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, KD of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (KD 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.

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag -T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.

Summary Helicobacter pyloriis a bacterial pathogen that colonizes the gastric niche of ~50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H. pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ~3000 H. pylori mutants revealed three non-cagPAI genes that affected NF-κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-κB, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded co-factor of T4SS function.

In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to alpha-D-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. alpha-D-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal D-Gal-(beta 1----3)-D-GalNAc, D-Gal and D-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited D-Gal, D-GlcNAc or sialic acid and D-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (alpha-D-Man), RCA-I (D-Gal), UEA-I (alpha-L-Fuc) and WGA (D-GlcNAc or sialic acid).