IDENTIFICATION OF SURFACE-EXPOSED OUTER-MEMBRANE ANTIGENS OF HELICOBACTER-PYLORI

Despite the potential significance of surface-localized antigens in th e colonization by and disease processes of Helicobacter pylori, few su ch components have been unequivocally identified and/or characterized. To further investigate the surface of this bacterium, monoclonal anti bodies (MAbs) to a sarcosine-insoluble outer membrane fraction prepare d from H. pylori NCTC 11637 were raised. MAbs were selected on the bas is of their surface reactivity to whole cells by enzyme-linked immunos orbent assay, immunofluorescence, and immunoelectron microscopy. By us e of this selection protocol, 14 surface-reactive MAbs were chosen. Th ese MAbs were used to identify six protein antigens (molecular masses, 80, 60, 51, 50, 48, and 31 kDa), all of which were localized within o r associated with the outer membrane. Two of the MAbs recognized the c ore region of lipopolysaccharide (LPS). Only these two anti-LPS MAbs a lso recognized the flagellar sheath, indicating a structural differenc e between the sheath and outer membrane. Three of the protein antigens (80, 60, and 51 kDa) were strain specific, while the other three antg ens 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-k Da 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 membra ne contamination is described. Sodium dodecyl sulfate-polyacrylamide g el electroporesis analysis showed that the protein content of the H. p ylori outer membrane is similar structurally to those of other species of Helicobacter but markedly different from those of taxonomically re lated Campylobacter spp. and Escherichia coli. H. pylori also appeared to lack peptidoglycan-associated proteins.