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Biomedical subjects

W Goebel

Publications and source records attributed to W Goebel.

At least 55 records · Page 3Linked to original sources
PubMed2000

Identification of immunodominant antigens from Helicobacter pylori and evaluation of their reactivities with sera from patients with different gastroduodenal pathologies.

Colonization of the gastric mucosa by Helicobacter pylori is the major cause of gastroduodenal pathologies in humans. Studying the outcome of the humoral immune response directed against this gastric pathogen may contribute substantially to vaccine development and to the improvement of diagnostic techniques based on serology. By using two-dimensional gel electrophoresis, 29 proteins from H. pylori G27 were identified which strongly react with sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. These antigens were characterized by mass spectrometry and proved to correspond to products of open reading frames predicted by the H. pylori genome sequence. The comparison of the antigenic patterns recognized by these sera revealed no association of specific H. pylori antigens with antibodies in patients with particular gastroduodenal pathologies.

Antibodies, Bacterial↗
PubMed2000

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: an electron microscopic study.

Internalization of Listeria monocytogenes into human brain microvascular endothelial cells (HBMEC) has recently been demonstrated to be dependent upon the inlB gene. In the present scanning electron microscopic study we show that L. monocytogenes efficiently interacts with the surface of HBMEC in an inlB-independent manner which is also different from invasion. The inlB-dependent invasion of HBMEC by L. monocytogenes is accompanied by intracellular multiplication, movement, and production of bacterium-containing protrusions. These protrusions extend from the cell surface without perturbation of any adjacent cellular membrane.

Bacterial Adhesion↗
PubMed2000

Listeria monocytogenes-infected human dendritic cells: uptake and host cell response.

Dendritic cells (DCs) are potent antigen-presenting cells and play a crucial role in initiation and modulation of specific immune responses. Various pathogens are able to persist inside DCs. However, internalization of the gram-positive bacterium Listeria monocytogenes into human DCs has not yet been shown. In the present study, we demonstrate that human monocyte-derived immature DCs can efficiently phagocytose L. monocytogenes. This uptake is independent of listerial adhesion factors internalin A and internalin B but requires cytoskeletal motion and factors present in human plasma. A major portion of internalized bacteria is found in membrane-bound phagosomes and is rarely free in the cytosol, as shown by transmission electron microscopy and by using an L. monocytogenes strain expressing green fluorescent protein when in the host cell cytosol. The infection caused maturation of the immature DCs into mature DCs displaying high levels of CD83, CD25, major histocompatibility complex class II, and the CD86 costimulator molecule. This effect appeared to be largely mediated by listerial lipoteichoic acid. Although L. monocytogenes infection is known to induce death in other cell types, infection of human DCs was found to induce necrotic but not apoptotic death in fewer than 20% of DCs. Therefore, the ability of DCs to act as effective antigen-presenting cells for listerial immunity is probably enhanced by their resistance to cell death, as well as their ability to rapidly differentiate into mature, immunostimulatory DCs upon encountering bacteria.

Antigens, CD↗
PubMed1999

Host cell signal transduction during Listeria monocytogenes infection.

The facultative intracellular bacterial pathogen Listeria monocytogenes invades and multiplies in many mammalian cell types. During the interaction with its host cells it strongly interferes with and modulates host cell functions. In the present review we summarize the current knowledge on the modulation of signal transduction pathways by secreted listerial products prior to bacterium-cell contact, during uptake, or while L. monocytogenes resides in the different intracellular compartments.

Animals↗
PubMed1999

Listeria monocytogenes infection of Caco-2 human epithelial cells induces activation of transcription factor NF-kappa B/Rel-like DNA binding activities.

The effect of Listeria monocytogenes infection on the cellular level of transcription factor NF-kappa B in the human epithelia-like cell line Caco-2 was investigated. Infection with L. monocytogenes or treatment with lipoteichoic acid induced the formation of three NF-kappa B-like DNA-protein complexes C1, C2, and C3, which were identified as containing either, RelA and p50, RelB and p50, or p50, respectively. NF-kappa B activation in L. monocytogenes-infected Caco-2 cells was distinct from NF-kappa B activation in infected P388D1 macrophages concerning the NF-kappa B complexes formed and the kinetics of the induction.

Caco-2 Cells↗
PubMed1999

Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua.

The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.

Animals↗
PubMed1999

Differential expression of Listeria monocytogenes virulence genes in mammalian host cells.

We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells. The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol. Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages. Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages. In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria. Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability. The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s).

Animals↗
PubMed1999

Introduction of protein or DNA delivered via recombinant Salmonella typhimurium into the major histocompatibility complex class I presentation pathway of macrophages.

Recombinant (r) Salmonella typhimurium aroA strains which display the hen egg ovalbumin OVA(257-264) peptide SIINFEKL in secreted form were constructed. In addition, attenuated rS. typhimurium pcDNA-OVA constructs harbouring a eukaryotic expression plasmid encoding complete OVA were used to introduce the immunodominant OVA(257-264) epitope into the major histocompatibility complex (MHC) class I presentation pathway. Both modes of antigen delivery (DNA and protein) by Salmonella vaccine carriers stimulated OVA(257-264)-specific CD8 T-cell hybridomas. An in vitro infection system was established that allowed both rSalmonella carrier devices to facilitate MHC class I delivery of OVA(257-264) by coexpression of listeriolysin (Hly) or by coinfection with rS. typhimurium Hlys (Hess J., Gentschev I., Miko D., Welzel M., Ladel C., Goebel W., Kaufmann S.H.E., Proc. Natl. Acad. Sci. USA 93 (1996) 1458-1463). Coexpression of Hly and coinfection with rS. typhimurium Hlys slightly improved MHC class I processing of OVA. Our data provide further evidence for the feasibility of attenuated, Hly-expressing rS. typhimurium carriers secreting heterologous antigens or harbouring heterologous DNA as effective vaccines for stimulating CD8 T cells in addition to CD4 T cells.

Amino Acid Sequence↗
PubMed1999

Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin.

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartaterich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca2+, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C-terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.

Adenylate Cyclase Toxin↗
PubMed1999

Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12.

Escherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N-terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA-controlled transcription started predominantly 72 bp upstream from clyA, as shown by primer extension. The corresponding putative promoter contains an unusual -10 sequence (TATGAAT) that is separated from a conventional -35 sequence by a GC-rich spacer. Site-directed deletion of the G in the -10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the G + C content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation-selective transmembrane pores that have a diameter of about 2.5-3 nm.

Bacterial Proteins↗
PubMed1999

Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR.

The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3' end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.

Bacterial Proteins↗
PubMed1999

Genomic subtraction identifies Salmonella typhimurium prophages, F-related plasmid sequences, and a novel fimbrial operon, stf, which are absent in Salmonella typhi.

Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.

Animals↗
PubMed1998

Th1 cells specific for a secreted protein of Listeria monocytogenes are protective in vivo.

In the present study we have investigated the role of the secreted p60 protein from Listeria monocytogenes as an Ag for CD4 T cells. The p60 protein is an abundant extracellular protein that is highly conserved within the members of the genus Listeria. Our results show that L. monocytogenes infection induces a potent p60-specific Th1 immune response. Remarkably, we found that p60-specific Th1 clones mediate significant protection against L. monocytogenes infection. For one p60-specific clone, the peptide epitope was defined. This clone recognized p60 301-312 (EAAKPAPAPSTN) in the context of the H-2Ad molecule. Despite the fact that acquired immunity against L. monocytogenes is primarily mediated by cytotoxic CD8 T lymphocytes, our data clearly demonstrate that secreted bacterial proteins are important CD4 T cell Ags and that Th1 clones specific for a secreted bacterial protein can contribute to the protection against an intracellular pathogen such as L. monocytogenes.

Amino Acid Sequence↗
PubMed1998

In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes.

Simultaneous in situ analysis of the structure and function of bacterial cells present within complex communities is a key for improving our understanding of microbial ecology. A protocol for the in situ identification of Listeria spp. using fluorescently tagged, rRNA-targeted oligonucleotide probes was developed. Ethanol fixation and enzymatic pretreatment with lysozyme and proteinase K were used to optimize whole cell hybridization of exponential phase and stationary phase Listeria spp. cells. In parallel, transcript probes carrying multiple digoxigenin molecules were combined with anti-digoxigenin Fab antibody fragments labeled with horseradish peroxidase to detect, via the catalytic deposition of fluorescein-tyramide, the iap-mRNA in single Listeria monocytogenes cells. The iap gene encodes the associated virulence factor p60. Application of the new signal amplification technique resulted in strong signals comparable in intensity to those obtained with fluorescently labeled rRNA-targeted oligonucleotide probes.

Bacterial Proteins↗
PubMed1998

Sequence comparison of the chromosomal regions encompassing the internalin C genes (inlC) of Listeria monocytogenes and L. ivanovii.

We have recently cloned and characterized the inlC gene of Listeria monocytogenes which belongs to the listerial internalin multigene family and codes for a 30-kDa secreted protein containing five consecutive leucine-rich repeats. Here, we show that in L. monocytogenes inlC is located between the rplS gene (encoding the 50S ribosomal protein L19), and the infC gene (encoding the translation initiation factor 3). By direct and inverse polymerase chain reactions (PCR), we cloned a 5.4-kb region containing a homologous gene (termed i-inlC) from L. ivanovii, the other pathogenic member of the genus Listeria. In this microorganism, the i-inlC gene is preceded by another internalin gene, i-inlD, which seems to be specific for L. ivanovii, as this gene could not be detected in L. monocytogenes by Southern hybridization with an i-inlD gene probe. The i-inlD gene also encodes a small secretory internalin (i-InlD), which shares extended homology with (i-)InlC. Upstream of i-inlD are genes for 23S rRNA and 5S rRNA, and two tRNA genes [Asn-tDNA (GTT) and Thr-tDNA(GTT)]. The 3' terminus of the Thr-tRNA gene appears to be the site of an insertion of a genetic element including i-inlC and i-inlD. A putative transcriptional regulator gene, the product of which contains the TetR family signature, is located downstream of i-inlC. This chromosomal position of the two inlC genes on their respective chromosomes may be due to horizontal transfer of this gene. Transcription of i-inlC and i-inlD is strictly dependent on the transcriptional activator PrfA, which regulates transcription of most of the known virulence genes (including inlC) of L. monocytogenes and of L. ivanovii.

Amino Acid Sequence↗
PubMed1998

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice.

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.

Amino Acid Sequence↗