Specificity of mycoparasite attachment to the host cell surface

1985 ◽  
Vol 63 (4) ◽  
pp. 772-778 ◽  
Author(s):  
M. S. Manocha
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Recognition ◽  
Binding Reaction ◽  
Choanephora Cucurbitarum ◽  
Host Cell Surface ◽  
Germ Tubes

The use of isolated cell wall fragments of Choanephora cucurbitarum (Berk. & Rav.) Thaxter (a host), and of Linderina pennispora Raper and Fennell (a nonhost), has provided not only a convenient method to quantify attachment of the parasite, Piptocephalis virginiana Leadbeater and Mercer, by the artificial inoculation and washing-off procedure, but also an excellent material for investigations on the molecular basis of specificity and host recognition. The parasite germ tubes are attached to the cell wall fragments of the host but not of the nonhost. Attachment was inhibited by the addition of sugars, chitobiose and chitotriose, and by treatment with acid or alkali indicating the involvement of proteins or glycoproteins in recognizing sugar residues at the cell surface. Both host and nonhost showed a positive binding reaction with fluorescent lectins specific for N-acetyl-D-glucosamine oligomer. The cell surface of the nonhost also contains D-galactose and N-acetyl-D-galactosamine residues as lectin binding sites. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of cell wall extracts of host and nonhost revealed four bands of glycoproteins common to both fungi and two were specific to the host.

Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

1997 ◽  
Vol 43 (7) ◽  
pp. 625-632 ◽  
Author(s):  
M. S. Manocha ◽  
D. Xiong ◽  
V. Govindsamy
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Western Blot ◽  
Host Cell ◽  
Immunofluorescence Microscopy ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Primary Antibody ◽  
Rabbit Igg ◽  
Host Cell Surface

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.

Cell surface characteristics of Mortierella species and their interaction with a mycoparasite

1984 ◽  
Vol 30 (3) ◽  
pp. 290-298 ◽  
Author(s):  
M. S. Manocha
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Characteristics ◽  
Protein Patterns ◽  
Periodic Acid Schiff ◽  
The Difference ◽  
Chitin And Chitosan

Cell surface characteristics of three Mortierella species differing in their response to a mycoparasite, Piptocephalis virginiana, were examined. Their cell wall composition was typical of mucoraceous fungi with chitin and chitosan as major polysaccharides. Electron microscopy revealed that the mycoparasite penetrated and formed haustoria in the hyphae of susceptible hosts, M. pusilla and M. isabellina. The failure of the parasite to establish contact and penetrate a hypha of the nonhost, M. candelabrum, was not due to cell wall thickness, rigidity, or chitin contents. Markedly different protein patterns obtained from crude alkali extracts of host and nonhost cell walls by sodium dodecyl sulfate – polyacrylamide gel electrophoresis might explain the difference in host and nonhost response to the mycoparasite. Whereas most of the bands differed only in intensity after staining with either Coomassie blue or periodic acid – Schiff reagent, there were two distinct bands of glycoproteins (76 000 and 74 000) observed in the host species which were absent in the nonhost species.

Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite

1991 ◽  
Vol 37 (5) ◽  
pp. 377-383 ◽  
Author(s):  
M. S. Manocha ◽  
Y. Chen
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Carbohydrate Binding ◽  
Protein Extract ◽  
Host Cell Wall ◽  
Host Cell Surface ◽  
Faint Band

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, of the mycoparasite Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 agglutination units/mg) compared with that of the nonhost extract (21 agglutination units/mg). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the crude extract of the host revealed four bands, a, b, c, and d, with respective Mr of 117 000, 100 000, 85 000 and 64 000; these bands except for a faint band c, were absent from the nonhost surface. Deletion of proteins b or c from the crude protein extract of the host significantly reduced its agglutinating activity. Proteins b and c, purified by a series of procedures, were shown to be glycoproteins with glucose and N-acetylglucosamine as major saccharides. The agglutinating activity of a mixture of pure proteins b and c was over 500 times that of either glycoprotein alone, suggesting an involvement of both glycoproteins in the agglutination process. Further characterization showed that the two glycoproteins were heat-resistant with respect to their agglutinin function, which could be totally inhibited by three sugars: arabinose, glucose and N-acetyglucosamine. It is suggested that glycoproteins b and c are the two subunits of a carbohydrate-binding agglutinin present at the host cell surface and involved in agglutination and attachment of the mycoparasite germ tubes. Key words: agglutinin, attachment, cell surface, sugars, glycoproteins, mycoparasitism.

Involvement of cell surface sugars in recognition, attachment, and appressorium formation by a mycoparasite

1990 ◽  
Vol 36 (11) ◽  
pp. 771-778 ◽  
Author(s):  
M. S. Manocha ◽  
Y. Chen ◽  
N. Rao
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Host Cell ◽  
Germ Tube ◽  
Lectin Binding ◽  
Fluorescein Isothiocyanate ◽  
Appressorium Formation ◽  
Obvious Effect ◽  
Surface Attachment ◽  
Host Cell Surface

Fluorescein isothiocyanate labeled lectin binding techniques have revealed differences in the distribution pattern of glycosyl residues at the cell wall level between fungi that are hosts and those that are nonhosts of the mycoparasite Piptocephalis virginiana, and at the protoplast level between compatible and incompatible hosts. The cell wall of the compatible hosts (Choanephora cucurbitarum and Mortierella pusilla) and an incompatible host (Phascolomyces articulosus), as well as that of the mycoparasite itself, contains glucose and N-acetylglucosamine. However, the cell wall of a nonhost (Mortierella candelabrum) tested positive with lectins specific for various sugars, including not only glucose and N-acetylglucosamine, but also fucose, N-acetylgalactosamine, and galactose. These latter sugars could also be exposed at the surfaces of hosts and of the mycoparasite, but only after mild treatment with proteinase or when grown in a liquid culture. Pretreatment of the mycoparasite with glucose and N-acetylglucosamine inhibited its attachment to the host cell surface, but had no obvious effect on appressorium formation. On the other hand, appressorium formation was inhibited by heat treatment of host cell wall fragments which still permitted attachment, thus indicating that the factors responsible for attachment and for appressorium formation are different. The protoplast surfaces of compatible hosts contained all the sugars listed above and these protoplasts could attach to the germ tube of the mycoparasite. Only lectins specific for N-acetylglucosamine and for glucose were bound at the protoplast surface of the incompatible host; these protoplasts did not attach to the mycoparasite germ tube. Key words: mycoparasite, appressorium formation, lectins, host cell surface, attachment, protoplast surface.

scholarly journals Lectin-binding components of normal granulocytes and leukaemic myeloid cells

1983 ◽  
Vol 213 (3) ◽  
pp. 661-670 ◽  
Author(s):  
F A Spring ◽  
D J Anstee
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Helix Pomatia ◽  
Sodium Dodecyl ◽  
Myeloid Cells ◽  
Neuraminidase Treatment ◽  
Leukaemic Cells

A panel of lectins was used to analyse glycoproteins of normal granulocytes and leukaemic myeloid cells. The glycoproteins of detergent-solubilized whole cells were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their lectin-binding properties determined by incubation of the fixed gels with radioiodinated lectins. Normal granulocytes and leukaemic myeloid cells in different stages of maturation possess a cell-surface sialic acid-rich glycoprotein of apparent mol.wt. 115 000 (GP115), that can be labelled by both the lactoperoxidase and periodate/NaB3H4 cell-surface labelling techniques. The sialoglycoprotein of leukaemic myeloblasts has a slightly lower apparent mol.wt., 112000 (GP112). After neuraminidase treatment before cell solubilization, both GP115 and GP112 bind the lectins from Arachis hypogaea (peanut) and Helix pomatia (snail) and have an increased apparent molecular weight of 125000. Two concanavalin A-binding glycoproteins of apparent mol.wts. 98000 and 90000 are present in leukaemic myeloblasts. Concanavalin A binding to these glycoproteins is decreased in more mature leukaemic cells and absent in granulocytes. As concanavalin A binding decreases in the maturer forms, there is a concomitant increase in the binding of Ricinus communis (castor bean) and Maclura aurantiaca (osage orange) lectins to these glycoproteins. Whole granulocytes, but not leukaemic myeloblasts, contain a major cell-surface concanavalin A binding glycoprotein of apparent mol.wt. 130000, which is labelled by the periodate/NaB3H4 technique. Concanavalin A binding to this glycoprotein increases as the morphology of leukaemic cells approaches that of mature granulocytes.

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum

1982 ◽  
Vol 152 (1) ◽  
pp. 298-305
Author(s):  
P Dehazya ◽  
R S Coles
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fusobacterium Nucleatum ◽  
Blue Staining ◽  
Sephadex Column ◽  
Dodecyl Sulfate ◽  
Triton X

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.

scholarly journals Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes

1981 ◽  
Vol 194 (1) ◽  
pp. 351-355 ◽  
Author(s):  
J G Salisbury ◽  
J M Graham
Keyword(s):  
Cell Surface ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
New Method

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.

scholarly journals A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane

1976 ◽  
Vol 153 (2) ◽  
pp. 265-270 ◽  
Author(s):  
M J A Tanner ◽  
D J Anstee
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Erythrocyte Membrane ◽  
Individual Pattern ◽  
Direct Demonstration ◽  
Staining Techniques ◽  
Membrane Components

1. A method which allows the characterization of lectin-binding components is described. This method should be useful in defining the nature and heterogeneity of these components in cell membranes. 2. The method, which we have used on erythrocyte “ghosts”, involves the fixation of “ghost” components after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and incubation with purified 125I-labelled lectins. 3. Each of the four lectins used shows an individual pattern of reactivity towards “ghosts” components. Band 3, the major membrane-penetrating glycoprotein, is bound by the lectins from Ricinus communis and Phaseolus vulgaris (phytohaemagglutinin) and by concanavalin A. The major erythrocyte sialoglycoprotein is bound by the lectins from R. communis, P. vulgaris and Maclura aurantiaca. 4. Three of the lectins displays binding for other membrane components, some of which are not demonstratable by conventional protein- and carbohydrate-staining techniques.

Cell wall associations of some antigenic proteins of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product "viili"

1986 ◽  
Vol 32 (2) ◽  
pp. 176-178 ◽  
Author(s):  
Raili Forsén ◽  
Teuvo Hentunen ◽  
Kaua Valkonen ◽  
Sirpa Kontusaari
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Cell Walls ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fermented Milk ◽  
Milk Product ◽  
Streptococcus Cremoris ◽  
Protein Components

Cell walls were isolated from mechanically disrupted cells of the slime-forming, encapsulated Streptococcus cremoris strains T5 and MLS96 by using sucrose gradient centrifugation as the last purification step. This cell wall isolation procedure was developed to obtain cell wall associated protein components. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed several polypeptide bands; the 50 kiloDalton band was major in strain T5 cell walls and the 26 and 30 kiloDalton bands were major in strain MLS96 cell walls. Both strains contained five antigenic polypeptides with molecular radius (Mr) values of 40, 47, 50, 54, and 70 kiloDaltons as analysed by immunoblotting and autoradiography. The polypeptides of strain MLS96 with molecular mass of 40 and 70 kiloDaltons reacted most strongly with homologous anti-whole cell serum. In addition, antigenic polypeptides with molecular mass of 100 and 160 kiloDaltons were also detected in strain T5.

scholarly journals Identification of the major lectin-binding surface proteins of human neutrophils and alveolar macrophages

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1624-1632
Author(s):  
NP Christiansen ◽  
KM Skubitz
Keyword(s):  
Cell Surface ◽  
Alveolar Macrophages ◽  
Cell Function ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Lectin Affinity Chromatography ◽  
Con A ◽  
Major Surface

Concanavalin A (Con A) and wheat germ agglutinin (WGA) are frequently used as stimuli of neutrophils and macrophages. While the effects of these lectins on cell function are presumably mediated by interaction with cell-surface molecules, the target structures on the cell surface involved are not well defined. We have used the techniques of lactoperoxidase catalyzed cell-surface iodination, lectin affinity chromatography, monoclonal antibody immunoprecipitation, and NaDodSO4- polyacrylamide gel electrophoresis to study the surface proteins of human neutrophils and alveolar macrophages that react with six lectins including Con A and WGA. We found that several major surface-labeled proteins of neutrophils bound Con A. Four of these proteins were identified by immunoprecipitation as members of the LFA-1/HMac- 1/gp150,95 adhesion glycoprotein family. Con A also bound CR1 and a 135- kd surface-labeled protein recognized by CD15 monoclonal antibodies. WGA also bound many of these proteins, but had a much lower avidity for CR1. All three of the major surface-labeled proteins of human alveolar macrophages bound to Con A, including the 183-kd mannose receptor and the 30-kd smoking-associated protein. WGA also bound the 183-kd macrophage protein, but not the 30-kd protein. These results should aid the understanding of studies using these lectins as stimuli.

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