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Biomedical subjects

W Goebel

Publications and source records attributed to W Goebel.

At least 37 records · Page 2Linked to original sources
PubMed2000

Escherichia coli alpha-hemolysin (HlyA) is heterogeneously acylated in vivo with 14-, 15-, and 17-carbon fatty acids.

alpha-Hemolysin (HlyA) is a secreted protein virulence factor observed in certain uropathogenic strains of Escherichia coli. The active, mature form of HlyA is produced by posttranslational modification of the protoxin that is mediated by acyl carrier protein and an acyltransferase, HlyC. We have now shown using mass spectrometry that these modifications, when observed in protein isolated in vivo, consist of acylation at the epsilon-amino groups of two internal lysine residues, at positions 564 and 690, with saturated 14- (68%), 15- (26%), and 17- (6%) carbon amide-linked side chains. Thus, HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures, and the substrate specificity of the HlyC acyltransferase appears to differ from that of the closely related CyaC acyltransferase expressed by Bordetella pertussis.

Acylation↗
PubMed2000

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes.

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.

Antigens, Bacterial↗
PubMed2000

Interaction of Listeria monocytogenes with the intestinal epithelium.

Listeria monocytogenes is a food-borne pathogen that must cross the intestinal epithelial barrier to reach its target organs. We have investigated the importance of M cells in translocation using an experimental mouse model and a novel, recently described in vitro coculture system that mimics the follicle-associated epithelium (FAE). Our data demonstrate that L. monocytogenes does not require, nor specifically use, M cells of the FAE to cross the gut. We also show that bacterial translocation is rapid and L. monocytogenes can attach very efficiently to exposed basal lamina of the small intestine indicating an important role for extracellular matrix proteins.

Animals↗
PubMed2000

Identification of immunogenic antigens of Helicobacter pylori via the Escherichia coli hemolysin secretion system(1).

We describe a new procedure allowing the generation and detection of immunogenic antigens from Helicobacter pylori via the hemolysin secretion apparatus of Escherichia coli. The gene (or gene fragment) encoding the H. pylori protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyA(s)). These fusion proteins are secreted efficiently by E. coli. This new approach allows the identification of immunodominant antigens by using sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. Three immunodominant antigens bearing the ureB (urease B-subunit), flaA (flagellin A-subunit), and an unknown ORF (HP0888) encoding an E. coli FecE analogous protein fused to hlyA(s) were identified and characterized.

Antigens, Bacterial↗
PubMed2000

Yersinia enterocolitica-mediated translocation of defined fusion proteins to the cytosol of mammalian cells results in peptide-specific MHC class I-restricted antigen presentation.

Yersinia enterocolitica delivers a set of effector proteins [Yersinia outer proteins (Yop)] into the cytosol of target cells to modulate host cell signal transduction pathways required for the extracellular survival of the bacterium. Secretion and subsequent translocation of Yop across the eukaryotic cell membrane are achieved via a type III secretion system. About 50 - 100 amino acids of the N terminus of Yop are required for chaperone-directed secretion and translocation. In this study, it is demonstrated by immunoblot analysis of Yersinia-infected cultured epithelial cells that one ot these proteins, YopE, can serve as a molecular carrier to deliver protein fragments of the heterologous p60 antigen of Listeria monocytogenes into the cytosol of target cells. T cell activation assays revealed that the observed type III-mediated antigen translocation led to a p60 peptide-specific MHC class I-restricted antigen presentation. Efficient translocation and antigen presentation were strictly dependent on the co-localized expression of hybrid YopE-p60 proteins and the YopE-specific chaperone SycE. These results suggest that the Yersinia type III secretion system may serve as an attractive tool for antigen delivery in Yersinia-based live vaccines to induce cellular immune responses.

Animals↗
PubMed2000

DNA vaccine delivery by attenuated intracellular bacteria.

Vaccination by intramuscular or intradermal injection of antigen-encoding DNA is a promising new approach leading to strong cellular and humoral immune responses. Since bone-marrow derived antigen presenting cells (APC) seem to induce these immune responses after migration to the spleen, it is desirable to deliver DNA vaccines directly to splenic APC. Recently, attenuated intracellular bacteria have been exploited for the introduction of DNA vaccine vectors into different cell types in vitro as well as in vivo and offer an attractive alternative to the direct inoculation of naked plasmid DNA.

Animals↗
PubMed2000

Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila.

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

Acanthamoeba↗
PubMed2000

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis.

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.

Administration, Oral↗
PubMed2000

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination.

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r)Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced 'transporter associated with antigen processing'-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both rSalmonella constructs revealed superior vaccine efficacy of the haemolytic rS. typhimurium Hlys construct as compared to the non-haemolytic rSalmonella Hlys(492) strain. In contrast, efficacy of i.v. vaccination with either rSalmonella strain did not significantly differ. Therefore, rSalmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.

Administration, Oral↗
PubMed2000

Bacterial replication in the host cell cytosol.

Intracellular bacteria in mammalian host cells can either live in a membrane-bound vacuole modified to support bacterial growth, or escape from the primary phagosome into the host cell cytoplasm. Phagosomal escape is best studied in Listeria monocytogenes in which a pore-forming cytolysin and two phospholipases are involved in the lysis of the phagosomal membrane. The mechanisms of and requirements for cytoplasmic growth are less clear but there is growing evidence that proficient replication of bacteria in the cytoplasmic compartment requires specific bacterial and cellular preconditions.

Animals↗
PubMed2000

PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex.

There is accumulating evidence that the coordinate transcription of the virulence genes in Listeria monocytogenes constitutes a very complex regulation mechanism which might require other factors in addition to PrfA. We previously described an unknown proteinaceous component from crude bacterial cell extracts, which, together with PrfA, formed a specific complex (CI) in electrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same height as CI. By using EMSA and DNaseI footprint experiments it could be shown that PrfA leads to an enhanced and specific binding of RNAP. Transcriptional activity of RNAP in vitro, using the actA promoter, was strictly dependent on PrfA.

Bacterial Proteins↗
PubMed2000

LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes.

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogenes EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes.

Actins↗
PubMed2000

Bacterial systems for the delivery of eukaryotic antigen expression vectors.

Attenuated bacterial strains allow the administration of recombinant vaccines via the mucosal surfaces. Whereas attenuated bacteria are generally engineered to express heterologous antigens, a novel approach employs intracellular bacteria for the delivery of eukaryotic antigen expression vectors (so-called DNA vaccines). This strategy allows a direct delivery of DNA to professional antigen-presenting cells (APC), such as macrophages and dendritic cells (DC), through bacterial infection. The bacteria used for DNA vaccine delivery either enter the host cell cytosol after phagocytosis by the APC, for example, Shigella and Listeria, or they remain in the phagosomal compartment, such as Salmonella. Both intracellular localizations of the bacterial carriers seem to be suitable for successful delivery of DNA vaccine vectors.

Antigen-Presenting Cells↗
PubMed2000

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG.

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.

Acyltransferases↗
PubMed2000

Novel bacterial systems for the delivery of recombinant protein or DNA.

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors. The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the alpha-hemolysin secretion system of Escherichia coli. The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen presenting cells (APC) like macrophages in vitro as well as in vivo.

Animals↗