Biomedical subjects

Steffen Backert

Publications and source records attributed to Steffen Backert.

At least 19 recordsLinked to original sources

PubMed2007

The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin.

Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of the CagA bacterial protein into gastric epithelial cells has been linked to an increased risk of peptic ulcer disease and gastric carcinoma. Upon translocation, CagA is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated CagA disrupts adhesion of AGS cells to the extracellular matrix. Owing to the inactivation of c-Src via CagA interaction, vinculin is dephosphorylated at tyrosine residues, 100 and 1065, by corresponding phosphatases. Vinculin dephosphorylation disturbs the interaction and recruitment of the actin-related protein 2/3 (Arp2/3) complex by p34Arc, resulting in a reduction of focal adhesion complexes. These defects can be mimicked by downregulating vinculin using RNA interference in non-infected cells. Tyrosine dephosphorylation of vinculin results in severe cellular deficiencies in cell-matrix adhesion, cell spreading and wound repair. We hypothesize that CagA-mediated inactivation of vinculin is a key step in the mechanism by which H. pylori induces damage to the gastric epithelium and represents an important step in disease development.

Antigens, Bacterial↗

PubMed2006

Conjugative transfer of chromosomally encoded antibiotic resistance from Helicobacter pylori to Campylobacter jejuni.

Many strains of Helicobacter pylori are naturally competent for transformation and able to transfer chromosomal DNA among different isolates using a conjugation-like mechanism. In this study, we sought to determine whether H. pylori can transfer DNA into Campylobacter jejuni, a closely related species of the Campylobacterales group. To monitor the transfer, a chromosomally encoded streptomycin resistance cassette prearranged by a specific mutation in the rpsL gene of H. pylori was used. Mating of the bacteria on plates or in liquid broth medium produced C. jejuni progeny containing the streptomycin marker. DNA transfer was unidirectional, from H. pylori to C. jejuni, and the progeny were genetically identical to C. jejuni recipient strains. DNase I treatment reduced but did not eliminate transfer, and DNase I-treated cell supernatants did not transform, ruling out phage transduction. Recombinants also did not occur when the mating bacteria were separated by a membrane, suggesting that DNA transfer requires cell-to-cell contact. Transfer of the streptomycin marker was independent of the H. pylori comB transformation system, the cag pathogenicity island, and another type IV secretion system called tfs3. These findings indicated that a DNase I-resistant, conjugation-like mechanism may contribute to horizontal DNA transfer between different members of the Campylobacteriales group. The significance of this DNA uptake by C. jejuni in the context of acquiring antibiotic resistance is discussed.

Anti-Bacterial Agents↗

PubMed2006

Helicobacter pylori HopH (OipA) and bacterial pathogenicity: genetic and functional genomic analysis of hopH gene polymorphisms.

BACKGROUND: Expression of the Helicobacter pylori outer membrane protein HopH is regulated by phase variation within a CT dinucleotide repeat motif of the hopH gene. METHODS: To investigate the importance of HopH for bacterial pathogenicity, we performed a detailed functional genomic and population-based genetic characterization of this contingency locus. RESULTS: Sequencing of hopH in H. pylori strains from 58 patients revealed that the hopH "on" genotype is linked to bacterial virulence determinants, such as the vacAs1, vacAm1, babA2, and, most strongly, cagA genotypes. hopH mutagenesis resulted in reduced bacterial adherence to gastric epithelia in vitro. Complementation of hopH in trans restored the adherence properties of hopH mutants. Although HopH has been previously linked to proinflammatory epithelial signaling, hopH mutagenesis did not alter epithelial interleukin-8 secretion in vitro. Comparative epithelial gene-expression profiling by cDNA microarrays revealed no significant differences between the wild-type-specific and hopH mutant-specific transcriptomes. By contrast, a large set of genes was differentially regulated in a cag pathogenicity island-dependent manner. CONCLUSION: An in-frame hopH gene may be linked to gastroduodenal diseases because of its association with other virulence factors or increased bacterial adherence and colonization. The strong linkage with cagA indicates that HopH may contribute to the fitness of cagA-positive strains in vivo.

Adhesins, Bacterial↗

PubMed2006

Type IV secretion systems and their effectors in bacterial pathogenesis.

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.

Animals↗

PubMed2005

Interplay of bacterial toxins with host defence: molecular mechanisms of immunomodulatory signalling.

Mucosal surfaces are protected by a first line of defence termed the innate immune system, which controls the resident microflora to effectively prevent microbial diseases. Inflammatory processes are activated as a consequence of a complex interplay between microbes and host target cells. Many bacterial pathogens have developed mechanisms to avoid host recognition. Some are even able to exploit the immune response through sophisticated interactions with the host signalling cascades in order to increase their replication and propagation. While in the past major emphasis on bacterial protein toxins was directed towards their cytotoxic role, it is increasingly evident that toxins can also modulate various cellular functions at non-cytolytic concentrations. Here, we review important aspects on the molecular mechanisms of immunomodulatory interference by bacterial toxins, with emphasis on the signalling induced in the intestinal epithelium.

Animals↗

PubMed2005

Helicobacter pylori induce neutrophil transendothelial migration: role of the bacterial HP-NAP.

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.

Bacterial Proteins↗

PubMed2005

NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein.

The Helicobacter pylori immunodominant protein, CagA, is associated with severe gastritis and carcinoma. Injection of CagA into gastric epithelial cells by type IV secretion leads to actin-cytoskeletal rearrangements and cell scattering. CagA has been reported to have no role in the induction of transcription factor NF-kappaB and IL-8, which are crucial determinants for chronic inflammation. Here, we provide several lines of evidence showing that CagA is able to induce IL-8 in a time- and strain-dependent manner. We also show that by exchanging specific cagA genes, high IL-8-inducing H. pylori strains could be converted into low inducing strains and vice versa. Our results suggest that IL-8 release induced by CagA occurs via a Ras-->Raf-->Mek-->Erk-->NF-kappaB signaling pathway in a Shp-2- and c-Met-independent manner. Thus, CagA is a multifunctional protein capable of effecting both actin remodeling and potentiation of chemokine release.

Actins↗

PubMed2005

Strain-specific expression profiles of virulence genes in Helicobacter pylori during infection of gastric epithelial cells and granulocytes.

Helicobacter pylori expresses a variety of known virulence-associated factors, whose expression is likely to be dependent on the ecological niche of this pathogen. Here, we compared the temporal changes in the level of virulence-associated gene transcription in H. pylori strains isolated from patients with different pathology. Our aim was to study the coordinated gene expression profiles of these virulence factors during infection of AGS gastric epithelial cells and granulocytes. Using real-time quantitative (TaqMan) RT-PCR, we determined the mRNA expression of cagA, ureA, napA, katA, vacAs1 and vacAs2 alleles in a time course up to 6 h. The expression profiles of the investigated genes vary according to the strain, and were mainly either upregulated or unchanged upon bacterial contact with AGS cells. In contrast, upon contact with granulocytes, the majority of the genes were repressed in H. pylori. The following major results were obtained: (i) genetically diverse H. pylori exhibit different mRNA expression profiles, (ii) the expression patterns were strain-specific and time-dependent and (iii) the regulation of expression profiles was host cell dependent. These data were statistically significant and suggest that contact with target cells leads to an active cross-talk between the pathogen and its host. The use of Taqman-PCR to analyse the expression of mRNA of a bacterial pathogen in response to a changing host environment enabled us to identify variable and strain-specific transcription profiles in a sensitive and reproducible manner.

Adenocarcinoma↗

PubMed2005

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.

Amino Acid Sequence↗

PubMed2005

Gene expression and protein profiling of AGS gastric epithelial cells upon infection with Helicobacter pylori.

Helicobacter pylori, one of the most common bacterial pathogens, colonizes the human stomach and causes a variety of gastric diseases. This pathogen elicits a range of phenotypic responses in infected cultured AGS gastric epithelial cells, including expression of proinflammatory genes and changes in the actin cytoskeleton. Some of these responses are mediated by the type IV secretion system (T4SS) encoded by the cag pathogenicity island. We have used two global approaches, namely 2-DE combined with PMF and cDNA expression array analyses, to study in both a comprehensive and quantitative manner the protein profile and the temporal patterns of mRNA accumulation in AGS cells upon infection with H. pylori and isogenic T4SS mutants. We identified 140 transcripts and detected 190 protein species that were differentially regulated upon infection. Infection with wild-type H. pylori induced expression of a variety of host genes and changes in protein pattern involved in transcriptional responses, cell shape regulation and signal transduction. Among them, some were differentially regulated in a cag PAI-dependent manner, as shown by both the proteomic and cDNA expression array approaches. While 2-DE and PMF allowed us to examine the protein profiles in the infected host, array analysis enabled us to demonstrate dynamic temporal changes in host gene expression profile. In conclusion, our combined application of the two global approaches provides further molecular details on how the host cell responds to infection by H. pylori and its isogenic T4SS mutants on both transcriptional and protein levels. The findings pinpoint host proteins such as serine/threonine and tyrosine kinases, transcription factors, cell cycle related components and actin cytoskeletal signaling molecules as potential targets of individual H. pylori virulence determinants. This study serves as a basis for future work on transcription and proteome analyses of the H. pylori infection model.

Adenocarcinoma↗

PubMed2005

Cortactin: an Achilles' heel of the actin cytoskeleton targeted by pathogens.

Cortactin is an actin-binding protein and a central regulator of the actin cytoskeleton. Importantly, cortactin is also a common target exploited by microbes during infection. Its involvement in disease development is exemplified by a variety of pathogenic processes, such as pedestal formation [enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC)], invasion (Shigella, Neisseria, Rickettsia, Chlamydia, Staphylococcus and Cryptosporidium), actin-based motility (Listeria, Shigella and vaccinia virus) and cell scattering (Helicobacter). Recent progress turns our attention to how cortactin function can be regulated by serine and tyrosine phosphorylation. This has an important impact on how pathogens abuse cortactin to modulate the architecture of the host actin cytoskeleton.

Actins↗

PubMed2005

Tyrosine-phosphorylated bacterial effector proteins: the enemies within.

The tyrosine phosphorylation of proteins has a central role during signal transduction in eukaryotes. Recent progress shows that tyrosine phosphorylation is also a common feature of several effector proteins translocated by bacterial type III and type IV secretion systems. The involvement of these secretion systems in disease development is exemplified by a variety of pathogenic processes: pedestal formation (Tir of EPEC and Citrobacter), cell scattering (CagA of Helicobacter), invasion (Tarp of Chlamydia) and possibly proinflammatory responses and cell proliferation (BepD-F of Bartonella). The discovery that different bacterial pathogens use this common strategy to subvert host-cell function suggests that more examples will soon emerge.

Antigens, Bacterial↗

PubMed2005

Conjugative plasmid DNA transfer in Helicobacter pylori mediated by chromosomally encoded relaxase and TraG-like proteins.

One of the striking characteristics of Helicobacter pylori is the extensive genetic diversity among clinical isolates. This diversity has been attributed to an elevated mutation rate, impaired DNA repair, DNA transfer and frequent recombination events. Plasmids have also been identified in H. pylori but it remained unknown whether conjugation can contribute to DNA transfer between clinical isolates. To examine whether H. pylori possesses intrinsic capability for conjugative plasmid transfer, shuttle vectors were introduced into H. pylori containing an oriT sequence of the conjugative IncPalpha plasmid RP4 but no mobilization (mob) genes. It was shown that these vectors could stably replicate and be mobilized among clinical H. pylori strains. It was also demonstrated that traG and relaxase (rlx) homologues carried on the H. pylori chromosome were important for plasmid transfer. Primer extension studies and mutagenesis further confirmed that the relaxase homologue rlx1 in H. pylori encodes a functional enzyme capable of acting on the RP4 oriT. Furthermore, the findings of this study indicate that traG and rlx1 act independently of the previously described type IV secretion systems, including that encoded by the cag pathogenicity island and the comB transformation apparatus, in mediating conjugative plasmid DNA transfer between H. pylori strains.

Bacterial Proteins↗

PubMed2005

Helicobacter pylori induces transendothelial migration of activated memory T cells.

Helicobacter pylori infection is associated with pronounced infiltration of granulocytes and lymphocytes into the gastric mucosa, resulting in active chronic gastritis that may develop into duodenal ulcer disease or gastric adenocarcinoma. Infiltrating T cells play a major role in the pathology of these diseases, but the signals involved in recruitment of T cells from blood to H. pylori-infected tissues are not well understood. We therefore examined H. pylori-induced T-cell transendothelial migration (TEM). The Transwell system, employing a monolayer of human umbilical vein endothelial cells, was used as a model to study TEM. H. pylori induced a significant T-cell migration, compared to spontaneous migration. CD4+ and CD8+ T cells migrated to the same extent in response to H. pylori, whereas there was significantly larger transmigration of memory T cells compared to naive T cells. Both H. pylori culture filtrate and urease induced migration, and the presence of the H. pylori cag pathogenicity island increased TEM. T-cell TEM was mediated by LFA-1-ICAM-1 interactions in accordance with an increased ICAM-1 expression on the endothelial cells after contact with H. pylori. Migrating T cells had increased expression of activation marker CD69 and chemokine receptors CXCR3, CCR4, and CCR9. Furthermore, T cells migrating in response to H. pylori secreted Th1 but not Th2 cytokines upon stimulation. In conclusion, our data indicate that live H. pylori and its secreted products contribute to T-cell recruitment to the gastric mucosa and that the responding T cells have an activated memory Th1 phenotype.

Cell Movement↗

PubMed2004

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin.

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.

Actins↗

PubMed2004

Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer.

Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.

Antigens, Bacterial↗

PubMed2004

Helicobacter pylori induces AGS cell motility and elongation via independent signaling pathways.

Helicobacter pylori induces motogenic and cytoskeletal responses in gastric epithelial cells. We demonstrate that these responses can be induced via independent signaling pathways that often occur in parallel. The cag pathogenicity island appears to be nonessential for induction of motility, whereas the elongation phenotype depends on translocation and phosphorylation of CagA.

Antigens, Bacterial↗

PubMed2003

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori.

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.

Bacterial Proteins↗