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Identification of a fimbriae-associated haemagglutinin from Prevotella intermedia
K Leung1, W E Nesbitt, M Okamoto
1College of Dentistry, University of Florida, Gainesville, FL 32610, USA.
Abstract:
Prevotella intermedia, a putative periodontopathic microorganism, possesses various types of fimbriae (surface appendages). Some of these surface structures mediate the adherence of the organism to several mammalian erythrocytes, resulting in the agglutination of the erythrocytes. Prevotella intermedia fimbriae were solubilized and separated by preparative SDS gel electrophoresis followed by preparative isoelectric focusing to determine which fimbrial component(s) were responsible for the haemagglutinating activity exhibited by these bacteria. Heat treatment of isolated fractions which exhibited haemagglutinating activity revealed the presence of two types of haemagglutinating activity which were either heat sensitive or resistant. Analysis of isolated fractions, which exhibited haemagglutinating activity that were heat labile, by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 3.8 kD peptide. The purified peptide had a pI of 4.8-5.0. Examination of fractions containing this peptide by electron microscopy showed the presence of fimbriae. Immunogold labelling showed that mouse antibodies raised against the 3.8 kD peptide bound specifically and extensively to P. intermedia fimbriae. It appears that this peptide is a fimbriae-associated haemagglutinin and may represent a major constituent of the fimbriae. Further, fractions exhibiting haemagglutinating activity that were heat resistant, which were recovered at a pH of 3.5 in preparative isoelectric focusing of fimbrial samples, did not possess any detectable major protein bands as shown by analytical gel electrophoresis. However, silver stained gels for the detection of lipopolysaccharide (LPS) revealed the presence of LPS-like components in these fractions. In addition, LPS isolated from whole cells showed a similar electrophoretic pattern and exhibited the haemagglutinating activity that was heat resistant. The results of this study strongly suggest that P. intermedia possesses the machinery to agglutinate erythrocytes, which may be a contributing factor in its colonization in vivo.
Insights
Prevotella intermedia uses a 3.8 kD peptide fimbrial component and lipopolysaccharide (LPS) to agglutinate erythrocytes, potentially aiding its colonization. This study identifies key factors in bacterial adherence and pathogenicity.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Prevotella intermedia is implicated in periodontitis.
- Bacterial fimbriae mediate adherence and colonization.
- Erythrocyte agglutination by bacteria can be a virulence factor.
Purpose of the Study:
- To identify the specific components of Prevotella intermedia fimbriae responsible for hemagglutinating activity.
- To differentiate between heat-labile and heat-stable hemagglutinins.
- To elucidate the role of fimbriae and other bacterial components in erythrocyte agglutination.
Main Methods:
- Solubilization and separation of Prevotella intermedia fimbriae using preparative SDS-PAGE and isoelectric focusing.
- Heat treatment of isolated fractions to assess hemagglutinating activity.
- Tricine-SDS-PAGE and electron microscopy to analyze peptide components and fimbrial structures.
- Immunogold labeling to confirm antibody binding to fimbriae.
- Isolation and analysis of lipopolysaccharide (LPS) for hemagglutinating activity.
Main Results:
- Two types of hemagglutinating activity were identified: heat-labile and heat-resistant.
- A 3.8 kD peptide, identified as a fimbriae-associated hemagglutinin, was responsible for heat-labile activity.
- Heat-resistant hemagglutinating activity was associated with lipopolysaccharide (LPS) components.
- The 3.8 kD peptide was a major constituent of Prevotella intermedia fimbriae.
Conclusions:
- Prevotella intermedia possesses both a peptide hemagglutinin and LPS that mediate erythrocyte agglutination.
- These agglutinating factors likely contribute to the bacterium's ability to colonize host tissues in vivo.
- Understanding these mechanisms is crucial for developing strategies against periodontitis-causing bacteria.