Helicobacter pylori Type IV Secretion Apparatus Exploits β1 Integrin in a Novel RGD-Independent Manner

Abstract

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, K_D of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (K_D of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation. Integrins are single transmembrane proteins present on almost all types of cells. They are composed of an α and a β subunit, which together form the ligand binding pocket, able to interact with extracellular matrix proteins. The best known binding domain on integrin ligands is the RGD domain. Many bacterial, but also viral pathogens exploit this ligand-binding domain to interact with integrins on the host cell. Helicobacter pylori, a common bacterial pathogen associated with gastric diseases, was recently added to this list. One of H. pylori's most important factors associated with gastric pathologies is the CagA protein. This protein is directly injected into host cells through the Cag Type IV Secretion System (cag-T4SS). Previous studies demonstrated that the cag-T4SS requires integrins for the injection (translocation) of CagA into cells. We provide evidence that three proteins, CagA, CagI and CagY, interact with integrins in an RGD-independent way. Additionally, our data point out that the Cag apparatus needs the physical capacity of a β1 integrin heterodimer to change from an active/extended conformation to a closed/bent conformation. This novel kind of integrin interaction opens a new way in which pathogens can use receptors on cells.