Persistent colonization of the stomach by strains of Helicobacter pylori is an important risk factor for the development of gastric cancer. More than 50 Helicobacter pylori genes are predicted to encode integral outer membrane proteins (OMPs), but there has been relatively little experimental investigation of the H. pylori cell-surface proteome. In this study, we used selective biotinylation, affinity purification, and mass spectrometric analysis to identify proteins localized to the surface of H. pylori, along with differential detergent extraction procedures to identify proteins localized to the outer membrane. Proteins that met multiple criteria for surface-exposed outer membrane localization included known adhesins as well as Cag proteins required for activity of the cag type IV secretion system, putative lipoproteins, and other proteins not previously recognized as cell-surface components. We identified sites of non-tryptic cleavage consistent with signal sequence cleavage, as well as several distinct classes of C-terminal motifs that may be relevant for protein localization. Upon addition of proteinase K to intact bacteria, the majority of Hop and Hom OMPs were highly susceptible to proteolysis, whereas Hor and Hof proteins were less susceptible. Analysis of the mass spectral data provided evidence that each of the protease-susceptible OMPs contains a large protease-susceptible extracellular domain exported beyond the outer membrane and a protease-resistant domain at the C-terminus with a predicted β-barrel structure. These features suggest that, similar to VacA, the protease-susceptible OMPs contain passenger domains that are exported through an autotransporter pathway. These results provide new insights into the repertoire of surface-exposed H. pylori proteins that may mediate bacteria-host interactions, as well as the cell-surface topology of these proteins.
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