Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa.
A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.
Mutharia
L M
LM
Hancock
R E
RE
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis*; D006090:Gram-Negative Bacteria / Q000276:immunology; D010447:Peptide Hydrolases / Q000494:pharmacology; D018272:Porins; D011549:Pseudomonas / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D018272:Porins; D010447:Peptide Hydrolases