[Development of genetic and molecular approaches for the diagnosis and study of the pathogenicity of Helicobacter pylori, agent of gastric inflammatory diseases].

Helicobacter pylori (H. pylori) is a small gram-negative bacillus, recently discovered, found in the stomach of patients with active chronic gastritis and duodenal ulcers. Production of a potent urease has been described as a trait common to all H. pylori so far isolated. To clarify the role of urease in the pathogenic process, as well as to engineer genetic tools useful for the diagnosis of H. pylori, we cloned the genes responsible for urease activity. A genomic library was constructed in Escherichia coli (E. coli) from the chromosomal DNA of the H. pylori strain 85P using a shuttle cosmind vector that we constructed in vitro capable of replicating both in E. coli and Campylobacter jejuni (C. jejuni). The genes responsible for the urease biosynthesis were cloned into E. coli host, then mobilized into C. jejuni where they were expressed. At least six different genes were shown to be required for the expression of the synthesis of an active enzyme; these genes belong to the same cluster and are regulated at the transcriptional level. The two genes encoding the two subunits of the urease enzyme were identified and sequenced; the products of these genes were compared to the other bacterial ureases. The genetic approach allowed to determine the amino-acid sequence of the most immunogenic antigens of H. pylori. In addition, it provides us with genetic tools: a 294-base pairs (bp) DNA fragment internal to one of the urease genes, was shown to be specific of H. pylori strains. This fragment was selectively amplified by polymerase chain reaction (P.C.R.) using two primers designed to target the urease region of all H. pylori isolates present in biological specimen. In addition, P.C.R. followed by direct DNA sequencing of the 294-bp amplified product was shown to be useful to identify and to distinguish between different H. pylori isolates.