Helicobacter pylori are curved Gram-negative bacteria that are now recognized as the cause of chronic superficial gastritis in humans, and this organism plays an important etiologic role in the pathogenesis of peptic ulcer disease and distal gastric adenocarcinoma. H. pylori colonizes the mucus layer overlying the gastric epithelium, where it is exposed to a wide range of pH values. Urease activity is required for H. pylori survival at low pH, and its presence is essential for colonization. However, little else is known about the mechanisms which allow H. pylori to survive in acidic environments. In previous studies, the investigators have used the strategy of subtractive RNA hybridization to identify an acid-inducible gene in H. pylori, wbcJ, that is essential for O-antigen expression and which contributes to acid survival of the organism. The long-term goal of this study is to isolate and identify additional acid-inducible factors in H. pylori which are required for establishment and maintenance of infection. The hypothesis of this study is that H. pylori possesses a regulated, inducible acid stress response system.
The specific aims are 1) to study the transcriptional regulation of the acid-inducible H. pylori gene, wbcJ and to characterize its role in LPS biosynthesis and structure, 2) to identify other H. pylori genes whose expression is increased in response to acidic pH, and 3) to isolate acid-inducible promoters in H. pylori. To accomplish the first objective, analyses will be done to characterize the promoter region and genetic organization of wbcJ and its co-transcribed genes. Transcriptional wbcJ fusions then will be constructed using xylE to examine the expression of wbcJ under different environmental conditions in vitro. The LPS structure of wbcJ mutants will be analyzed to determine the role of this gene in H. pylori LPS biosynthetic pathways. The method of subtractive RNA hybridization will be used to isolate H. pylori genes whose expression is increased during growth at acidic pH. Genes identified by this approach will be cloned and disrupted using insertional mutagenesis, which will permit the study of their role in H. pylori acid survival and colonization. As an alternate strategy to identifying acid-regulated genes, we will employ the technique of differential fluorescence induction to enrich for promoters that are up-regulated following exposure to acidic pH, utilizing GFP and a fluorescence-activated cell sorter. The proposed work will further elucidate the mechanisms used by H. pylori to colonize and persist within the gastric environment and will lead to improved understanding of how H. pylori causes disease.
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