| Helicobacter pylori colonization of the mouse gastric mucosa: the entner-doudoroff pathway and development of a promoter-trapping system (2003) | |||||||||||||||
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| Helicobacter pylori is a microaerophilic, Gram-negative bacterium that colonizes the mucous layer of the human stomach. Colonization results in chronic gastritis, which increases the risk of peptic ulcer disease and gastric carcinoma. Our present understanding of the bacterial factors that might affect the course of disease is largely incomplete. This dissertation looked at two aspects of colonization. The first was the analysis of a metabolic pathway, the Entner-Doudoroff (ED) pathway. The objectives were to confirm the presence of 6-phosphogluconate dehydratase (6PGD), the key enzyme in the ED pathway, in H. pylori, to create a mutant strain and to use the mutant strain in mouse colonization experiments. Results indicated that 6-phosphogluconate dehydratase was active in H. pylori. Two ED-negative strains were created by insertional mutagenesis of 6PGD and mice were inoculated with wild-type and/or mutant H. pylori strains. All strains colonized but colonization was lower for both mutant strains compared to the parental strains. The minimum infectious dose was 100-1000-fold lower for the wild-type than for the mutants. Surprisingly, co-inoculation experiments revealed that wild-type H. pylori did not displace the mutant strain. Conservation of the ED pathway in H. pylori and the fact that colonization was diminished by loss of 6PGD suggests that the ED pathway has some function in H. pylori metabolism. Although our data show that it is not vital for colonization, it may have some other function in colonization or transmission of the organism among hosts. The second focus of this dissertation was the development of a technique, in vivo expression technology (IVET), to screen the H. pylori genome for genes that may affect colonization. The objectives in this project were to develop an IVET plasmid for H. pylori and methods for testing and screening an H. pylori promoter library. The IVET plasmid was created so that promoter fragments could be inserted upstream of a promoterless ureB gene and contained regions for recombination into the genome. This IVET plasmid can be used to test promoters for in vivo activity. Methods for generating a promoter library and screening bacteria for promoter activity in vitro were also developed. | |||||||||||||||
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Cited publications (44) | |||||||||||||||