Project 1 Summary H. pylori is the strongest known risk factor for gastric cancer. One H. pylori determinant that augments cancer risk is the cag type IV secretion system (T4SS) which exports an oncoprotein, CagA, into host epithelial cells. A host molecule that influences gastric cancer in conjunction with H. pylori is -catenin, which tightly regulates stem cell homeostasis; correspondingly, aberrant -catenin signaling within a susceptible stem cell population may lower the threshold for carcinogenesis. A specific stem cell marker in the stomach is Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1), a pan-ERBB regulator, and targeted activation of -catenin within Lrig1+ cells leads to the development of hyperplasia, hyperproliferation, and high-grade dysplasia in the stomach. Our group has now infected mouse and human gastroids to demonstrate that H. pylori can activate - catenin and drive proliferation, expansion, and functional activation of Lrig1+ cells in a cag T4SS-dependent manner. Another cag T4SS-dependent carcinogenic response occurs within the context of iron deficiency and we have shown that iron depletion enhances the ability of H. pylori to colonize the gastric stem cell niche, to activate -catenin, and induce injury in mice. In studies performed with Core A, we demonstrated that iron depletion accelerates carcinogenesis in H. pylori-infected Mongolian gerbils in a cagA-dependent manner. With Project 3, we demonstrated that H. pylori harvested from iron-deficient gerbils exhibit an enhanced capacity to assemble cag T4SS-associated pili, translocate CagA, and induce expression of proinflammatory cytokines. Proteomics and metabolomics studies performed with Cores B and C and whole genome sequencing performed with Projects 2 and 3 have identified a focused subset of differentially expressed proteins, metabolites, and genetic mutations, respectively, among H. pylori strains isolated from iron-deficient versus iron-normal gerbils as well as humans with premalignant lesions. Our hypothesis is that specific interactions between H. pylori and Lrig1 progenitor cells contribute to augmentation in cancer risk conferred by cag+ strains within the context of iron deficiency. Thus, our specific aims are to: 1. Utilize mouse models and gastroids to define the role of Lrig1 and new effectors in regulating oncogenic epithelial responses to H. pylori cag+ carcinogenic strains. 2. Define the role of iron deficiency and Lrig1 in pathogenesis using H. pylori-infected gastroids and - catenin over-expressing mouse models. 3. Utilize rodent models and H. pylori mutant strains to inform mechanistic studies focused on microbial virulence constituents within the context of iron deficiency. !