Identification of surface-exposed outer membrane antigens of Helicobacter pylori

Abstract

Despite the potential significance of surface-localized antigens in the colonization by and disease processes ofHelicobacterpylori, 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 51and 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. pyloni also appeared to lack peptido2lvcan-associatedLproteins.

A role for the bacterial outer membrane in the pathogenesis of Helicobacter pylori infection

“…The H. pylori outer membrane has been shown to contain porin proteins [21]. These ubiquitous proteins in Gram-negative bacteria form water-¢lled channels across the membrane and play a role in selective permeability [17].…”

“…These ubiquitous proteins in Gram-negative bacteria form water-¢lled channels across the membrane and play a role in selective permeability [17]. Unusually resistant to heating in strong detergents, porins exhibit a di¡erent M r during SDS-PAGE, depending upon the temperature of solubilization [17,21] Several heat-mod-i¢able proteins were observed in the membrane vesicles when electrophoresed (Fig. 3).…”

In Vitro Aging of Helicobacter pylori: Changes in Morphology, Intracellular Composition and Surface Properties

“…Specific antigens are expressed by the coccoid form in vivo and therefore the change in morphology from rod-shape to the coccoid form should not be seen as a degenerative transformation [37]. Six different surface-exposed outer membrane antigens from rod-shaped H. pylori have been examined by Doig et al [38]. The protein content was shown to be structurally similar within the genus Helicobacter, but differed from the other bacterial genera tested.…”

Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pylori

“…The subcellular fractions of H. pylori were obtained as described previously [47]. Briefly, H. pylori was grown for 48 h on Columbia agar plates containing 7% (v ⁄ v) horse blood.…”