1 was overexpressed, micafungin tolerance was increased in the wild-type strain and partial restoration of the drug tolerance was observed in the ,slt2 background. It was also demonstrated that echinocandin-class antifungals were more effective against C. glabrata under acidic conditions or when used concurrently with the chitin synthesis inhibitor nikkomycin Z. Finally, in a mouse model of disseminated candidiasis, the deletion and overexpression of C. glabrata SLT2 resulted in mild decreases and increases, respectively, in the CFUs from murine organs compared with the wild-type strain. These fundamental data will help in further understanding the mechanisms of cell wall stress response in C. glabrata and developing more effective treatments using echinocandin antifungals in clinical settings. [source] Characterization and functional analysis of the ,-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensisFEMS YEAST RESEARCH, Issue 1 2009Nadya Da Silva Castro Abstract The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, ,-1,3-glucanosyltransferase 3 (PbGel3p) is presented here. PbGel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis. The recombinant PbGel3p was overexpressed in Escherichia coli, and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1, mutant. The results indicated that PbGel3p is a cell wall-associated protein that probably works as a ,-1,3-glucan elongase capable of mediating fungal cell wall integrity. [source] Pleiotropic phenotypes caused by an opal nonsense mutation in an essential gene encoding HMG-CoA reductase in fission yeastGENES TO CELLS, Issue 6 2009Yue Fang Schizosaccharomyces pombe genome contains an essential gene hmg1+ encoding the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Here, we isolated an allele of the hmg1+ gene, hmg1-1/its12, as a mutant that showed sensitivities to high temperature and to FK506, a calcineurin inhibitor. The hmg1-1 allele contained an opal nonsense mutation in its N-terminal transmembrane domain, yet in spite of the mutation a full-length protein was produced, suggesting a read-through termination codon. Consistently, overexpression of the hmg1-1 mutant gene suppressed the mutant phenotypes. The hmg1-1 mutant showed hypersensitivity to pravastatin, an HMGR inhibitor, suggesting a defective HMGR activity. The mutant treated with FK506 caused dramatic morphological changes and showed defects in cell wall integrity, as well as displayed synthetic growth phenotypes with the mutant alleles of genes involved in cytokinesis and cell wall integrity. The mutant exhibited different phenotypes from those of the disruption mutants of ergosterol biosynthesis genes, and it showed normal filipin staining as well as showed normal subcellular localization of small GTPases. These data suggest that the pleiotropic phenotypes reflect the integrated effects of the reduced availability of ergosterol and various intermediates of the mevalonate pathway. [source] Genetic and functional interaction between Ryh1 and Ypt3: two Rab GTPases that function in S. pombe secretory pathwayGENES TO CELLS, Issue 3 2006Yi He We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1+ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1+ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling. [source] The linkage between cell wall metabolism and fruit softening: looking to the futureJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 8 2007Ariel R Vicente Abstract The softening that accompanies ripening of commercially important fruits exacerbates damage incurred during shipping and handling and increases pathogen susceptibility. Thus, postharvest biologists have studied fruit softening to identify ways to manage ripening and optimise fruit quality. Studies, generally based on the premise that cell wall polysaccharide breakdown causes ripening-associated softening, have not provided the insights needed to genetically engineer, or selectively breed for, fruits whose softening can be adequately controlled. Herein it is argued that a more holistic view of fruit softening is required. Polysaccharide metabolism is undoubtedly important, but understanding this requires a full appreciation of wall structure and how wall components interact to provide strength. Consideration must be given to wall assembly as well as to wall disassembly. Furthermore, the apoplast must be considered as a developmentally and biochemically distinct, dynamic ,compartment', not just the location of the cell wall structural matrix. New analytical approaches for enhancing the ability to understand wall structure and metabolism are discussed. Fruit cells regulate their turgor pressure as well as cell wall integrity as they ripen, and it is proposed that future studies of fruit softening should include attempts to understand the bases of cell- and tissue-level turgor regulation if the goal of optimising softening control is to be reached. Finally, recent studies show that cell wall breakdown provides sugar substrates that fuel other important cellular pathways and processes. These connections must be explored so that optimisation of softening does not lead to decreases in other aspects of fruit quality. Copyright © 2007 Society of Chemical Industry [source] Kin1 is a plasma membrane-associated kinase that regulates the cell surface in fission yeastMOLECULAR MICROBIOLOGY, Issue 5 2010Angela Cadou Summary Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1-GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1, null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue-sensitive allele kin1-as1. Kin1 inhibition primarily promotes delocalization of plasma membrane-associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen-activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen-activated protein kinase components reduces kin1, hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall. [source] Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombeMOLECULAR MICROBIOLOGY, Issue 4 2008Ma Antonia Villar-Tajadura Summary Schizosaccharomyces pombe Rho2 GTPase regulates ,-D-glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase-activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor-stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N-terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this GTPase. Accordingly, rga2, cells contain more ,-D-glucan and therefore partially suppress the thermosensitivity of mok1,664 cells, which have a defective ,-D-glucan synthase. Additionally, genetic interactions and biochemical analysis suggest that Rga2 regulates Rho2,Pck2 interaction and might participate in the regulation of the MAPK cell integrity pathway. [source] Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2pMOLECULAR MICROBIOLOGY, Issue 6 2008Franziska Hutzler Summary The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N -glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N -glycan to be directly involved in Mid2p sensing. [source] Identification of an essential gene responsible for d -Asp incorporation in the Lactococcus lactis peptidoglycan crossbridgeMOLECULAR MICROBIOLOGY, Issue 6 2006Patrick Veiga Summary Bacteria such as Lactococcus lactis have d -aspartate (d -Asp) or its amidated derivative d -asparagine (d -Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing d -amino acids in their PG crossbridge, but absent from those that instead insert l -amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate l -Ser- l -Ala or l -Ala- l -Ala in the PG crossbridge. Our results show that (i) yxbA encodes d -Asp ligase responsible for incorporation of d -Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of d -Asp may be complemented by l -Ser- l -Ala or l -Ala- l -Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having l -amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG side-chain composition. [source] The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin functionMOLECULAR MICROBIOLOGY, Issue 5 2003Peter R. Kraus Summary Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37°C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a ,-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall. [source] The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculansNEW PHYTOLOGIST, Issue 4 2008Estelle Remy Summary ,,Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. ,,The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. ,,The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. ,,It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans. [source] Banana (Musa spp.) as a model to study the meristem proteome: Acclimation to osmotic stressPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2007Sebastien C. Carpentier Abstract Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using non-parametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop. [source] | |||||||||||||