Biomedical subjects

Thomas F Meyer

Publications and source records attributed to Thomas F Meyer.

At least 37 records · Page 2Linked to original sources

PubMed2005

CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells.

Neisseria gonorrhoeae is a gram-negative bacterial pathogen which infects the human mucosal epithelium. An early critical event in neisserial infection is the type IV pilus-mediated adherence to the host cell. The PilC protein, located on the pilus tip, has earlier been identified as the major pilus adhesin. Previous studies suggested that the cell surface protein CD46 is a pilus receptor for Neisseria. We investigated the role of CD46 in pilus-mediated gonococcal infection of epithelial cells. Differences in binding efficiencies of piliated gonococci as well as purified pilus adhesin PilC2 on human epithelial cell lines did not correlate to the level of surface-expressed CD46. Additionally, no binding of piliated gonococci or PilC2 protein was observed on CD46-transfected CHO and MDCK cells. Furthermore, specific down-regulation of CD46 expression in human epithelial cell lines by RNA interference did not alter the binding efficiency of piliated gonococci or purified PilC2 protein, although other CD46-dependent processes, such as measles virus infection and C3b cleavage, were significantly reduced. These data support the notion that pilus-mediated gonococcal infection of epithelial cells can occur in a CD46-independent manner, thus questioning the function of CD46 as an essential pilus receptor for pathogenic neisseriae.

Animals↗

PubMed2005

Analysis of cell type-specific responses mediated by the type IV secretion system of Helicobacter pylori.

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.

Animals↗

PubMed2005

Gain and loss of multiple genes during the evolution of Helicobacter pylori.

Sequence diversity and gene content distinguish most isolates of Helicobacter pylori. Even greater sequence differences differentiate distinct populations of H. pylori from different continents, but it was not clear whether these populations also differ in gene content. To address this question, we tested 56 globally representative strains of H. pylori and four strains of Helicobacter acinonychis with whole genome microarrays. Of the weighted average of 1,531 genes present in the two sequenced genomes, 25% are absent in at least one strain of H. pylori and 21% were absent or variable in H. acinonychis. We extrapolate that the core genome present in all isolates of H. pylori contains 1,111 genes. Variable genes tend to be small and possess unusual GC content; many of them have probably been imported by horizontal gene transfer. Phylogenetic trees based on the microarray data differ from those based on sequences of seven genes from the core genome. These discrepancies are due to homoplasies resulting from independent gene loss by deletion or recombination in multiple strains, which distort phylogenetic patterns. The patterns of these discrepancies versus population structure allow a reconstruction of the timing of the acquisition of variable genes within this species. Variable genes that are located within the cag pathogenicity island were apparently first acquired en bloc after speciation. In contrast, most other variable genes are of unknown function or encode restriction/modification enzymes, transposases, or outer membrane proteins. These seem to have been acquired prior to speciation of H. pylori and were subsequently lost by convergent evolution within individual strains. Thus, the use of microarrays can reveal patterns of gene gain or loss when examined within a phylogenetic context that is based on sequences of core genes.

Evolution, Molecular↗

PubMed2005

The anti-inflammatory compound curcumin inhibits Neisseria gonorrhoeae-induced NF-kappaB signaling, release of pro-inflammatory cytokines/chemokines and attenuates adhesion in late infection.

Neisseria gonorrhoeae (Ngo) is a Gram-negative pathogenic bacterium responsible for an array of diseases ranging from urethritis to disseminated gonococcal infections. Early events in the establishment of infection involve interactions between Ngo and the mucosal epithelium, which induce a local inflammatory response. Here we analyzed the molecular mechanism involved in the Ngo-induced induction of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), and IL-8. We identified the immediate early response transcription factor nuclear factor kappaB (NF-kappaB) as a key molecule for the induction of cytokine release. Ngo-induced activation of direct upstream signaling molecules was demonstrated for IkappaB kinase alpha and beta (IKKalpha and IKKbeta) by phosphorylation of IkappaBalpha as a substrate and IKK autophosphorylation. Using dominant negative cDNAs encoding kinase-dead IKKalpha, IKKbeta, and NF-kappaB-inducing kinase (NIK), Ngo-induced NF-kappaB activity was significantly inhibited. Curcumin, the yellow pigment derived from Curcuma longa, inhibited IKKalpha, IKKbeta and NIK, indicating its strong potential to block NF-kappaB-mediated cytokine release and the innate immune response. In addition to the inhibition of Ngo-induced signaling, curcumin treatment of cells completely abolished the adherence of bacteria to cells in late infection, underlining the high potential of curcumin as an anti-microbial compound without cytotoxic side effects.

Anti-Inflammatory Agents↗

PubMed2004

A global approach combining proteome analysis and phenotypic screening with RNA interference yields novel apoptosis regulators.

Global approaches like proteome or transcriptome analyses have been performed extensively to identify candidate genes or proteins involved in biological and pathological processes. Here we describe the identification of proteins implicated in the regulation of apoptosis using proteome analysis and the functional validation of targets by RNA interference. A high-throughput platform for the validation of synthetic small interfering RNAs (siRNAs) by quantitative real-time PCR was established. Genes of the identified factors were silenced by automated siRNA transfection, and their role in apoptotic signaling was investigated. Using this strategy, nine new modulators of apoptosis were identified. A subsequent detailed study demonstrated that hepatoma-derived growth factor (HDGF) is required for TNFalpha-induced release of pro-apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.

Apoptosis↗

PubMed2004

Transcription profiling analysis of the mechanisms of vaccine-induced protection against H. pylori.

Development of a vaccine against H. pylori is regarded as desirable alternative to the current antibiotic therapy regimens. Mice immunized with an attenuated recombinant Salmonella typhimurium expressing H. pylori urease subunits A&B have dramatically reduced bacterial loads after a single dose. The mechanism(s) of protection against this largely extra-cellular pathogen are not fully understood. The aim of this study was to identify genes that were regulated specifically in response to immunization, in order to gain a broader picture of the immune response in the immunized gastric epithelium. Gene expression in RNA isolated from the gastric mucosa of immunized and infected Balb/c mice was compared with that in infected only mice at 1, 3, and 14 days after challenge with a mouse-adapted strain of H. pylori. We show that infection with H. pylori causes an immediate reaction in vivo, which was clearly divided into acute and chronic phases, and further that the transcriptional response in the H. pylori infected and immunized gastric mucosa is unique. Analysis of gene expression patterns at day 14 post-infection suggested not only the beginning of a lymphocytic infiltrate, but of an integrated epithelial response characterized by increased expression of genes controlling cell cycle and turnover. This observation was confirmed in independent experiments. The global approach has brought new insights to the effect of immunization on the gastric epithelium and has led us to propose a new multi-factorial model for the mechanisms underlying vaccine-induced protection.

Animals↗

PubMed2004

Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins.

Pathogenic Neisseria express type IV pili (tfp), which have been shown to play a central role in the interactions of bacteria with their environment. The regulation of piliation thus constitutes a central element in bacterial life cycle. The PilC proteins are outer membrane-associated proteins that have a key role in tfp biogenesis since PilC-null mutants appear defective for fibre expression. Moreover, tfp are also subjected to retraction, which is under the control of the PilT nucleotide-binding protein. In this work, we bring evidence that fibre retraction involves the translocation of pilin subunits to the cytoplasmic membrane. Furthermore, by engineering meningococcal strains that harbour inducible pilC genes, and with the use of meningococcus-cell interaction as a model for the sequential observation of fibre expression and retraction, we show that the PilC proteins regulate PilT-mediated fibre retraction.

Adenosine Triphosphatases↗

PubMed2004

Identification of candidate antigens for serologic detection of Helicobacter pylori-infected patients with gastric carcinoma.

Helicobacter pylori colonizes the stomach of almost half the world population and is a causative agent of gastric carcinomas and duodenal ulcers. Only a small fraction of infected people will develop these severe illnesses and a predictive test to identify people at high risk would greatly benefit disease management. Our study aimed to identify conserved bacterial antigens that may be useful for the development of such a diagnostic test. High-resolution immunoproteomics by 2-dimensional electrophoresis of H. pylori 26695 proteins was carried out with sera from infected patients with either duodenal ulcer (n=30) or gastric carcinoma (n=30), 2 clinically divergent conditions. According to their antigen recognition patterns clear groups of patients were identified. Although this classification did not correspond to the clinical status, it may be correlated to other bacterial or host factors that influence the outcome of infection. In general antigen recognition patterns were found to be highly variable, however by utilizing powerful image analysis and statistical tests the recognition of 14 antigenic protein species was found to differ significantly (p<0.01) between both diseases. Particular protein species of GroEL, HyuA, GroES and AtpA appear to be useful surrogate markers for gastric carcinoma detection and consequently should be considered for further prospective studies to assess their predictive value. For one protein species of AtpA, evidence was found that different post-translational modifications may confer different immunogenicities.

Adenocarcinoma↗

PubMed2004

Web-accessible proteome databases for microbial research.

The analysis of proteomes of biological organisms represents a major challenge of the post-genome era. Classical proteomics combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) for the identification of proteins. Novel technologies such as isotope coded affinity tag (ICAT)-liquid chromatography/mass spectrometry (LC/MS) open new insights into protein alterations. The vast amount and diverse types of proteomic data require adequate web-accessible computational and database technologies for storage, integration, dissemination, analysis and visualization. A proteome database system (http://www.mpiib-berlin.mpg.de/2D-PAGE) for microbial research has been constructed which integrates 2-DE/MS, ICAT-LC/MS and functional classification data of proteins with genomic, metabolic and other biological knowledge sources. The two-dimensional polyacrylamide gel electrophoresis database delivers experimental data on microbial proteins including mass spectra for the validation of protein identification. The ICAT-LC/MS database comprises experimental data for protein alterations of mycobacterial strains BCG vs. H37Rv. By formulating complex queries within a functional protein classification database "FUNC_CLASS" for Mycobacterium tuberculosis and Helicobacter pylori the researcher can gather precise information on genes, proteins, protein classes and metabolic pathways. The use of the R language in the database architecture allows high-level data analysis and visualization to be performed "on-the-fly". The database system is centrally administrated, and investigators without specific bioinformatic competence in database construction can submit their data. The database system also serves as a template for a prototype of a European Proteome Database of Pathogenic Bacteria. Currently, the database system includes proteome information for six strains of microorganisms.

Bacterial Proteins↗

PubMed2004

Helicobacter pylori vaccine development based on combined subproteome analysis.

Effective vaccines could provide long-term solutions to many important infectious diseases, however, vaccine development has been hampered by the slow identification of protective antigens. Proteomics provides global information about relevant antigen properties and thus might be ideally suited for identifying promising vaccine antigen subsets. Helicobacter pylori proteomics data are stored in a proteomics database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). In this review, we describe how a combined Helicobacter subproteome analysis resulted in the rapid identification of novel, highly protective antigens. This illustrates the great potential of pathogen proteomics for vaccine development.

Antigens, Bacterial↗

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

Action and reaction: Chlamydophila pneumoniae proteome alteration in a persistent infection induced by iron deficiency.

Chlamydophila pneumoniae is an obligate intracellular pathogen implicated in a variety of acute and chronic diseases. Long-term infections are associated with a persistent life stage, in which bacteria can stay for years. They are less accessible to antibiotic treatment but still prone to sustain an inflammatory response. Different in vitro models have been established to mimic and characterize chlamydial persistency. For C. pneumoniae and Chlamydia trachomatis, altered metabolic activities and changed antigenic profiles compared to acute infections have been reported. Most studies including transcriptome and proteome analyses describe persistency induced by IFNgamma treatment. Here, we use iron depletion of the infected cell culture that also leads into persistent infection. We describe differently regulated proteins found by subtractive proteome analysis comparing two early stages of infection with and without addition of the iron chelator deferoxamine-mesylate. While only one bacterial protein was up-regulated during iron deficiency up to 24 h post infection (p.i.), 11 were found to be up-regulated and eight to be down-regulated from 24-48 h p.i. Two down-regulated proteins could be identified by peptide mass fingerprinting as thioredoxin reductase and chromosome partitioning protein (ParB). The latter is involved in chromosome segregation. Thus, using a comparative approach we identified on a proteome level down-regulation of ParB in persistent chlamydial forms, which is in agreement with previous results describing changes in cell division and atypical altered morphology of persistent Chlamydiae.

Amino Acid Sequence↗

PubMed2004

Proteomic and gene profiling approaches to study host responses to bacterial infection.

Infectious disease is the result of an intimate relationship between the pathogen and host, which involves cross-talk. After an initial flood of mainly descriptive reports on the influence of acute bacterial infection on cells, transcriptome and proteome studies are now becoming more refined in their approach, and are shedding light on the role of pathogen-specific mechanisms/structures in pathogenesis. In addition, studies of gene expression in vivo have shed new light on how the host influences the niche occupied by bacteria. Elegant refinements to proteomics using beads coated with bacterial invasins, or purifying subcellular fractions are producing a picture of invasion specific processes. Such approaches combined with modern functional genomics technologies such as RNAi represent the next phase in understanding host-bacteria interactions.

Animals↗

PubMed2004

From the inside out--processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.

Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae. Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram-negative bacterial proteins designated autotransporters. This prediction, however, was never experimentally supported, nor was the function of PmpD known. Here, using 1D and 2D PAGE we demonstrate that PmpD is processed into two parts, N-terminal (N-pmpD), middle (M-pmpD) and presumably third, C-terminal part (C-pmpD). Based on localization of the external part on the outer membrane as shown by immunofluorescence, immuno-electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N-pmpD translocates to the surface of bacteria where it non-covalently binds other components of the outer membrane. We propose that N-pmpD functions as an adhesin, as antibodies raised against N-pmpD blocked chlamydial infectivity in the epithelial cells. In addition, recombinant N-pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL-8 release in a dose-dependent manner. These results demonstrate that N-PmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation.

Amino Acid Sequence↗

PubMed2004

Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori.

The flagellar system of Helicobacter pylori, which comprises more than 40 mostly unclustered genes, is essential for colonization of the human stomach mucosa. In order to elucidate the complex transcriptional circuitry of flagellar biosynthesis in H. pylori and its link to other cell functions, mutants in regulatory genes governing flagellar biosynthesis (rpoN, flgR, flhA, flhF, HP0244) and whole-genome microarray technology were used in this study. The regulon controlled by RpoN, its activator FlgR (FleR) and the cognate histidine kinase HP0244 (FleS) was characterized on a genome-wide scale for the first time. Seven novel genes (HP1076, HP1233, HP1154/1155, HP0366/367, HP0869) were identified as belonging to RpoN-associated flagellar regulons. The hydrogenase accessory gene HP0869 was the only annotated non-flagellar gene in the RpoN regulon. Flagellar basal body components FlhA and FlhF were characterized as functional equivalents to master regulators in H. pylori, as their absence led to a general reduction of transcripts in the RpoN (class 2) and FliA (class 3) regulons, and of 24 genes newly attributed to intermediate regulons, under the control of two or more promoters. FlhA- and FlhF-dependent regulons comprised flagellar and non-flagellar genes. Transcriptome analysis revealed that negative feedback regulation of the FliA regulon was dependent on the antisigma factor FlgM. FlgM was also involved in FlhA- but not FlhF-dependent feedback control of the RpoN regulon. In contrast to other bacteria, chemotaxis and flagellar motor genes were not controlled by FliA or RpoN. A true master regulator of flagellar biosynthesis is absent in H. pylori, consistent with the essential role of flagellar motility and chemotaxis for this organism.

Bacterial Proteins↗

PubMed2004

Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC).

Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.

Adhesins, Escherichia coli↗

PubMed2004

Lack of stage-specific proteins in coccoid Helicobacter pylori cells.

Helicobacter pylori exists in two distinct forms, rod shaped or coccoid, in stomachs of infected patients. Based on in vitro proteome comparisons, there are no detectable coccoid-specific proteins, which argues against the specific adaptation of coccoid Helicobacter to distinct biological functions, such as enhanced persistence or transmission to other hosts.

Adaptation, Physiological↗

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↗