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

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.

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

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
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 ◽  
Content Type ◽  
Animal Pathogens

ABSTRACT The 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. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatus. 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 at 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 ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice. IMPORTANCE β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina 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 understanding them. In this study, we showed 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 indicate 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 members of the subphylum Pezizomycotina.

scholarly journals Characterization of Monolaurin Resistance in Enterococcus faecalis

2007 ◽  
Vol 73 (17) ◽  
pp. 5507-5515 ◽  
Author(s):  
Muriel Dufour ◽  
Janet M. Manson ◽  
Philip J. Bremer ◽  
Jean-Pierre Dufour ◽  
Gregory M. Cook ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Wall Structure ◽  
Transmission Electron ◽  
Serious Infections ◽  
Autolytic Activity

ABSTRACT There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 μg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

Hydrophobic cell wall protein glycosylation by the pathogenic fungus Candida albicans

1994 ◽  
Vol 40 (4) ◽  
pp. 266-272 ◽  
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.

scholarly journals The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

2002 ◽  
Vol 68 (4) ◽  
pp. 2018-2025 ◽  
Author(s):  
Hitoshi Shimoi ◽  
Kazutoshi Sakamoto ◽  
Masaki Okuda ◽  
Ratchanee Atthi ◽  
Kazuhiro Iwashita ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Repetitive Sequences ◽  
Alcoholic Beverage ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
A Cell ◽  
Characteristic Sequences ◽  
Foam Forming ◽  
Almost All

ABSTRACT Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.

scholarly journals Probiotic potential and safety characterization of Enterococcus hirae G24 isolated from indigenous raw goat milk

2020 ◽  
pp. 218-226
Author(s):  
Kamni Rajput ◽  
Ramesh Chandra Dubey
Keyword(s):  
Cell Surface ◽  
Pathogenic Bacteria ◽  
Cell Surface Hydrophobicity ◽  
Raw Milk ◽  
Surface Hydrophobicity ◽  
Rrna Gene ◽  
Enterococcus Hirae ◽  
Bacterial Cultures ◽  
Probiotic Potential

In this paper, an investigation on lactic acid bacterial isolates from ethnic goat raw milk samples were examined for their probiotic potential and safety parameters. For this purpose, isolated bacterial cultures were screened based on certain parameters viz., sugar fermentation, tolerance to temperature, salt, low pH, bile salts, and phenol resistance. After that, these bacterial cultures were more estimated in vitro for auto-aggregation, cell surface hydrophobicity, response to simulated stomach duodenum channel, antibiotic resistance, and antimicrobial activity. Besides, probiotic traits show the absence of gelatinase and hemolytic activity supports its safety. The isolate G24 showed good viability at different pH, bile concentration, phenol resistance and response to simulated stomach duodenum passage but it did not show gelatinase and hemolytic activities. Isolate G24 was susceptible to amikacin, carbenicillin, kanamycin, ciprofloxacin, co-trimazine, nitrofurantoin, streptomycin, and tetracycline. Isolate G24 also exhibited antimicrobial action against five common pathogenic bacteria, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogens, and Salmonella typhimurium. It displayed the maximum auto-aggregation, cell surface hydrophobicity to different hydrocarbons. Following molecular characterization the isolate G24 was identified as Enterococcus hirae with 16S rRNA gene sequencing and phylogeny. E. hirae G24 bears the excellent properties of probiotics.

Lactobacilli Isolated from Chicken Intestines: Potential Use as Probiotics

1999 ◽  
Vol 62 (3) ◽  
pp. 252-256 ◽  
Author(s):  
C. GUSILS ◽  
A. PÉREZ CHAIA ◽  
S. GONZÁLEZ ◽  
G. OLIVER
Keyword(s):  
Cell Surface ◽  
Mixed Culture ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Probiotic Properties ◽  
Culture Studies ◽  
Salmonella Gallinarum ◽  
Feed Formulation ◽  
Lactobacillus Animalis

Lactobacillus strains were tested for their in vitro probiotic properties. Cell surface hydrophobicity was found to be very high for Lactobacillus fermentum subsp. cellobiosus and Salmonella Gallinarum; high values could indicate a greater ability to adhere to epithelial cells. Studies on Lactobacillus animalis indicated relative cell surface hydrophobicities smaller than those of L. fermentum subsp. cellobiosus and L. fermentum. L. animalis and Enterococcus faecalis were able to coaggregate with L. fermentum subsp. cellobiosus and L. fermentum, respectively, but not with Salmonella Gallinarum. After mixed-culture studies for determining suitable growth behavior, the pair of strains L. animalis plus L. fermentum subsp. cellobiosus was selected for an attempted challenge against Salmonella Gallinarum. Double and triple mixed-culture studies indicated that selected lactobacillus strains were able to retain their beneficial characteristics in the presence of Salmonella Gallinarum such as presence of lectins, production of antimicrobial compounds, and ability to grow and compete. The selected microorganisms can be considered as potential ingredients for a chicken probiotic feed formulation intended to control salmonellosis and also improve poultry sanitation.

scholarly journals Development of Selenized Lactic Acid Bacteria and their Selenium Bioaccummulation Capacity

Fermentation ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. 91
Author(s):  
Gabriela Krausova ◽  
Antonin Kana ◽  
Ivana Hyrslova ◽  
Iva Mrvikova ◽  
Miloslava Kavkova
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Cell Surface ◽  
Sodium Selenite ◽  
Antioxidant Properties ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
In Vivo Studies ◽  
Dpph Method

Selenized lactic acid bacteria (LAB) represent potentially safe and effective sources of selenium (Se), essential for human health, as lactic acid fermentation improves Se bioavailability and reduces its toxicity. LAB are generally recognized as safe (GRAS) and widely used in fermented dairy products. To facilitate selenized LAB implementation as a functional food, we developed and characterized new Se-enriched strains based on the food industry commercial strains Streptococcus thermophilus CCDM 144 and Enterococcus faecium CCDM 922A as representatives of two LAB genera. We evaluated Se bioaccumulation capacity, Se biotransformation and growth ability in the presence of different sodium selenite concentrations (0–50 mg/L), and antioxidant properties (2, 2-diphenyl-1-picrylhydrazyl (DPPH) method) and cell surface hydrophobicity between Se-enriched and parental strains in vitro. Sodium selenite addition did not negatively influence growth of either strain; thus, 50 mg/L was chosen as the optimal concentration based on strain accumulation capacity. Selenization improved the antioxidant properties of both strains and significantly increased their cell surface hydrophobicity (p < 0.05). To our knowledge, this represents the first report of Se-enriched strain hydrophobicity as well as the first on Se speciation in families Enterococcaceae and Streptococcaceae. Moreover, both tested strains demonstrated good potential for Se-enrichment, providing a foundation for further in vitro and in vivo studies to confirm the suitability of these Se-enriched strains for industrial applications.

scholarly journals Microenvironment ofMycobacterium smegmatisCulture to Induce Cholesterol Consumption Does Cell Wall Remodeling and Enables the Formation of Granuloma-Like Structures

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Ana Cristina Doria dos Santos ◽  
Victor Hugo de Souza Marinho ◽  
Pedro Henrique de Aviz Silva ◽  
Barbarella de Matos Macchi ◽  
Mara Silvia Pinheiro Arruda ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Complete Medium ◽  
Alternative Source ◽  
Cholesterol Supplementation ◽  
Mycobacterial Cell Wall ◽  
Bacteria Culture ◽  
Phosphatidylinositol Mannosides

Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as anin vitroexperimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenicMycobacterium smegmatisin MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDaandLAM37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H2O2. The infection of J774 macrophages withM. smegmatis,after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.

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