SUBSTRATE SELECTION AND EFFECTOR GATING IN THE \u3ci\u3eHELICOBACTER PYLORI\u3c/i\u3e CAG TYPE IV SECRETION SYSTEM

Helicobacter pylori is a recognized carcinogen and gastric colonization by strains that harbor the cag type IV secretion system (1) is the strongest known risk factor for stomach malignancy. Gastric adenocarcinoma is the fourth leading cause of cancer-related deaths world-wide ( \u3e 700,000 deaths annually), with H. pylori directly contributing to the development of more than one million new cases of cancer per year accounting for 5.5% of all malignancies. H. pylori exploits cag T4SS activity to alter the mucosal microenvironment by delivering diverse immunostimulatory cargo into target gastric epithelial cells. Currently, the mechanisms by which the cag T4SS transports substrates across the bacterial envelope are undefined. The work presented here provides mechanistic insight into H. pylori cag T4SS structure and function. Using techniques to monitor uncontrolled effector release, I show that the pilin ortholog CagC plays a critical role in regulating cargo release to the bacterial cell surface, suggesting that CagC forms a gating apparatus that governs substrate transport across the outer membrane. This work demonstrates that cagC is required for trans-kingdom DNA conjugation (2, 3) and provides evidence that translocated DNA is double-stranded and exposed to the extracellular milieu prior to entering host cells, supporting a two-step DNA secretion model. CagC interacts with components predicted to comprise the cag T4SS inner membrane-embedded apparatus, as well as CagX and CagY within the periplasmic ring complex (PRC). Structural analyses revealed intermolecular π-π interactions among aromatic and positively charged residues formed between adjacent CagX subunits within asymmetric regions of the PRC. I identified CagX as a novel DNA binding protein that strongly and preferentially interacts with dsDNA and demonstrate that CagX π-π stacking coordinates substrate selection and enables trans-kingdom DNA conjugation without disrupting translocation of protein and peptidoglycan effector molecules. Collectively, these studies suggest a model whereby architectural symmetry mismatch exposes CagX π-π interfaces within the PRC to facilitate DNA transit through the cag T4SS translocation channel. Furthermore, this work identified several additional components that regulate effector release across the bacterial outer membrane, providing insight into substrate gating mechanisms in the cag T4SS translocation channel. Finally, I demonstrate that APEX2 proximity labeling approaches can be adapted to analyze cag T4SS architecture. These studies are expected to uncover the composition of novel apparatus complexes that assemble to deliver diverse effector molecules into gastric epithelial cells