In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir

ABSTRACT J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline β-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5′ region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.

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Hydrophobic cell wall protein glycosylation by the pathogenic fungus Candida albicans

Canadian Journal of Microbiology ◽

10.1139/m94-043 ◽

1994 ◽

Vol 40 (4) ◽

pp. 266-272 ◽

Cited By ~ 26

Author(s):

Kevin C. Hazen ◽

Pati M. Glee

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Surface ◽

Molecular Mass ◽

Pathogenic Fungus ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Protein Glycosylation ◽

Yeast Cells ◽

Cell Wall Proteins

Cell surface hydrophobicity influences adhesion and virulence of the opportunistic fungal pathogen Candida albicans. Previous studies have shown that cell surface hydrophobicity is due to specific proteins that are exposed on hydrophobic cells but are masked by long fibrils on hydrophilic cells. This observation suggests that hydrophobic cell wall proteins may contain little or no mannosylation. In the present study, the glycosylation levels of three hydrophobic cell wall proteins (molecular mass range between 36 and 40 kDa) derived from yeast cells were examined. One hydrophilic protein (90 kDa) was also tested. Various endoglycosidases (endoglycosidase F – N-glycosidase F, O-glycosidase, β-mannosidase, N-glycosidase F), an exoglycosidase (α-mannosidase), and trifluoromethane sulfonic acid were used to deglycosylate the proteins. All four proteins were reactive to the lectin concanavalin A, demonstrating that they were mannoproteins. However, gel electrophoresis of the control and treated proteins revealed that mannosyl groups of hydrophobic proteins were less than 2 kDa in size, while the mannosyl group of the hydrophilic protein had a molecular mass of approximately 20 kDa. These results suggest that unlike many hydrophilic proteins, hydrophobic proteins may have low levels of glycosylation. Changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface.Key words: Candida albicans, cell wall, mannoproteins, hydrophobicity, fibrils.

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An RpoB Mutation Confers Dual Heteroresistance to Daptomycin and Vancomycin in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00437-10 ◽

2010 ◽

Vol 54 (12) ◽

pp. 5222-5233 ◽

Cited By ~ 108

Author(s):

Longzhu Cui ◽

Taisuke Isii ◽

Minoru Fukuda ◽

Tomonori Ochiai ◽

Hui-min Neoh ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Surface ◽

Ribosomal Proteins ◽

Laboratory Strain ◽

Genome Comparison ◽

Nucleotide Position ◽

Nucleotide Polymorphisms ◽

Gene Encoding

ABSTRACT We have previously reported the establishment of a Staphylococcus aureus laboratory strain, 10*3d1, having reduced susceptibility to daptomycin and heterogeneous vancomycin-intermediate S. aureus (VISA) phenotype. The strain was generated in vitro by serial daptomycin selection (Camargo, I. L., H. M. Neoh, L. Cui, and K. Hiramatsu, Antimicrob. Agents Chemother. 52:4289-4299, 2008). Here we explored the genetic mechanism of resistance in the strain by whole-genome sequencing and by producing gene-replaced strains. By genome comparison between 10*3d1 and its parent methicillin-resistant Staphylococcus aureus (MRSA) strain N315ΔIP, we identified five nonsynonymous single nucleotide polymorphisms (SNPs). One of the five mutations was found in the rpoB gene encoding the RNA polymerase β subunit. The mutation at nucleotide position 1862 substituted the 621st alanine by glutamic acid. The replacement of the intact rpoB with the mutated rpoB, designated rpoB(A621E), conferred N315ΔIP with the phenotypes of reduced susceptibility to daptomycin and hetero-VISA. The rpoB(A621E)-mediated resistance conversion was accompanied by a thickened cell wall and reduction of the cell surface negative charge. Being consistent with these phenotypic changes, microarray data showed that the expression of the dlt operon, which increases the cell surface positive charge, was enhanced in the rpoB(A621E) mutant. Other remarkable findings of microarray analysis of the rpoB(A621E) mutant included repression of metabolic pathways of purine, pyrimidine, arginine, the urea cycle, and the lac operon, enhancement of the biosynthetic pathway of vitamin B2, K1, and K2, and cell wall metabolism. Finally, mutations identified in rplV and rplC, encoding 50S ribosomal proteins L22 and L3, respectively, were found to be associated with the slow growth, but not with the phenotype of decreased susceptibility to vancomycin and daptomycin, of 10*3d1.

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Biosynthesis of β-(1→5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

10.1101/814756 ◽

2019 ◽

Author(s):

Yuria Chihara ◽

Yutaka Tanaka ◽

Minoru Izumi ◽

Daisuke Hagiwara ◽

Akira Watanabe ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Aspergillus Fumigatus ◽

Pathogenic Fungus ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Wall Structure ◽

Fungal Cell ◽

Animal Pathogens

ABSTRACTThe pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. Here, we clarified the entire biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatgus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from the strains ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans, and GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidia formation ability as well as increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immune-compromised mice.IMPORTANCEβ-(1→5)-galactofuranosyl residues are widely distributed in the subphylum Pezisomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for their understanding. In this study, we show that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicates that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the subphylum Pezisomycotina.

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Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody

Microbiology ◽

10.1099/mic.0.043851-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3645-3659 ◽

Cited By ~ 26

Author(s):

David A. Coleman ◽

Soon-Hwan Oh ◽

Xiaomin Zhao ◽

Lois L. Hoyer

Keyword(s):

Candida Albicans ◽

Cell Surface ◽

Spatial Localization ◽

Host Cells ◽

Yeast Cells ◽

Cell Surface Antigens ◽

Heterogeneous Distribution ◽

Germ Tubes

Despite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo.

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Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

Applied and Environmental Microbiology ◽

10.1128/aem.00824-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5519-5526 ◽

Cited By ~ 29

Author(s):

Li Zhang ◽

Shuli Liang ◽

Xinying Zhou ◽

Zi Jin ◽

Fengchun Jiang ◽

...

Keyword(s):

Cell Wall ◽

Pichia Pastoris ◽

Cell Surface ◽

Signal Sequence ◽

Recombinant Expression ◽

Cell Wall Proteins ◽

Heterologous Proteins ◽

Content Type ◽

Yeast Cell Surface

ABSTRACTGlycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different usesin vitro. In the present study, the genome ofPichia pastorisGS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and matureCandida antarcticalipase B (CALB). The expression of fusion proteins on the cell surface ofP. pastorisGS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface ofP. pastorisGS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis ofp-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins inP. pastorisGS115, which can be used to display heterologous proteins on the yeast cell surface.

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Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization

Applied and Environmental Microbiology ◽

10.1128/aem.68.7.3385-3391.2002 ◽

2002 ◽

Vol 68 (7) ◽

pp. 3385-3391 ◽

Cited By ~ 23

Author(s):

Tiina Nakari-Setälä ◽

Joana Azeredo ◽

Mariana Henriques ◽

Rosário Oliveira ◽

José Teixeira ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Surface ◽

Surface Properties ◽

Hydrophobic Interactions ◽

Surface Characteristics ◽

Yeast Cells ◽

Phase System ◽

Two Phase ◽

Cell Surface Properties

ABSTRACT The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C12-18EO5.

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Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri

Microbiology ◽

10.1099/mic.0.043265-0 ◽

2010 ◽

Vol 156 (11) ◽

pp. 3368-3378 ◽

Cited By ~ 108

Author(s):

Donald A. MacKenzie ◽

Faye Jeffers ◽

Mary L. Parker ◽

Amandine Vibert-Vallet ◽

Roy J. Bongaerts ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Binding Proteins ◽

Lactobacillus Reuteri ◽

Surface Protein ◽

Cell Aggregation ◽

Spontaneous Mutant ◽

Vertebrate Hosts

Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867i), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins, although present in many L. reuteri isolates, show a high genetic heterogeneity among strains.

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Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

10.1101/2021.07.23.451969 ◽

2021 ◽

Author(s):

Pei Yi Choo ◽

Charles Wang ◽

Michael VanNieuwenhze ◽

Kimberly Kline

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Enterococcus Faecalis ◽

General Rule ◽

Cell Wall Synthesis ◽

Lipid Ii ◽

Growing Cell ◽

Temporal Localization

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.

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The Yeast-Phase Virulence Requirement for α-Glucan Synthase Differs among Histoplasma capsulatum Chemotypes

Eukaryotic Cell ◽

10.1128/ec.00214-10 ◽

2010 ◽

Vol 10 (1) ◽

pp. 87-97 ◽

Cited By ~ 52

Author(s):

Jessica A. Edwards ◽

Elizabeth A. Alore ◽

Chad A. Rappleye

Keyword(s):

Cell Wall ◽

Histoplasma Capsulatum ◽

Yeast Cells ◽

Dependent Manner ◽

Glucan Synthase ◽

Content Type ◽

I Factor ◽

Yeast Phase

ABSTRACTHistoplasma capsulatumstrains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype IIHistoplasmavirulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucanin vitro, yet they remain fully virulentin vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishesAGS1expression. Nonetheless,AGS1mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced duringin vivogrowth despite its absencein vitro. To directly test whetherAGS1contributes to chemotype I strain virulence, we preventedAGS1function by RNA interference and by insertional mutation. Loss ofAGS1function in chemotype I does not impair the cytotoxicity ofags1(−) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, theags1(−) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate thatAGS1expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a uniqueHistoplasmachemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

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