Factors affecting the molecular structure and the agglutinating ability of concanavalin A and other lectins.
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Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature. It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer-tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4 degrees up to 37 degrees C). It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin-A-induced agglutination was previously found to be temperature-sensitive. These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and nott only to modification of cell surface properties. (+info)
Mutations in the lspA1 and lspA2 genes of Haemophilus ducreyi affect the virulence of this pathogen in an animal model system.
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Haemophilus ducreyi 35000HP contains two genes, lspA1 and lspA2, whose predicted protein products have molecular weights of 456,000 and 543,000, respectively (C. K. Ward, S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen, J. Bacteriol. 180:6013-6022, 1998). We have constructed three H. ducreyi 35000HP mutants containing antibiotic resistance cartridges in one or both of the lspA1 and lspA2 open reading frames. Western blot analysis using LspA1- and LspA2-specific monoclonal antibodies indicated that the wild-type parent strain 35000HP expressed LspA1 protein that was readily detectable in culture supernatant fluid together with a barely detectable amount of LspA2 protein. The lspA2 mutant 35000HP.2 expressed LspA1 protein that was detectable in culture supernatant fluid and no LspA2 protein. In contrast, the H. ducreyi lspA1 mutant 35000HP.1, which did not express the LspA1 protein, expressed a greater quantity of the LspA2 protein than did the wild-type parent strain. The lspA1 lspA2 double mutant 35000HP.12 expressed neither LspA1 nor LspA2. The three mutant strains adhered to human foreskin fibroblasts and to a human keratinocyte cell line in vitro at a level that was not significantly different from that of the wild-type strain 35000HP. Lack of expression of the LspA1 protein by both the lspA1 mutant and the lspA1 lspA2 double mutant was associated with an increased tendency to autoagglutinate. When evaluated in the temperature-dependent rabbit model for chancroid, the lspA1 lspA2 double mutant was substantially less virulent than the wild-type strain 35000HP. The results of these studies indicated that H. ducreyi requires both the LspA1 and LspA2 proteins to be fully virulent in this animal model for experimental chancroid. (+info)
Environmental isolates of Citrobacter braakii that agglutinate with Escherichia coli O157 antiserum but do not possess the genes responsible for the biosynthesis of O157 somatic antigen.
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While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum. The five C. braakii isolates gave positive results when a sensitive dot-ELISA was performed with E. coli O157 monoclonal antibody. Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C. braakii isolates. Apart from weak reactions with two or three of the DNA probes, all the C. braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis. These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E. coli (STEC) specially the serotype O157: H7. Therefore, it appears that the serological cross-reaction between C. braakii and E. coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C. braakii and E. coli O157. (+info)
Variants of hamster fibroblasts resistant to Ricinus communis toxin (ricin).
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1. Variant baby-hamster kidney (BHK) cell lines were isolated that grow in the presence of high concentrations of ricin, the toxic lectin of castor beans (Ricinus communis). The variant lines were independently derived from several cultures of normal BHK cells which had been exposed to the mutagen, methyl-N-nitro-N-nitrosoguanidine, before selection by ricin. 2. The cell lines maintain a high degree of resistance to ricin after growth in lectin-free medium for prolonged periods and therefore exhibit stable phenotypes that are different from normal BHK cells. 3. A preliminary classification of the phenotypes was made. Several cell lines bind normal amounts of 125I-labelled ricin, whereas other bind the lectin poorly. 4. A loss of surface receptors for two other lectins, R. communis RCA and Axinella polyploides, which have specificities similar to ricin, was also found in some but not all of the cell lines showing decreased surface concentrations of ricin receptors. 5. The binding to the ricin-resistant cells of lectins of different sugar specificity, namely Lens culinaris lectin and concanavalin A, was similar to, or higher than, to normal BHK cells. 6. Several of the ricin-resistant cell lines were shown to be cross-resistant to the weak cytotoxicity of Phaseolus vulgaris lectin. By contrast, some cell lines were more sensitive to concanavalin A than were normal BHK cells. (+info)
The immunoglobulin D-binding protein MID from Moraxella catarrhalis is also an adhesin.
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The Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is a 200-kDa outer membrane protein displaying a unique and specific affinity for human IgD. MID is found in the majority of M. catarrhalis strains. In the present paper, we show that MID-expressing M. catarrhalis strains agglutinate human erythrocytes and bind to type II alveolar epithelial cells. In contrast, M. catarrhalis isolates with low MID expression levels and two mutants deficient in MID, but with readily detectable UspA1 expression, do not agglutinate erythrocytes and have a 50% lower adhesive capacity. To examine the adhesive part of MID, the protein was dissected into nine fragments covering the entire molecule. The truncated MID proteins were expressed in Escherichia coli, purified, and used for raising polyclonal antibodies in rabbits. Interestingly, by using recombinant fragments, we show that the hemagglutinating and adhesive part of MID is localized within the 150-amino-acid fragment MID(764-913). In addition, antibodies against full-length MID, MID(764-913), or a 30-amino-acid consensus sequence (MID(775-804)) inhibited adhesion to alveolar epithelial cells. Antibodies against UspA1, an outer membrane protein expressed in essentially all M. catarrhalis strains, also inhibited adhesion, suggesting that both MID and UspA1 are needed for optimal attachment to epithelial cells. Taken together, in addition to MID-dependent IgD binding, we have demonstrated that the outer membrane protein MID is a novel adhesin that would be a suitable target for a future vaccine against M. catarrhalis. (+info)
Roles of sexual cell agglutination in yeast mass mating.
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The agalpha1 mutant MAT alpha cells specifically lack the cell surface alpha-type sexual agglutination substance, which is also called alpha-agglutinin. Because the mutant cells (MATalpha agalpha1) can not form aggregates with MATa cells, MATalpha agalpha1 cells are unable to mate with MATa cells when they are co-inoculated in a liquid medium, and the mating is attenuated on solid medium. The attenuated mating ability shown in the previous studies gave us a vague idea about a physiological function of the sexual agglutinability. In order to solve the question, mating behavior of MATalpha agalpha1 cells was investigated here under conditions where the contact between MATa and MAT alpha cells is assisted by physical methods. A synthetic mutation agalpha1::URA3 was constructed and used as well as agalpha1-1 for this study to ensure the genetic defect. When a mixture of MATa and MAT alpha cells was kept on filter membrane placed on relatively dry agar medium, even agalpha1::URA3 mutant cells mated as efficiently as the wild type (AGalpha1) cells did. On filter membrane placed on moist agar medium, agalpha1 mutants mated 10-fold less efficiently than wild type cells did. The mutant cells mated 10000-time less efficiently than the wild type cells in a pellet formed by brief low speed centrifugation. In contrast, the wild type MATalpha cells mated well under all conditions tested. Under the pellet condition, a mixture of MATa and MATalpha AG alpha1 cells formed an extended and cotton-like pellet while a mixture of MATa and MATalpha agalpha1 cells formed a compact and tight pellet. These results suggest that sexual cell agglutination contributes not only to cell contact between MATa and MAT alpha cells thereby stabilizing a-alpha cell pairs, but also to construction of a uniquely organized ultra structure favorable for zygote formation and subsequent growth of diploid cells. The mating specific extended pellet formation was observed also in 4 pairs of a and alpha strains in ascosporogenous yeast genera Hansenula and Pichia. (+info)
Glucosyltransferase production by Streptococcus sanguis 804 (NCTC 10904).
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Streptococcus sanguis 804 (NCTC 10904) was grown ih batch culture at constant pH. and the glucosyltransferase activity of the supernatant was assayed over a 40-h growth period. The optimum pH for enzyme production was 7.0 to 7.2. During growth of the culture, three reproducible phases of enzyme activity were observed. The polysaccharides synthesized during each of these phases were characterized as dextran-like glucans by analysis of acid hydrolysates, gas-liquid chromatography, and a specific aggregation technique. The glucans were studied further by infrared spectroscopy, enzymic degradation, and periodate oxidation. Differences in the proportions of alpha-(1 leads to 3)- and alpha-(1 leads to 6)-linkages were observed. The results suggest that glucan synthesis by S. sanguis involves a multienzyme system. (+info)
Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic-adherent Staphylococcus epidermidis: evidence for functional relation to intercellular adhesion.
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The initial attachment and the accumulation of Staphylococcus epidermidis on polymer surfaces in multilayered cell clusters embedded in amorphous slime, which together lead to the plastic-adherent phenotype detected by the adherence assay used in this study, have been proposed to be major virulence factors of these bacteria. An antigen specific for plastic-adherent S. epidermidis strains was detected by an indirect immunofluorescence test using absorbed antiserum raised against the strongly plastic-adherent S. epidermidis 1457. A coagglutination assay was established, which allowed the quantitation of the antigen in bacterial extracts under different physiologic growth conditions. Expression of the antigen and of plastic adherence depended significantly on the presence of glucose in the growth medium. Parallel to increased plastic adherence, a 32- to 64-fold increase in the amount of the antigen was detected in bacterial extracts of cells grown in tryptone soya broth (TSB) compared with that in extracts of cells grown in TSB lacking glucose. A parallel time-dependent increase of plastic adherence and expression of the antigen was observed after stimulation by glucose of stationary-phase cultures of plastic-adherent S. epidermidis strains grown in TSB lacking glucose. The antigen consisted most probably of polysaccharide, because its immunologic reactivity was completely abolished by periodate oxidation but was resistant to protease digestion. A significant proportion of cells of plastic-adherent as compared with nonadherent S. epidermidis strains grown in TSB were located in large cell clusters exceeding 50 cells, which completely disintegrated after periodate oxidation of the cell preparations. Periodate oxidation of adherent bacterial films in situ led to release of the adherent cells from the plastic surface. These results strongly indicate a functional relation of the antigen to adherence of S. epidermidis to polymer surfaces, most probably by mediating intercellular adhesion of cells leading to accumulation in multilayered cell clusters. (+info)