Molecular dissection of protein–protein interactions between integrin α5β1 and the Helicobacter pylori Cag type IV secretion system

Abstract

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 ), CagY C-terminal domain (CagY ) and integrin α5β1 ectodomain (α5β1 ) or headpiece domain (α5β1 ). We found that CagI, CagA, CagL and CagY but not CagY were able to interact with α5β1 with affinities similar to the one observed for α5β1 interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagY 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 interacted with the α5β1 . Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagY , 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.

Specific high affinity interaction of Helicobacter pylori CagL with integrin αVβ6 promotes type IV secretion of CagA into human cells

“…By and large, these results are consistent with our previous results from competitive binding studies . However, the low apparent affinity of CagL for α 5 β 1 is at odds with previous SPR studies that yielded K d values of 90 n m or 183 n m and 39 n m for the clasped and unclasped ectodomain preparations of α 5 β 1 , respectively . Therefore, we checked whether the immobilized α 5 β 1 is functional in our assay using a fibronectin titration as positive control.…”

“…The lower affinity that we found for a 5 b 1 is at odds with published data. Right now, we have no clear explanation for the big difference between the EC 50 value that we measured and published K d values for CagL and integrin a 5 b 1 [9,38]. Different protein constructs and preparations for integrin and CagL, different assays and different buffer conditions represent potential causes.…”

“…The integrin b 1 subunit and integrin a 5 b 1 in particular were consistently found to be important for CagA translocation [9,27] and for induction of IL-8 secretion [25,28]. This requirement for a 5 b 1 does not contradict our finding that CagL binding to a 5 b 1 is weak in vitro, because three other cagPAI proteins, CagA, CagI and CagY, were also found to interact with the integrin b 1 subunit in yeast two hybrid assays [27,49] or to directly bind purified integrin a 5 b 1 or its ectodomain in pull-down and SPR studies [27,38]. In another recent in vitro study it was shown that H. pylori binding to immobilized a 5 b 1 integrin under shear flow was dependent on CagY, while CagI or CagL were not required [51].…”

“…Meanwhile, however, the picture has become more complex. Several other cagT4SS proteins were found to directly interact with integrin a 5 b 1 [27,38,49]. Likewise, additional CagL receptors were found, namely integrins a V b 3 and a V b 5 [20,24].…”

Coating Nanoparticles with Gastric Epithelial Cell Membrane for Targeted Antibiotic Delivery against Helicobacter pylori Infection

“…For example, H. pylori were shown to bind with integrin β1 (CD29) in gastric epithelia cells. [28, 29] An increased expression of CD29 correlated with enhanced invasion of the bacteria. In addition, the fucosylated Lewis blood group antigens (Leb) on gastric epithelia cells are also known as receptors for H. pylori binding mediated through bacterial adhesin BabA.…”