"Pathogenesis of Helicobacter pylori Infection"

Veterans Health Administration, Decatur PA

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Abstract

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach. The presence of H. pylori in the stomach is associated with an increased risk of gastric cancer and peptic ulcer disease. The World Health Organization has classified H. pylori as a type I carcinogen, and gastric cancer is the fourth leading cause of cancer-related death worldwide. H. pylori genomes contain more than 50 genes that are predicted to encode outer membrane proteins. Six H. pylori genes encode non-identical OMPs designated as FecA- or FrpB-like proteins. These six proteins are predicted to function as TonB-dependent transporters (TBDTs). TBDTs are outer membrane proteins that bind and transport substrates through the outer membrane, using an energy-dependent process requiring an inner membrane complex (TonB-ExbB-ExbD) that transduces energy (proton motive force) to the outer membrane. Relatively little experimental work has been done to analyze the functions of the six putative H. pylori TBDTs, but one is reported to have a role in nickel acquisition. In preliminary studies, we have mutated H. pylori genes encoding each of the six putative TBDTs and genes encoding each of two non-identical TonB proteins. Our preliminary experimental results confirm that one of the TBDTs (FrpB3) has a role in nickel acquisition and support our hypothesis that TonB-dependent processes contribute to H. pylori iron acquisition. In addition, our experimental results indicate that these proteins contribute to H. pylori fitness in vitro. The long- term goals of this work are to understand the molecular mechanisms that allow H. pylori to persistently colonize the human gastric mucosa, understand the molecular mechanisms by which H. pylori infection leads to the development of gastric cancer or peptic ulceration, and develop effective strategies for the prevention of these diseases. To achieve these long-term goals, we seek to understand the actions of proteins that are localized on the surface of H. pylori and understand their roles in H. pylori acquisition of essential nutrients. The overall hypothesis of this proposal is that FecA- and FrpB-like proteins have important roles in H. pylori nutrient metal acquisition and contribute to H. pylori fitness in vivo, thereby influencing the capacity of H. pylori to colonize the stomach and cause gastric disease. The specific aims are (i) to define the roles of FecA- and FrpB-like proteins in H. pylori nutrient acquisition and fitness in vitro, and (ii) to define the roles of FecA- and FrpB-like proteins in vivo. To accomplish Aim 1, we will further define the roles of FecA- and FrpB-like proteins and TonB proteins in H. pylori nutrient metal acquisition; systematically analyze the regulation of fecA-like, frpB-like, and tonB genes; and analyze multiple properties of mutant strains (harboring mutations in fecA-like, frpB-like, or tonB genes) compared to wild-type and complemented mutant strains. To accomplish Aim 2, we will experimentally infect Mongolian gerbils and mice with wild-type H. pylori strains, mutant strains (harboring mutations in fecA-like, frpB- like, or tonB genes), and complemented mutant strains. We will assess the in vivo fitness of the wild-type and mutant strains under varying conditions, including animals receiving diets containing altered concentrations of iron or nickel, and we will test the hypothesis that the mutant strains differ from the wild-type strain in capacity to cause gastric disease. Finally, we will use ICP-MS and laser ablation (LA-ICP-MS) methods to define the effects of H. pylori on metal concentrations and the spatial distribution of metals in gastric tissue. Collectively, these experiments will provide an understanding of the functions of FecA- and FrpB-like proteins and TonB- dependent processes in nutrient metal acquisition, the roles of these proteins in H. pylori colonization of the stomach, and their roles in gastric disease.