Excision and transfer of an integrating and conjugative element in a bacterial species with high recombination efficiency.

Horizontal transfer of mobile genetic elements, such as integrating and conjugative elements (ICEs), plays an important role in generating diversity and maintaining comprehensive pan-genomes in bacterial populations. The human gastric pathogen Helicobacter pylori, which is known for its extreme genetic diversity, possesses highly efficient transformation and recombination systems to achieve this diversity, but it is unclear to what extent these systems influence ICE physiology. In this study, we have examined the excision/integration and horizontal transfer characteristics of an ICE (termed ICEHptfs4) in these bacteria. We show that transfer of ICEHptfs4 DNA during mating between donor and recipient strains is independent of its conjugation genes, and that homologous recombination is much more efficient than site-specific integration into the recipient chromosome. Nevertheless, ICEHptfs4 excision by site-specific recombination occurs permanently in a subpopulation of cells and involves relocation of a circularization-dependent promoter. Selection experiments for excision indicate that the circular form of ICEHptfs4 is not replicative, but readily reintegrates by site-specific recombination. Thus, although ICEHptfs4 harbours all essential transfer genes, and typical ICE functions such as site-specific integration are active in H. pylori, canonical ICE transfer is subordinate to the more efficient general DNA uptake and homologous recombination machineries in these bacteria.

The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA.

Helicobacter pylori infects half of the world's population, and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high-resolution structures of several HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori-specific antimicrobial agents.

Quantitative analysis of CagA type IV secretion by Helicobacter pylori reveals substrate recognition and translocation requirements.

Bacterial type IV secretion systems are protein transporters with a remarkable diversity of substrates and substrate recognition mechanisms. Type IV-secreted proteins often contain C-terminal secretion signals, but may also require other regions for recognition as secretory substrates, or for full secretion efficiency. For example, type IV secretion of CagA, a major pathogenicity factor of the human gastric pathogen Helicobacter pylori, depends on a C-terminal signal and on N-terminal protein regions. To examine the involvement of individual CagA regions for type IV secretion efficiency, we have established and evaluated a ß-lactamase-dependent reporter system which allows quantitative determination of translocation into host cells. For validation, we used this reporter system to obtain quantitative data for type IV secretion of CagA variants with sequential C-terminal truncations. Alanine-scanning mutagenesis of the CagA C-terminus revealed that none of the characteristic charged residues in this region is necessary for type IV secretion. Translocation rates measured for CagA variants with N-terminal deletions show that CagA does not have an N-terminal signal sequence, but requires its N-terminal domain for efficient secretion. Finally, we provide evidence that only newly synthesized CagA protein is translocated, supporting a model in which type IV secretion is coupled to protein biosynthesis.

Characterization of the translocation-competent complex between the Helicobacter pylori oncogenic protein CagA and the accessory protein CagF.

CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nM affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with µM affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA.

Multiple pathways of plasmid DNA transfer in Helicobacter pylori.

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.

CagI is an essential component of the Helicobacter pylori Cag type IV secretion system and forms a complex with CagL.

Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly.

The coupling protein Cagbeta and its interaction partner CagZ are required for type IV secretion of the Helicobacter pylori CagA protein.

Bacterial type IV secretion systems are macromolecule transporters with essential functions for horizontal gene transfer and for symbiotic and pathogenic interactions with eukaryotic host cells. Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject its effector protein CagA into gastric cells. This protein translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it has been linked to cancer development. Interactions of CagA with host cell proteins have been studied in great detail, but little is known about the molecular details of CagA recognition as a type IV secretion substrate or of the translocation process. Apart from components of the secretion apparatus, we previously identified several CagA translocation factors that are either required for or support CagA translocation. To identify protein-protein interactions between these translocation factors, we used a yeast two-hybrid approach comprising all cag pathogenicity island genes. Among several other interactions involving translocation factors, we found a strong interaction between the coupling protein homologue Cagß (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagß, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagß and CagA and between CagZ and Cagß, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.

Helicobacter pylori type IV secretion apparatus exploits beta1 integrin in a novel RGD-independent manner.

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 alpha5beta1 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 beta1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to alpha5beta1 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 beta1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of beta1 integrin-specific monoclonal antibodies directed against various defined beta1 integrin epitopes, such as the PSI, the I-like, the EGF or the beta-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of beta1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the beta1 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.

Integrin subunit CD18 Is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin.

Helicobacter pylori infection is associated with gastritis, ulcerations, and gastric adenocarcinoma. H. pylori secretes the vacuolating cytotoxin (VacA), a major pathogenicity factor. VacA has immunosuppressive effects, inhibiting interleukin-2 (IL-2) secretion by interference with the T cell receptor/IL-2 signaling pathway at the level of calcineurin, the Ca2+-calmodulin-dependent phosphatase. Here, we show that VacA efficiently enters activated, migrating primary human T lymphocytes by binding to the beta2 (CD18) integrin receptor subunit and exploiting the recycling of lymphocyte function-associated antigen (LFA)-1. LFA-1-deficient Jurkat T cells were resistant to vacuolation and IL-2 modulation, and genetic complementation restored sensitivity to VacA. VacA targeted human, but not murine, CD18 for cell entry, consistent with the species-specific adaptation of H. pylori. Furthermore, expression of human integrin receptors (LFA-1 or Mac-1) in murine T cells resulted in VacA-mediated cellular vacuolation. Thus, H. pylori co-opts CD18 as a VacA receptor on human T lymphocytes to subvert the host immune response.

The Helicobacter pylori CagF protein is a type IV secretion chaperone-like molecule that binds close to the C-terminal secretion signal of the CagA effector protein.

Type IV secretion systems are common bacterial macromolecule transporters that have been adapted to various functions, such as effector protein translocation to eukaryotic cells, nucleoprotein transfer to bacterial or eukaryotic cells, and DNA transport into and out of bacterial cells. Helicobacter pylori, the causative agent of bacterial gastritis, peptic ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject the CagA protein into host cells, thereby altering gene expression profiles and the host cell cytoskeleton. The molecular mechanism of CagA recognition as a type IV substrate is only poorly understood, but seems to be more complex than that of other type IV secretion systems. Apart from 14 essential components of the secretion apparatus, CagA translocation specifically requires the presence of four additional Cag proteins. Here we show that the CagA-binding protein CagF is a secretion chaperone-like protein that interacts with a 100 aa region that is adjacent to the C-terminal secretion signal of CagA. The interaction between CagA and CagF takes place at the bacterial cytoplasmic membrane, and is independent of a functional type IV secretion apparatus and other cag-encoded factors. Our data indicate that CagF binding precedes recognition of the C-terminal CagA translocation signal, and that both steps are required to recruit CagA to the type IV translocation channel.

Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.

Helicobacter pylori (Hp) vacuolating cytotoxin VacA induces cellular vacuolation in epithelial cells. We found that VacA could efficiently block proliferation of T cells by inducing a G1/S cell cycle arrest. It interfered with the T cell receptor/interleukin-2 (IL-2) signaling pathway at the level of the Ca2+-calmodulin-dependent phosphatase calcineurin. Nuclear translocation of nuclear factor of activated T cells (NFAT), a transcription factor acting as a global regulator of immune response genes, was abrogated, resulting in down-regulation of IL-2 transcription. VacA partially mimicked the activity of the immunosuppressive drug FK506 by possibly inducing a local immune suppression, explaining the extraordinary chronicity of Hp infections.