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The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein cytotoxin-associated gene A (CagA) into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the interaction of the cagT4SS pilus with the integrin ectodomain are still poorly understood. In this study, we have used surface plasmon resonance (SPR) to generate a comprehensive analysis of the protein-protein interactions between purified CagA, CagL, CagI, CagY repeat domain II (CagY RRII ), CagY C-terminal domain (CagY B 10 ) and integrin α5β1 ectodomain (α5β1 E ) or headpiece domain (α5β1 HP ). We found that CagI, CagA, CagL and CagY B 10 but not CagY RRII were able to interact with α5β1 E with affinities similar to the one observed for α5β1 E interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagY B 10 affinities for the receptor. Furthermore, CagI did not interact with integrin unless the receptor was in open conformation. CagI, CagA but not CagL and CagY B 10 interacted with the α5β1 HP . Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagY B 10 , and CagA and CagI. Altogether, our data map the network of interactions between host-cell α5β1 integrin and the cagT4SS proteins and suggest that activation of the receptor promotes interactions with the secretion apparatus and possibly CagA injection., (© 2017 Federation of European Biochemical Societies.)