No abstract

Infiltration of neutrophils and monocytes into the gastric mucosa is a hallmark of chronic gastritis caused by Helicobacter pylori. Certain H. pylori strains nonopsonized stimulate neutrophils to production of reactive oxygen species causing oxidative damage of the gastric epithelium. Here, the contribution of some H. pylori virulence factors, the blood group antigen-binding adhesin BabA, the sialic acid-binding adhesin SabA, the neutrophil-activating protein HP-NAP, and the vacuolating cytotoxin VacA, to the activation of human neutrophils in terms of adherence, phagocytosis, and oxidative burst was investigated. Neutrophils were challenged with wild type bacteria and isogenic mutants lacking BabA, SabA, HP-NAP, or VacA. Mutant and wild type strains lacking SabA had no neutrophil-activating capacity, demonstrating that binding of H. pylori to sialylated neutrophil receptors plays a pivotal initial role in the adherence and phagocytosis of the bacteria and the induction of the oxidative burst. The link between receptor binding and oxidative burst involves a G-proteinlinked signaling pathway and downstream activation of phosphatidylinositol 3-kinase as shown by experiments using signal transduction inhibitors. Collectively our data suggest that the sialic acid-binding SabA adhesin is a prerequisite for the nonopsonic activation of human neutrophils and, thus, is a virulence factor important for the pathogenesis of H. pylori infection.

The possible interaction of the neutrophil-activating protein of Helicobacter pylori with target cell glycoconjugates was investigated by the binding of 125 I-labeled recombinant protein to glycosphingolipids from human neutrophils in solid phase assays. Thereby, a distinct binding of the neutrophil-activating protein to four bands in the acid glycosphingolipid fraction from human neutrophils was detected, whereas no binding to the non-acid glycosphingolipids or polyglycosyl ceramides from these cells was obtained. When using glycosphingolipids not present in the cell membrane of human neutrophils, it was found that the neutrophilactivating protein also bound to sulfated glycosphingolipids as sulfatide and sulfated gangliotetraosyl ceramide. Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources suggested that in human granulocytes, the neutrophil-activating protein of H. pylori preferentially recognizes glycoconjugates with a terminally unsubstituted NeuAc␣3Gal␤4GlcNAc␤3Gal␤4GlcNAc␤ sequence.

SUMMARY The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood-group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive but binding is restored by pH neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions; changes during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA’s extraordinary reversible acid-responsiveness enables tight mucosal bacterial adherence while at the same time allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutations and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, and BabA’s adaptive evolution contributes importantly to H. pylori persistence and to overt gastric disease.

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.