| dc.description.abstract | Helicobacter pylori strains containing the cag pathogenicity island (PAI) are associated with the development of gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a secreted effector protein (CagA) and a type IV secretion system (Cag T4SS). The Cag T4SS outer membrane core complex (OMCC) is composed of an outer membrane cap (OMC), a periplasmic ring (PR), and a stalk. The OMC contains multiple copies of CagY/VirB10, CagX/VirB9, CagT/VirB7, CagM, and Cag3. The PR contains only CagY and CagX, and the stalk's composition is undefined. The OMC contains a ring-like structure known as the antenna region, which is formed by 14 CagY (VirB10) proteins. Individual CagY components of the antenna region (antenna projections, AP) contain two α-helices connected by an unstructured loop. This dissertation reports an investigation of the role of the CagY AP in Cag T4SS function, along with an investigation of its role in OMCC assembly. We found that a H. pylori CagY(∆AP) mutant lacks Cag T4SS activity, and mutants with shortened CagY AP loop lengths exhibit reduced T4SS activity. To test the hypothesis that CagY AP interactions with the outer membrane contribute to OMCC assembly, we analyzed the composition and structure of OMCCs isolated from the CagY(∆AP) mutant. Mass spectrometry analyses indicated that these OMCCs contained all 5 protein components; in contrast, Cag T4SS complexes isolated from a ΔcagT mutant lacked CagT and Cag3, and complexes from a ΔcagM mutant lacked CagM, CagT, and Cag3. Cryo-EM analysis indicated that the OMCCs isolated from the CagY(∆AP) mutant have a structure nearly identical to that of wild-type OMCCs; in contrast, OMCCs from ΔcagT or ΔcagM mutants have disorganized OMCs but stable PRs. These results indicate that CagY and CagX components are sufficient for maintaining a stable PR, but a well-structured OMC requires the presence of CagM and CagT. Collectively, these results provide new insights into functional properties of CagY in Cag T4SS activity and the roles of individual OMCC components in Cag T4SS structural organization. | |
