Functional and intricate interaction network connecting <i>Helicobacter pylori</i> Cag type 4 secretion system surface proteins with outer membrane proteins HopQ and HopZ

The <i>Helicobacter pylori cag</i> pathogenicity island (<i>cag</i>PAI) encodes a complex virulence-associated type IV secretion system (CagT4SS). Recently, structural detail on the CagT4SS has been substantially improved by Cryo-EM. However, important structural and functional information is still missing. In the present study, we followed the hypothesis that <i>H. pylori</i> T4SS external proteins may form a surface-exposed assembly, together with non-CagT4SS proteins, which may be essential for T4SS function. Using interaction screens followed by biochemical and functional characterization, we have enhanced the knowledge about functional protein-protein interactions of the CagT4SS extracellular proteins. This comprises newly identified interactions of CagT4SS surface proteins, including the VirB2 homolog CagC, the VirB5 homolog CagL and CagN, with outer membrane proteins HopQ and HopZ. We have further quantitated direct, pH dependent, interactions of T4SS surface proteins with HopZ and HopQ, with host cell factors CEACAM and integrin, and self-interactions of both HopZ and HopQ. Utilizing chromosomal tag insertions in <i>H. pylori</i>, we detected surface-exposed colocalization of HopQ with T4SS components in the absence or, for HopQ, also in the presence of human gastric epithelial cells. Functionally antagonistic roles of HopQ and HopZ were uncovered in early proinflammatory human epithelial cell activation by the T4SS. In summary, we identified a network of interactions between <i>H. pylori</i> outer membrane proteins and CagT4SS surface proteins that are functionally relevant for T4SS-dependent transport processes. This study provides a valuable resource guiding future studies to refine structure and mechanistic roles of the surface-exposed portions of the CagT4SS.