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Fractionation and partial characterization of the hemagglutinating and bacterial aggregating adhesins of Bacteroides gingivalis

University of British Columbia

In order to characterize the surface components responsible for hemagglutination and bacterial aggregation, the outer membrane complex of Bacteroides gingivalis W12 was isolated. It was found to contain both hemagglutinating and bacterial aggregating activity. Examination of the membrane material by biochemical analysis, SDS-polyacrylamide gel electrophoresis and immunological means revealed that the crude outer membrane preparation contained three major proteins and a lipopolysaccharide (LPS) population which displayed size heterogeneity. At least two membrane proteins as well as the LPS were found to be antigenically active - using immun-blot analysis. Using gel chromatography and an LPS disaggregating buffer the membrane material was separated into two fractions. An accompanying separation of the two adherence- activities was observed. The first membrane fraction, containing mostly protein and carbohydrate material was found to contain the bacterial aggregating activity. This fraction also contained a"smooth" IPS population. The second membrane fraction consisted of "rough"type LPS, protein and loosely bound lipid and was found to contain the hemagglutinating activity. Further investigation revealed that bacterial aggregating activity was blocked by the presence of D-galactose (14 mM) , D-glucosamine (23 mm) and N-acetylglucoscmine (23 mm) and by dialysed iitmune rabbit serum. Dialysed non-immune rabbit serum did not block this activity. Bacterial aggregation was not removed by chloroform-methanol extraction or destroyed by proteolytic treatment. Pronase treatment was found in fact to enhance bacterial aggregating activity on a dry weight basis. Based on these observations it appeared that the bacterial aggregating adhesin was either the capsular polysaccharide or "smooth" type LPS. Hemagglutinating activity was blocked by the presence of N-acetyl-galactosamine (23 mM) and bovine brain ganglioside (312 ug/ml). As well, dialysed immune and non-immune serum were found to inhibit this activity, while β-mercaptoethanol and dithiothreitol were found to enhance bacterial aggregation. Both protease digestion and chloroform-methanol extraction destroyed hemagglutinating activity which suggested that the hemagglutinin was either a protein or a lipid-protein complex.

Text

2010-05-10

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http://hdl.handle.net/2429/24554

Microbiology and Immunology

University of British Columbia

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