Type IV Protein Secretion in Helicobacter pylori

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PROJECT SUMMARY (ABSTRACT) Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach. H. pylori colonization of the stomach results in chronic gastric mucosal inflammation and is a strong risk factor for gastric cancer and duodenal or gastric ulceration. Gastric cancer is the third leading cause of cancer-related death worldwide, and H. pylori has been classified as a type I carcinogen by the World Health Organization. The H. pylori CagA protein is secreted through a type IV secretion system (T4SS), enters gastric cells, and causes alterations in cellular signaling associated with malignant transformation. CagA and components of the Cag T4SS are encoded by genes within a chromosomal region known as the cag pathogenicity island (PAI), which is present in some H. pylori strains but not others. The incidence of symptomatic gastroduodenal disease (gastric cancer or peptic ulceration) is higher among individuals infected with cag PAI-positive strains than among those infected with cag PAI-negative strains. The molecular architecture and protein composition of the H. pylori Cag T4SS differ substantially from corresponding features of T4SSs found in other bacterial species. The overarching long-term goal of this research is to develop a better understanding of the molecular mechanisms by which H. pylori causes gastric disease. During the previous funding period, we isolated a transmembrane core complex of the Cag T4SS containing five proteins encoded by the cag PAI, described three main structural features of the complex (outer membrane cap, periplasmic ring and stalk), and built partial models of three proteins within the complex. The aims of the current proposal are i) to build a complete structural model of the five Cag T4SS core complex components; ii) to define structure-function relationships for CagY (a core complex component predicted to span from the inner membrane to the outer membrane), elucidating its role in CagA recruitment and T4SS activity; and (iii) to define actions of the Cag T4SS in vivo. Methods will include single particle cryo-electron microscopy analysis of the T4SS core complex, specialized techniques for genetic manipulation of H. pylori, cell culture- based assays of Cag T4SS activity, and an animal model of H. pylori-induced gastric cancer. These studies will provide important advances in our understanding of the molecular mechanisms by which H. pylori infection can lead to gastric cancer and other gastric diseases. On a broader scope, these studies will increase our understanding of bacterial secretion systems and the delivery of bacterial virulence factors into host cells, as well as molecular mechanisms underlying microbe-induced carcinogenesis.