Background and Questions. The Helicobacter pylori cag pathogenicity island (cagPAI) is an important virulence factor of the chronic gastric pathogen H. pylori and encodes a complex type IV secretion system (CagT4SS). Our earlier work has helped us to generate hypotheses concerning exposed, variable outer proteins that can interact with host factors [1,2,3]. Recently, structural information of the CagT4SS has been substantially improved by cryo-EM [4]. However, important details, in particular on the outer membrane complex (OMC) and on protein interactions within the OMC and the surface complex of the CagT4SS, consisting of T4SS outer proteins, are missing. This also concerns functional details on putative active and inactive conformations of the export complex, and on the transport functions in general.

Methods and Results. Using bacterial two hybrid system (BACTH) and biophysical characterization methods, we have enhanced our knowledge on protein-protein interactions in the CagT4SS outer proteins. This includes interactions of outer membrane proteins, of the VirB2 homolog CagC [1] and of the outer protein CagN [5] of yet unknown function. We have determined and quantitated homo-dimerization of CagC and CagN, elucidated novel interactions of all tested proteins by BACTH, and confirmed them using protein purification and interaction analysis, for instance by Octet biolayer interferometry analysis.

Conclusions. Novel interactions of H. pylori outer membrane proteins and CagT4SS outer proteins have been found and further characterized using biochemical and biophysical methods under different conditions. This will help to refine structural and functional details of the CagT4SS transport and translocation complex and machinery, also in contact with the human cell.

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