Publications (7)68.8 Total impact
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Article: Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori.
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ABSTRACT: 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.PLoS ONE 01/2012; 7(9):e45623. · 4.09 Impact Factor -
Article: CagI is an essential component of the Helicobacter pylori Cag type IV secretion system and forms a complex with CagL.
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ABSTRACT: 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.PLoS ONE 01/2012; 7(4):e35341. · 4.09 Impact Factor -
Article: The coupling protein Cagbeta and its interaction partner CagZ are required for type IV secretion of the Helicobacter pylori CagA protein.
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ABSTRACT: 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 Cagβ (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagβ, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagβ and CagA and between CagZ and Cagβ, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.Infection and immunity 09/2010; 78(12):5244-51. · 4.21 Impact Factor -
Article: Helicobacter pylori type IV secretion apparatus exploits beta1 integrin in a novel RGD-independent manner.
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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 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.PLoS Pathogens 12/2009; 5(12):e1000684. · 9.13 Impact Factor -
Article: Integrin subunit CD18 Is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin.
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ABSTRACT: 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.Cell host & microbe 02/2008; 3(1):20-9. · 13.02 Impact Factor -
Article: 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.
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ABSTRACT: 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.Microbiology 10/2007; 153(Pt 9):2896-909. · 3.06 Impact Factor -
Article: Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.
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ABSTRACT: 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.Science 09/2003; 301(5636):1099-102. · 31.20 Impact Factor
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- PLoS ONE (2)
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Institutions
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2007–2012
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Ludwig-Maximilian-University of Munich
- Max-von-Pettenkofer Institute for Hygiene and Medical Microbiology
München, Bavaria, Germany
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2003
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Max von Pettenkofer-Institut
München, Bavaria, Germany
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