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Cell Wall Proteins (cell + wall_protein)
Selected AbstractsDynamics of cell wall structure in Saccharomyces cerevisiaeFEMS MICROBIOLOGY REVIEWS, Issue 3 2002Frans M Klis Abstract The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein,polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers' yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed. [source] Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 8 2009Mariska Lilly Abstract The yeast Saccharomyces cerevisiae was genetically modified to assemble a minicellulosome on its cell surface by heterologous expression of a chimeric scaffoldin protein from Clostridium cellulolyticum under the regulation of the phosphoglycerate kinase 1 (PGK1) promoter and terminator regulatory elements, together with the ,-xylanase 2 secretion signal of Trichoderma reesei and cell wall protein 2 (Cwp2) of S. cerevisiae. Fluorescent microscopy and Far Western blot analysis confirmed that the Scaf3p is targeted to the yeast cell surface and that the Clostridium thermocellum cohesin domain is functional in yeast. Similarly, functionality of the C. thermocellum dockerin domain in yeast is shown by binding to the Scaf3 protein in Far Western blot analysis. Phenotypic evidence for cohesin,dockerin interaction was also established with the detection of a twofold increase in tethered endoglucanase enzyme activity in S. cerevisiae cells expressing the Scaf3 protein compared with the parent strain. This study highlights the feasibility to future design of enhanced cellulolytic strains of S. cerevisiae through emulation of the cellulosome concept. Potentially, Scaf3p-armed yeast could also be developed into an alternative cell surface display strategy with various tailor-made applications. [source] Immunoreactivity of peptides generated by limited proteolysis of 71-kDa cell wall protein of Mycobacterium tuberculosis H37Ra using PLG-microparticlesLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2000N. Dhiman Peptide mapping by limited proteolysis of a highly protective 71-kDa cell wall-associated protein of Mycobacterium tuberculosis H37Ra was carried out in order to identify key protective determinants within the native protein. The 71-kDa protein, which had an isoelectric point of 4·25, was digested into eight major bands at 48 h using trypsin and pepsin at equal enzyme to protein ratios (pH 5·5). The in vitro lymphocyte reactivity of individual peptides suggested P1, P2 and P5 to be significantly immunoreactive in mice immunized with native 71-kDa-polylactide-coglyeolide (PLG); however, the reactivity was significantly lower than that of the native 71-kDa protein. Immunization of mice with a pooled fraction (upper fraction-71 kDa) of more immunoreactive peptides (consisting of P1 and P2) did not further boost their immunoreactivity. However, P1 and P2 exhibited comparable or even higher lymphocyte proliferation in human tuberculous and control subjects. These data suggest distinct antigenic specificities in humans and mice and further substantiate the use of the 71-kDa protein or its peptides P1 and P2 as potential vaccine candidates for tuberculosis. [source] NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 6 2009Wouter De Jong Summary Streptomycetes have a complex morphogenetic programme culminating in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. We here compared whole genome expression of a wild-type colony of Streptomyces coelicolor forming aerial hyphae and spores with that of the chp null mutant that forms few aerial structures. This revealed that expression of 244 genes was significantly altered, among which genes known to be involved in development. One of the genes that was no longer expressed in the ,chpABCDEFGH mutant was nepA, which was previously shown to be expressed in a compartment connecting the substrate mycelium with the sporulating parts of the aerial mycelium. We here show that expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall. Germination of spores of a nepA deletion mutant was faster and more synchronous, resulting in colonies with an accelerated morphogenetic programme. Crucially, spores of the ,nepA mutant also germinated in water, unlike those of the wild-type strain. Taken together, NepA is the first bacterial structural cell wall protein that is important for maintenance of spore dormancy under unfavourable environmental conditions. [source] NPP1, a Phytophthora -associated trigger of plant defense in parsley and ArabidopsisTHE PLANT JOURNAL, Issue 3 2002Guido Fellbrich Summary Activation of non-cultivar-specific plant defense against attempted microbial infection is mediated through the recognition of pathogen-derived elicitors. Previously, we have identified a peptide fragment (Pep-13) within a 42-kDa cell wall transglutaminase from various Phytophthora species that triggers a multifacetted defense response in parsley cells. Many of these oomycete species have now been shown to possess another cell wall protein (24 kDa), that evoked the same pattern of responses in parsley as Pep-13. Unlike Pep-13, necrosis-inducing Phytophthora protein 1 (NPP1) purified from P. parasitica also induced hypersensitive cell death-like lesions in parsley. NPP1 structural homologs were found in oomycetes, fungi, and bacteria, but not in plants. Structure,activity relationship studies revealed the intact protein as well as two cysteine residues to be essential for elicitor activity. NPP1-mediated activation of pathogen defense in parsley does not employ the Pep-13 receptor. However, early induced cellular responses implicated in elicitor signal transmission (increased levels of cytoplasmic calcium, production of reactive oxygen species, MAP kinase activation) were stimulated by either elicitor, suggesting the existence of converging signaling pathways in parsley. Infiltration of NPP1 into leaves of Arabidopsis thaliana Col-0 plants resulted in transcript accumulation of pathogenesis-related (PR) genes, production of ROS and ethylene, callose apposition, and HR-like cell death. NPP1-mediated induction of the PR1 gene is salicylic acid-dependent, and, unlike the P. syringae pv. tomato DC3000(avrRpm1)-induced PR1 gene expression, requires both functional NDR1 and PAD4. In summary, Arabidopsis plants infiltrated with NPP1 constitute an experimental system that is amenable to forward genetic approaches aiming at the dissection of signaling pathways implicated in the activation of non-cultivar-specific plant defense. [source] Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using ,-agglutinin for production of oral vaccines ,BIOTECHNOLOGY PROGRESS, Issue 2 2010Jamie L. Wasilenko Abstract Yeast is an ideal organism to express viral antigens because yeast glycosylate proteins more similarly to mammals than bacteria. Expression of proteins in yeast is relatively fast and inexpensive. In addition to the convenience of production, for purposes of vaccination, yeast has been shown to have natural adjuvant activity making the expressed proteins more immunogenic when administered along with yeast cell wall components. Development of genetic systems to display foreign proteins on the surface of yeast via fusion to glycosylphosphatidylinositol-anchored (GPI) proteins has further simplified the purification of recombinant proteins by not requiring harsh treatments for cellular lysis or protein purification. We have expressed the hemagglutinin protein from a highly pathogenic avian influenza (HPAI) virus [A/Egret/HK/757.2/02], subtype H5N1, on the surface of the yeast strain Pichia pastoris, as an anchored C-terminal fusion with the Saccharomyces cerevisiae GPI-anchored cell wall protein, ,-agglutinin. Surface expression of the hemagglutinin fusion protein was demonstrated by immunofluorescence microscopy. Functionally, the fusion protein retained hemagglutinin agglutinating activity, and oral vaccination with the yeast resulted in production of virus neutralizing antibodies. This study represents the first steps in the generation of a yeast-based vaccine for protection against highly pathogenic strains of avian influenza. Published 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] The localization change of Ybr078w/Ecm33, a yeast GPI-associated protein, from the plasma membrane to the cell wall, affecting the cellular functionFEMS MICROBIOLOGY LETTERS, Issue 1 2003Hiromichi Terashima Abstract The YBR078W/ECM33 gene of Saccharomyces cerevisiae encodes a glycosylphosphatidylinositol (GPI)-attached protein and its disruptant strain exhibited a temperature-sensitive (ts) growth defect. A HA-tagged Ybr078w protein, which complemented the ts growth phenotype of the ybr078w, strain, was predominantly located on the plasma membrane by GPI anchoring. To examine the requirement of the GPI anchoring on the plasma membrane for the function, the ,-minus region of Ybr078w was replaced with those of Ydr534c/Fit1 and Ynl327w/Egt2, which are known as GPI-dependent cell wall proteins. The replacement induced the change in localization of the mutant proteins from the plasma membrane to the cell wall and the mutant proteins lost the function to complement the ts cell growth defect of the ybr078w, strain. In addition, a similar result was obtained in a mutant protein, where the authentic SKKSK sequence at the ,-5 to ,-1 site of Ybr078w was replaced with a synthetic ISSYS sequence. It is concluded that the GPI anchoring on the plasma membrane is required for the Ybr078w function. [source] Dynamics of cell wall structure in Saccharomyces cerevisiaeFEMS MICROBIOLOGY REVIEWS, Issue 3 2002Frans M Klis Abstract The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein,polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers' yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed. [source] Minisatellites in Saccharomyces cerevisiae genes encoding cell wall proteins: a new way towards wine strain characterisationFEMS YEAST RESEARCH, Issue 4-5 2004Paola Marinangeli Abstract With the aim of developing new tools for the characterisation of wine yeasts, by means of databases available on-line we scanned the genome of Saccharomyces cerevisiae in search of potentially polymorphic targets. As we have previously observed for SED1, we found that other genes coding for cell wall proteins contain minisatellite-like sequences. A polymerase chain reaction (PCR) survey of SED1 and three of these others, namely AGA1, DAN4 and HSP150, in a population of wild S. cerevisiae demonstrated that these genes are highly polymorphic in length and represent a sink of unexplored genetic variability. The primer pairs designed on the gene open reading frames yield stable and repeatable amplification profiles that show a level of resolution that allows the clear discriminate between different strains. These can therefore be utilised for PCR-based typing of S. cerevisiae. [source] 71 Proteomics of haematococcus pluvialis: new opportunities for study of genomics of a non-sequenced speciesJOURNAL OF PHYCOLOGY, Issue 2003Q. Hu The green alga, Haematococcus pluvialis, has become a model organism for commercial production of the high-value carotenoid astaxanthin. H. Pluvialis has also drawn significant scientific attention because fundamental biological questions relating to the massive cellular accumulation of astaxanthin have to be addressed in order to improve the yield and quality of the algal biomass. However, research has been impeded by the lack of molecular background information on this non-sequenced species. A combination of classical biochemistry with a state-of-the-art proteomic approach was used to address these questions. This was possible by taking advantage of information already available for homologous genes/gene-products in organisms whose genomes have been sequenced. The approach involved isolation of subsets of the proteome from subcellular compartments/organelles of an organism by one- or two-dimensional electrophoresis (1-DE or 2-DE) and their identification by N-terminal sequencing and peptide mass fingerprinting (PMF), involving matrix-assisted laser desorption/ionization and time-of-flight (MALDI-TOF) mass spectrometry coupled with bioinformatics. Based upon the information obtained from the combined methods, expression and physiological functions of specific genes/encoded proteins may be deduced. Examples include profiling of cell wall proteins, biogenesis and protein composition of lipid bodies, and expression patterns of soluble proteins under stress conditions. Advantages and limitations of the method for non-sequenced organisms and for cross-species protein identification will also be discussed. [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] Flocculation, adhesion and biofilm formation in yeastsMOLECULAR MICROBIOLOGY, Issue 1 2006Kevin J. Verstrepen Summary Yeast cells possess a remarkable capacity to adhere to abiotic surfaces, cells and tissues. These adhesion properties are of medical and industrial relevance. Pathogenic yeasts such as Candida albicans and Candida glabrata adhere to medical devices and form drug-resistant biofilms. In contrast, cell,cell adhesion (flocculation) is a desirable property of industrial Saccharomyces cerevisiae strains that allows the easy separation of cells from the fermentation product. Adhesion is conferred by a class of special cell wall proteins, called adhesins. Cells carry several different adhesins, each allowing adhesion to specific substrates. Several signalling cascades including the Ras/cAMP/PKA and MAP kinase (MAPK)-dependent filamentous growth pathways tightly control synthesis of the different adhesins. Together, these pathways trigger adhesion in response to stress, nutrient limitation or small molecules produced by the host, such as auxin in plants or NAD in mammals. In addition, adhesins are subject to subtelomeric epigenetic switching, resulting in stochastic expression patterns. Internal tandem repeats within adhesin genes trigger recombination events and the formation of novel adhesins, thereby offering fungi an endless reservoir of adhesion properties. These aspects of fungal adhesion exemplify the impressive phenotypic plasticity of yeasts, allowing them to adapt quickly to stressful environments and exploit new opportunities. [source] The distribution and expression of a biotrophy-related gene, CIH1, within the genus ColletotrichumMOLECULAR PLANT PATHOLOGY, Issue 4 2000Sarah E. Perfect During the biotrophic phase of the infection process of the hemibiotrophic anthracnose fungus Colletotrichum lindemuthianum, an intracellular hypha develops within epidermal cells of its host, Phaseolus vulgaris. This is followed by the formation of secondary hyphae during the necrotrophic phase. Previous work using a monoclonal antibody, UB25, has identified a glycoprotein that is specific to the interfacial matrix that forms between the wall of the intracellular hypha and the invaginated host plasma membrane. The gene encoding the protein identified by UB25 was cloned by immunoscreening and designated CIH1. The predicted amino acid sequence revealed a proline-rich glycoprotein, and biochemical evidence suggested that it formed a cross-linked structure at the biotrophic interface. Although CIH1 is a fungal gene, its product has several similarities to plant cell wall proteins. In this paper, we have surveyed the distribution and expression of CIH1 within the genus Colletotrichum, encompassing both necrotrophic and hemibiotrophic species. The results show that homologues of the CIH1 gene are present in all the Colletotrichum species tested. Northern blot studies of the time course of the infection process in planta have shown that CIH1 is expressed by both C. lindemuthianum in bean and C. trifolii in alfalfa during the biotrophic phase of fungal development. Immunofluorescence labelling of infected epidermal strips with UB25 revealed that the intracellular hyphae formed by C. destructivum as it infects alfalfa were specifically labelled in a similar way to those formed by C. lindemuthianum in bean. Northern and Western analysis showed that CIH1 was also expressed by C. lindemuthianum in vitro, though not constitutively. Overall, the evidence supports a role for CIH1 in biotrophy within the genus Colletotrichum. [source] Impact of the transcriptional regulator, Ace2, on the Candida glabrata secretomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2010David A. Stead Abstract Candida glabrata is a major fungal pathogen of humans, and the virulence of C. glabrata is increased by inactivation of the transcription factor, Ace2. Our previous examination of the effects of Ace2 inactivation upon the intracellular proteome suggested that the hypervirulence of C. glabrata ace2 mutants might be caused by differences in the secretome. Therefore in this study we have characterised the C. glabrata secretome and examined the effects of Ace2 inactivation upon this extracellular proteome. We have identified 31 distinct proteins in the secretome of wild-type C. glabrata cells by MS/MS of proteins that were precipitated from the growth medium and enriched by affinity chromatography on concanavalin A. Most of these proteins are predicted to be cell wall proteins, cell wall modifying enzymes and aspartyl proteinases. The endochitinase Cts1 and the endoglucanase Egt2 were not detected in the C. glabrata secretome following Ace2 inactivation. This can account for the cell separation defect of C. glabrata ace2 cells. Ace2 inactivation also resulted in the detection of new proteins in the C. glabrata secretome. The release of such proteins might contribute to the hypervirulence of ace2 cells. [source] Isolation and proteomic alalysis of cell wall-deficient Haematococcus pluvialis mutantsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2005Sheng-Bing Wang Abstract The green alga Haematococcus pluvialis has a plant-like cell wall consisting of glycoproteins and cellulose that is modified during the cell cycle and under various conditions. These features allow Haematococcus to be used as a model organism for studying cell wall biology. Development of the Haematococcus model is hampered by the absence of mutants that could provide insight into the biosynthesis and assembly of wall components. Haematococcus mutants (WM#537 and WM#2978) (WM#wall mutant) with defective cell walls were obtained by chemical mutagenesis. WM#537 features a secondary wall of considerably reduced thickness, whereas WM#2978 possesses a somewhat reduced secondary wall with little intervening space between the wall and plasmalemma. 2-DE revealed that a majority of the cell wall proteins were present in the wild-type and mutant cell walls throughout the cell cycle. PMF identified 55 wall protein orthologs from these strains, including a subset of induced proteins known to be involved in wall construction, remodeling, and defense. Down-regulation of certain wall proteins in the two mutants was associated with the wall defects, whereas overexpression of other proteins may have compensated for the defective walls in the two mutants. [source] | ||||||||||||||||||||||