Molecular characterization of the mechanisms of adhesion of Helicobacter pylori, a model of environmental regulation of bacterial attachment

grantor: University of TorontoIncubation of acid-treated 'Helicobacter pylori' with lipid receptors 'in vitro', showed a change in the pattern of binding usually observed at neutral pH. The major lipid species recognized at pH 2.0 and pH 2.5, which is the pH of the stomach lumen, were sulfatides (sulfogalactosylceramide, SGC), one of the most abundant glycolipids of the human stomach mucus. The suspicion that the SGC binding of 'H. pylori' was a stress induced response is supported by the finding of a similar change in binding specificity when the organisms were briefly exposed to heat-shock treatment. Following stress stimulus, the change in glycolipid binding specificity was prevented by the inclusion of protein synthesis inhibitors, or by incubation of acid-treated 'H. pylori' with anti-Hsp antibodies. Expression of Hsps in surface extracts of 'H. pylori', and bacterial surface reactivity with anti-Hsp antibodies, correlated with the change in glycolipid binding specificity. Previous experiments in our laboratory which showed binding of heat shock proteins members of the Hsp70 family to sulfatide, led the investigation of the role of the 'H. pylori' Hsp70 (DnaK) in the acid stress induction of SGC binding. The 'H. pylori' DnaK gene was cloned and sequenced, and its level of transcription and translation following acid stress determined. A single transcript, of approximately 1.9 kb in size, not detected under non stress conditions, was induced in 'H. pylori', exposed to 42°C and pH 2.5. Analysis of metabolically labeled proteins, induced by incubation of 'H. pylori' at acidic conditions (pH 2.5), showed a typical post-stress pattern of protein synthesis. Two major electrophoretic bands induced by these conditions corresponded to the heat shock proteins Hsp60 (GroEL) and DnaK. Preliminary characterization of PE-binding adhesins which correspond to a second phase of 'H. pylori' colonization, that is the attachment to the epithelial cells surface and the entry into intercellular spaces, identified several proteins including the enzymes catalase and urease, as well as a 62 kDa protein with an N-terminal sequence similar to an internal sequence present in bovine serum albumin (BSA) and TB66, a protein identified in 'Mycobacterium tuberculosis'.Ph.D