Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.
We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.
Bentley
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Department of Biological Sciences, University of Notre Dame, Indiana 46556.
Klebba
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology*; D007163:Immunosorbent Techniques; D008070:Lipopolysaccharides / Q000502:physiology*; D051379:Mice; D018272:Porins; D011489:Protein Denaturation; D012486:Salmonella typhimurium / Q000276:immunology*; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D008070:Lipopolysaccharides; D018272:Porins