Structural Studies of Helicobacter Pyloris Cag Type IV Secretion System

The Gram-negative bacterium, Helicobacter pylori, persistently infects the stomachs of over half of the human population. The cag Pathogenicity Island (cag PAI) of the Helicobacter pylori has been associated with severe gastric diseases including gastric cancer. The cag PAI houses the type IV secretion system (T4SS) used by the bacterium to secrete CagA, a protein associated with carcinogenesis. Considerable structural biology work has been done to characterize the T4SS outer membrane core complex (OMCC). The OMCC is composed of a 14- fold symmetrical outer membrane cap (OMC) and a 17-fold symmetrical periplasmic ring (PR), and stalk that has yet to be structurally or compositionally characterized. Sub-3.5 Å density maps determined using single particle cryo-electron microscopy are available for the OMC and PR. While these structures allowed for molecular models to be built for these parts of the OMCC, there is a lack of structural information for the stalk and the regions bridging the connections between the OMC, PR, and stalk. Additionally, there is little understanding of how the Cag T4SS facilitates translocation of CagA across the bacterial inner and outer membranes. I have improved both the Cag T4SS purification and vitrification steps, increasing particle yield and our ability to visualize the stalk in the vitrified ice. Using these advances, I will determine the structure and protein identity of the stalk, map connections between the OMCC sub-regions, and structurally and functionally characterize actively translocating Cag T4SSs. The results of this research will provide the first structural description of the Cag T4SS OMCC and molecular snapshots of translocating T4SSs. This will allow me to propose a structure-based rationale for how CagA is transported from the H. pylori cytoplasm into host cells. This work will also prove beneficial in studying other pathogenic bacterial T4SSs such as Legionella pneumophila, Bordetella pertussis, and Bartonella species, thus having a broader impact on medical microbiology. Project Number: 1F32AI186525-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Wilhelm Salmen | Institution: UNIVERSITY OF MICHIGAN AT ANN ARBOR, ANN ARBOR, MI | Award Amount: $80,224 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F04-S (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F32AI18652501A1