UBC Theses and Dissertations
Immunochemistry of Pseudomonas aeruginosa outer membrane proteins Mutharia, Lucy Muthoni
The immunochemistry and conservation of Pseudomonas aeruginosa outer membrane components was studied. Conservation of the major outer membrane proteins of P. aeruginosa was demonstrated by the strong similarities in the SDS-polyacrylamide gel profiles of outer membrane polypeptides from 47 different strains of P. aeruginosa. Immunological cross-reactivity was demonstrated for proteins F, H2 and I among 17 serotype strains of P. aeruginosa, using a rabbit polyclonal anti-outer membrane serum. In order to study the conservation of specific antigenic sites among P. aeruginosa strains and other gram-negative bacteria, monoclonal antibodies specific for proteins F, H2 and lipopolysaccharide (LPS) were isolated. Monoclonal antibodies were used to study the antigenic conservation of the different regions of LPS. These antibodies demonstrated strong conservation of the lipid A region of LPS among all tested gram-negative bacteria, less conservation of the P. aeruginosa rough core and great heterogeneity of the LPS O-antigen. In contrast, protein H2 was shown to be highly conserved among the fluorescent Pseudomonads by the reactivity of the monoclonal antibody MA1-6. Monoclonal antibodies specific for protein F showed two distinct specificities suggesting that there were at least two distinct antigenic sites on the protein. Antibodies MA4-4, MA4-2, MA2-10 and MA4-10 interacted with protein F in the outer membranes of all tested P. aeruginosa strains as well as P. syringae and P. putida strains. These antibodies also interacted with defined proteolytic peptides of protein F. The epitope recognized by these antibodies was destroyed by 2-mercaptoethanol and cyanogen bromide treatment. In contrast, antibody MA5-8 interacted only with P. aeruginosa strains, with two cyanogen bromide-derived peptides of the protein and with the 2-mercaptpethanol treated or untreated protein. None of these antibodies interacted with a protein F deficient P. aeruginosa strain.
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