Biomedical subjects
Publications and source records attributed to R J Doyle.
It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover. Peptidoglycan turnover would cause a turgor on the outer membrane, causing the outer membrane to bulge and finally bleb. Mechanical motion would then shear the blebs into the culture medium.
Cell suspensions of Streptococcus sobrinus can be aggregated by high molecular-weight alpha-1,6 glucans. The aggregation depends on the fidelity of a cell wall-bound, glucan-binding lectin (GBL). It is thought that the lectin may play a part in the sucrose-dependent accretion of streptococci in dental plaques. Results showed that the anionic detergent, sodium dodecyl sulphate (SDS) was a potent inhibitor of the lectin. When cells were incubated in SDS and washed to remove the detergent, lectin activity was diminished. Following incubation of the cells with SDS in the presence of glucan T-10, a low molecular-weight alpha-1,6 glucan, the loss of activity was less pronounced, suggesting that the glucan afforded partial protection against denaturation. Urea and guanidine hydrochloride were good inhibitors of the lectin, but, unlike SDS, were not able to inhibit it irreversibly, except at very high concentrations. Cationic detergents, such as cetylpyridinium bromide (and chloride), also irreversibly denatured the streptococcal lectin, but were not as effective as SDS in abolishing its activity. The results suggest that alpha-1,6 glucan stabilizes the GBL of S. sobrinus, rendering it more resistant to the effect of chaotropes. This may be one reason why dental plaques tend to resist detergents in dentrifices.
Lectins of various specificities were examined for interaction with strains of oral streptococci of various species. The lectins were capable of binding galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine, mannose, fucose and sialic acid. Lectin reactivities were strain-dependent in that some members of a species, but not others, could be aggregated by certain lectins. Proteolysis and extraction with hot water, guanidine hydrochloride and sodium dodecyl sulphate tended to increase the reactivity of the streptococci with lectins but did not change the recognition patterns of the bacteria. Sonication, in contrast, tended to reduce the ability of streptococci to be agglutinated by lectins. Furthermore, lectin reactivities were dependent on the growth conditions, as evidenced by changes in lectin titres following streptococcal growth in sub-inhibitory concentrations of citrate, fluoride or antibiotics. It is likely that lectins could be useful tools for epidemiological studies and to probe strain-dependent and growth-dependent surface characteristics of viridans streptococci.
Type 1 fimbriae are the most common adhesive organelles of Escherichia coli. Because of their virtual ubiquity, previous epidemiological studies have not found a correlation between the presence of type 1 fimbriae and urinary tract infections (UTIs). Recently it has become clear that type 1 fimbriae exhibit several different phenotypes, due to allelic variation of the gene for the lectin subunit, FimH, and that these phenotypes are differentially distributed among fecal and UTI isolates. In this study, we have analyzed in more detail the ability of isogenic, recombinant strains of E. coli expressing fimH genes of the predominant fecal and UTI phenotypes to adhere to glycoproteins and to uroepithelial cells. Evidence was obtained to indicate that type 1 fimbriae differ in their ability to recognize various mannosides, utilizing at least two different mechanisms. All FimH subunits studied to date are capable of mediating adhesion via trimannosyl residues, but only certain variants are capable of mediating high levels of adhesion via monomannosyl residues. The ability of the FimH lectins to interact with monomannosyl residues strongly correlates with their ability to mediate E. coli adhesion to uroepithelial cells. In this way, it would be possible for certain phenotypic variants of type 1 fimbriae to contribute more than others to virulence of E. coli in the urinary tract.
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Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.
The Gram stain, the most important stain in microbiology, was described more than a century ago. Only within the past decade, however, has an understanding of its mechanism emerged. It now seems clear that the cell wall of Gram-positive microorganisms is responsible for retention of a crystal violet:iodine complex. In Gram-negative cells, the staining procedures damage the cell surface resulting in loss of dye complexes. Gram-positive microorganisms require a relatively thick cell wall, irrespective of composition, to retain the dye. Therefore, Gram-stainability is a function of the cell wall and is not related to chemistry of cell constituents. This review provides a chronology of the Gram stain and discusses its recently discovered mechanism.
Tetranitromethane (TNM), a protein nitrating reagent, was shown to be effective in killing Gram-positive and Gram-negative bacteria, bacterial endospores and fungi. The killing action was greater in alkaline media, where nitration was most effective. TNM has a high affinity for hydrophobic solvents, and it is suggested that the reagent killed micro-organisms by nitrating critical membrane proteins.
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In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli, E. coli DH5 alpha cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized component(s) on the transformant outer surface.
The glucan-binding lectin (GBL) of Streptococcus sobrinus is cell associated, enabling the bacteria to be aggregated by alpha-1,6 glucans. Glucans, such as amylose, pullulan, laminarin and nigeran, have no affinity for the lectin. High molecular weight alpha-1,6 glucans (dextrans) readily aggregate the bacteria, whereas low molecular weight glucans inhibit the aggregation brought about by the high molecular weight species. Methylated glucan T-2000 (an alpha-1,6 glucan with an average molecular weight of 2 x 10(6) Da) aggregated the bacteria very poorly when the extent of methylation (DS, or degree of substitution) was high, and less poorly when the DS was low. Similarly, methylated low molecular weight alpha-1,6 glucan was a poor inhibitor of aggregation induced by the high molecular weight glucan T-2000. Because the methylation occurred primarily on the hydroxyl of C-2, it is suggested that the hydroxyl is needed for formation of the lectin-glucan complex. It appears that the GBL is not only stereospecific in interaction with glucans, but also regio-specific, interacting only with the underivatized alpha-1,6-glucan.
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The inhibition of saliva-induced oral streptococcal aggregation with anti-sera (anti-A, anti-B, anti-AB and anti-B treated with galactose), normal human serum (NHS), blood group-specific lectins (UEA-I, HBA, GPA, BSI-B4, GS-I), non-specific blood group lectins (MPA, SBA) and carbohydrates (galactose, N-acetylgalactosamine, L-fucose) was studied. Streptococcal species and strains included S. mutans 318, S. mutans 10449, S. mutans NG-8, S. salivarius and S. cricetus HS-6. The saliva was obtained from three subjects with secretor status (2 blood group B persons, 1 blood group A person). The data obtained from experiments performed with S. mutans 10449 and S. mutans NG-8 suggest the involvement of the H-antigenic determinant in the aggregation mechanism of the first strain and of the group B determinant for the second strain. The aggregation of S. salivarius only by B saliva might be related to a galactose-specific lectin on this strain and to some properties of its cell surface (hydrophobicity and the fibrillar surface layer). S. cricetus HS-6 aggregation was inhibited in different degrees by all the inhibitors used. The results demonstrate that interactions between oral streptococci and salivary components depend on the strain and species and on the individual saliva samples.
Several antibiotics, at subinhibitory concentrations, caused an increase in the ability of Streptococcus sobrinus to bind alpha-1,6-glucans, whereas other antibiotics decreased glucan binding. In every case, glucan binding was inversely proportional to cell surface hydrophobicity. High levels of glucan-binding activity resulted in low levels of hydrophobicity, whereas low levels of glucan binding caused high levels of cellular hydrophobicity. The results show that low concentrations of antibiotics may modulate lectin and hydrophobin adhesins in streptococci.
The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.
Tetranitromethane, a protein nitrating agent, was tested for its ability to disinfect surfaces from viruses. Different surfaces on commercially available pocket calculators were pretreated with either the Indiana strain of vesicular stomatitis virus or the Herts' strain of Newcastle disease virus. The calculators surfaces were then sprayed with either tetranitromethane or control solutions. The calculators were incubated for 30 min at ambient temperature, and then the surfaces were wiped with sterile swabs. The swabs were placed into test tubes containing phosphate-buffered saline. Samples of the phosphate-buffered saline were then titered on appropriate cell lines by plaque assay. The results indicated that the amount of vesicular stomatitis virus and Newcastle disease virus recovered from the tetranitromethane-treated surfaces was dramatically decreased compared to the amount of virus recovered from control-treated surfaces. These data suggest that tetranitromethane may be useful to disinfect surfaces from both enveloped and non-enveloped RNA viruses.
The peptidoglycan sacculus serves as a mechanical framework for the cell walls of most eubacteria and largely determines cell shape. The notion that the structure is a rigid shell is contradicted by findings that peptidoglycan can expand and contract. Thus, the sacculus functions as an elastic, flexible, polyionic, amphoteric, restraining network.