Identification of surface-exposed outer membrane antigens of Helicobacter pylori.
Despite the potential significance of surface-localized antigens in the colonization by and disease processes of Helicobacter pylori, few such components have been unequivocally identified and/or characterized. To further investigate the surface of this bacterium, monoclonal antibodies (MAbs) to a sarcosine-insoluble outer membrane fraction prepared from H. pylori NCTC 11637 were raised. MAbs were selected on the basis of their surface reactivity to whole cells by enzyme-linked immunosorbent assay, immunofluorescence, and immunoelectron microscopy. By use of this selection protocol, 14 surface-reactive MAbs were chosen. These MAbs were used to identify six protein antigens (molecular masses, 80, 60, 51, 50, 48, and 31 kDa), all of which were localized within or associated with the outer membrane. Two of the MAbs recognized the core region of lipopolysaccharide (LPS). Only these two anti-LPS MAbs also recognized the flagellar sheath, indicating a structural difference between the sheath and outer membrane. Three of the protein antigens (80, 60, and 51 kDa) were strain specific, while the other three antigens were present in other strains of H. pylori. Both the 51- and 48-kDa antigens were heat modifiable and likely are porins. A conserved 31-kDa protein may represent another species of porin. A method involving sucrose density ultracentrifugation and Triton extraction that allows the preparation of H. pylori outer membranes with minimal inner membrane contamination is described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein content of the H. pylori outer membrane is similar structurally to those of other species of Helicobacter but markedly different from those of taxonomically related Campylobacter spp. and Escherichia coli. H. pylori also appeared to lack peptidoglycan-associated proteins.
Doig
P
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Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Trust
T J
TJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D016480:Helicobacter pylori / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins