Helicobacter pylori is the causative agent of chronic gastric inflammation, peptic and duodenal ulcers. The long-term objective of the proposed work is to elucidate the molecular and cellular mechanisms by which H. pylori elicits and modulates a chronic inflammatory response which fails to clear the infection. Our prime focus is on proinflammatory agent(s) produced by the bacterial and the role of neutrophil activation in damage to gastric tissue. Recently we have identified, purified and characterized a neutrophil-activating protein (HP-NAP), a cytokine homologue, from H. pylori which acts directly on neutrophils, up regulating CD11b/CD18 and eliciting an oxidative burst response. We have cloned and sequenced the gen napA, encoding this protein and developed an expression system to produce recombinant HP-NAP.
Our Specific Aims are: 1) to identify functional regions within the 15 K subunit of HP-NAP by constructing deletion mutants by insertion of a CAT (chloramphenicol resistance) gene cassette and by reverse-PCR and by molecular mimicry using synthetic peptides representing HP-NAp amino acid sequences; 2) to identify individual amino acids responsible for neutrophil activation (a) by testing synthetic peptides containing amino acid substitutions within the putative functional regions and (b) constructing point mutations using site-direct mutagenesis PCR, employing the alanine scanning technique. Mutants will be expressed as fusion proteins in E. coli and tested for loss of activity; 3) to identify the neutrophil receptor for HP-NAP and the amino acid sequence composing the receptor-binding motif of HP-NAP; binding assays will be used to compare native and mutant recombinant HP-NAP; synthetic peptides based on the HP- NAP amino acid sequence will used to define a specific binding antagonist for HP-NAP; 4) to identify protein phosphorylation events, which are inherent to signal transduction in neutrophils, with the goal of identifying the protein kinases activated by HP-NAP binding and their target protein substrates; results of these experiments are expected to provide clues as to the identity of the signal transduction pathway(s) activated by HP-NAP. Ultimately this work should provide the means for blocking HP-NAP activity, thus preventing tissue damage produced by activated neutrophils in H. pylori gastritis.
