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W Goebel

Publications and source records attributed to W Goebel.

At least 73 records · Page 4Linked to original sources
PubMed1998

Host cell signalling during Listeria monocytogenes infection.

Macrophages and other mammalian cells respond to infection with Listeria monocytogenes by the transient or persistent activation of host cell signal transduction pathways. In addition, L. monocytogenes infection influences expression of various host cell genes, some of which may hinder or favour bacterial replication. The observed host cell responses vary with the different subcellular locations inhabited by L. monocytogenes during its intracellular life cycle.

Animals↗
PubMed1998

Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes.

Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).

Animals↗
PubMed1998

Differential interaction of the transcription factor PrfA and the PrfA-activating factor (Paf) of Listeria monocytogenes with target sequences.

The interaction of the purified PrfA transcription factor with the regulatory sequences located upstream of the PrfA-dependent listeriolysin (hly) and internalin (inlA) genes was studied in the presence and in the absence of Paf (PrfA-activating factor)-containing extracts. It is shown that PrfA protein is able to bind, independently of additional factors, to a 109bp DNA fragment including the entire hly promoter sequence with the anticipated PrfA binding site ('PrfA-box'). PrfA alone, but not in combination with Paf, can also bind to a shorter target sequence of 28 bp comprising essentially the PrfA-box of the hly promoter. The addition of a Paf-containing extract does not lead to significant protein binding to these two hly target sequences in the absence of PrfA but converts the complex (CIII) consisting of PrfA and the 109 bp hly DNA fragment to a slower migrating PrfA-Paf-DNA complex (CI). Incubation of cell-free extracts of wild-type Listeria monocytogenes with the 109 bp DNA fragment leads to the formation of CI. The addition of polyclonal PrfA antibodies causes a supershift of CIII. Purified PrfA and PrfA-Paf also bind to a DNA fragment containing the PrfA-dependent promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence. In contrast to the hly target DNA, the inlA promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA-Paf to the inlA target DNA. DNase I footprint experiments show that purified PrfA protects sequences of dyad symmetry previously proposed as PrfA binding sites in the hly and in the inlA promoter regions.

Bacterial Proteins↗
PubMed1998

A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice.

Several large, cell wall-associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i-inlFE. The two proteins encoded by this locus, i-InlE and i-InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i-inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i-InlE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-inlE mRNA and i-InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-inlF and i-inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfA-boxes') were identified in the promoter regions of i-inlF and i-inlE. Non-polar i-inlE and i-inlF deletion mutants and an i-inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 x 107 bacteria per mouse. These data suggest an important role for i-InlE and i-InlF in L. ivanovii virulence.

Amino Acid Sequence↗
PubMed1998

Suppression of major histocompatibility complex class I and class II gene expression in Listeria monocytogenes-infected murine macrophages.

Macrophage cells play a central role during infection with Listeria monocytogenes by both providing a major habitat for bacterial multiplication and presenting bacterial antigens to the immune system. In this study, we investigated the influence of L. monocytogenes infection on the expression of MHC class I and class II genes in two murine macrophage cell lines. Steady-state levels of I-Abeta chain mRNA were decreased in both resting J774A.1 and P388D1 macrophages infected with L. monocytogenes whereas reduction of H-2K mRNA was only observed in P388D1 cells. In addition, L. monocytogenes suppressed induction of MHC class I and class II mRNAs in response to gamma-interferon as well as the maintenance of the induced state in activated P388D1 macrophages. Exposure to the non-pathogenic species L. innocua or a deletion mutant of L. monocytogenes, which lacks the lecithinase operon, did not cause a reduction in H-2K and I-Abeta mRNA levels nor suppress expression of Ia antigens. Inhibition of MHC gene expression may represent an important part of the cross-talk between L. monocytogenes and the macrophage that probably influences the efficiency of a T cell-mediated immune response and thus the outcome of a listerial infection.

Animals↗
PubMed1998

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells.

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.

Adult↗
PubMed1998

Delivery of the p67 sporozoite antigen of Theileria parva by using recombinant Salmonella dublin: secretion of the product enhances specific antibody responses in cattle.

The p67 sporozoite antigen of Theileria parva has been fused to the C-terminal secretion signal of Escherichia coli hemolysin and expressed in secreted form by attenuated Salmonella dublin aroA strain SL5631. The recombinant p67 antigen was detected in the supernatant of transformed bacterial cultures. Immunization trials in cattle revealed that SL5631 secreting the antigen provoked a 10-fold-higher antibody response to p67 than recombinant SL5631 expressing but not secreting p67. Immunized calves were challenged with a 80% lethal dose of T. parva sporozoites and monitored for the development of infection. Two of three calves immunized intramuscularly with the p67-secreting SL5631 strain were found to be protected, whereas only one of three animals immunized with the nonsecreting p67-expressing SL5631 strain was protected. This is the first demonstration that complete eukaryotic antigens fused to the C-terminal portion of E. coli hemolysin can be exported from attenuated Salmonella strains and that such exported antigens can protect cattle against subsequent parasite challenge.

Animals↗
PubMed1998

Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA.

Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.

Bacterial Proteins↗
PubMed1997

Involvement of MAP-kinases and -phosphatases in uptake and intracellular replication of Listeria monocytogenes in J774 macrophage cells.

In this study we show that protein tyrosine kinases and also protein tyrosine phosphatases are involved in the uptake of Listeria monocytogenes by J774 macrophages to a different extent than in the uptake of inert latex beads. In addition, protein tyrosine kinases are necessary for the intracellular growth and survival of L. monocytogenes. The expression of the MAP kinase phosphatase MKP-1, a protein tyrosine phosphatase, is induced upon infection, and phagocytosis of L. monocytogenes by J774 cells overexpressing the MKP-1 protein is reduced compared to control cells. The decreased phagocytosis of L. monocytogenes as a result of the MKP-1 overexpression in J774 macrophages suggests that the activation of the MAP kinase(s) ERK-1 and/or ERK-2 is an essential requirement for the uptake of L. monocytogenes by J774 macrophages.

Animals↗
PubMed1997

Listeria monocytogenes-infected human umbilical vein endothelial cells: internalin-independent invasion, intracellular growth, movement, and host cell responses.

The interaction of Listeria monocytogenes with human umbilical vein endothelial cells was studied. We show that L. monocytogenes invades human umbilical vein endothelial cells independently of internalin A, internalin B, internalin C, and ActA. L. monocytogenes replicates efficiently inside the cells and moves intracellularly by the induction of actin polymerization. We further show that L. monocytogenes-infection of human umbilical vein endothelial cells induces interleukin-6 and interleukin-8 expression during the first 6 h of infection. The expression of MCP-1 and the adhesion molecules VCAM-1 and ICAM-1 was not altered under the experimental conditions used here.

Actins↗
PubMed1997

Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-kappaB (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IkappaBalpha and IkappaBbeta degradation.

As previously reported, Listeria monocytogenes infection of P388D1 macrophages results in a rapid induction of NF-kappaB DNA-binding activity. Here we show that this induction of NF-kappaB activity occurs in a biphasic mode: first, a transient, IkappaBalpha degradation-dependent phase of activity, also induced by the nonvirulent species Listeria innocua, which is mediated by binding of the bacteria to the macrophage, or by adding Listeria-derived lipoteichoic acid to the macrophage; the second persistent phase of activation is only markedly induced when the bacteria enter the cytoplasm of the host cell and express the virulence genes plcA and plcB, encoding two phospholipases. We suggest that products of the enzymatic activity of phospholipases directly interfere with host cell signal transduction pathways, thus leading to persistent NF-kappaB activation via persistent IkappaBbeta degradation.

Animals↗
PubMed1997

Listeria monocytogenes infection of HeLa cells results in listeriolysin O-mediated transient activation of the Raf-MEK-MAP kinase pathway.

In this study we investigated the effect of Listeria monocytogenes infection on the activation of the Raf-MEK-MAP kinase pathway in eukaryotic host cells. HeLa cell infection with L. monocytogenes EGD resulted in a rapid, but transient, phosphorylation of the MAP kinases erk-1 and erk-2, a transient phosphorylation of the MAP kinase kinase MEK-1, and a transient activation of the MAP kinase kinase kinase Raf. In parallel to the transient phosphorylation of the MAP kinases, we detected induced expression of the MAP kinase phosphatase MKP-1. Additionally we present evidence that listeriolysin O is the inducing agent for activation of the Raf-MEK-MAP kinase pathway.

Bacterial Toxins↗
PubMed1997

Complementation of Listeria seeligeri with the plcA-prfA genes from L. monocytogenes activates transcription of seeligerolysin and leads to bacterial escape from the phagosome of infected mammalian cells.

Infection experiments have shown that the avirulent species Listeria seeligeri invaded the enterocyte-like cell line Caco-2 with low efficiency but was unable to escape from the phagosome. Introduction of the listeriolysin gene (hly) from L. monocytogenes into L. seeligeri via a recombinant plasmid did not change these characteristics. No measurable transcription of this gene or of the structurally intact chromosomal seeligerolysin gene (lso) was detected. Transformation with a plasmid carrying the bicistronically transcribed plcA-prfA genes from L. monocytogenes resulted in the efficient expression of the plasmid-encoded transcription activator PrfA, a readily detectable synthesis of seeligerolysin and the escape of the bacteria from the phagosome of infected mammalian cells, followed by intracytoplasmic multiplication.

Animals↗
PubMed1997

The Listeria monocytogenes iap gene as an indicator gene for the study of PrfA-dependent regulation.

The iap gene of Listeria monocytogenes encodes the extracellular protein p60, which possesses a murein hydrolase activity necessary for septum separation. We constructed L. monocytogenes EGD strains harbouring plasmids that carry the iap gene under the control of the PrfA-regulated promoters of the L. monocytogenes genes hly, mpl, and actA. After insertional inactivation of the chromosomal iap gene in L. monocytogenes EGD, p60 synthesis was strictly dependent on PrfA. Elevated temperature (40 degrees C) enhanced synthesis of p60 in L. monocytogenes when the iap gene was under the control of the hly promoter; this appeared to be associated with increased synthesis of PrfA at this temperature. Synthesis of p60 in L. monocytogenes was significantly lower when the iap gene was placed under the control of the actA or the mpl promoter. Transcription of the iap gene was repressed in L. monocytogenes in the presence of PrfA when iap expression was under the control of the prfA promoter P2. Under the control of the hly promoter the gene produced low levels of secreted p60 in the presence of low amounts of PrfA, and this in turn led to the generation of long listerial cell filaments consisting of bacteria that had failed to separate. Overexpression of p60 in the presence of high levels of PrfA caused formation of single cells, which showed reduced viability depending on the level of secreted p60. These data suggest that the iap gene may be a valuable tool for monitoring virulence gene regulation by PrfA under in vivo conditions, without disturbing the integrity of the infected host cells.

Bacterial Proteins↗
PubMed1997

In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli.

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3'-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3'-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.

Bacterial Proteins↗
PubMed1997

Responses by murine macrophages infected with Listeria monocytogenes crucial for the development of immunity to this pathogen.

Macrophages and other mammalian cells respond to infection with Listeria monocytogenes (L. monocytogenes) by the transient or persistent activation of host cell signal transduction pathways. In addition, L. monocytogenes infection influences expression of various host cell genes, such as stress genes, genes from the MHC I and II complex, cytokine genes, and cytokine receptor genes. The possible influences of the different host cell responses on the outcome of an L. monocytogenes infection in vitro as well as for the development of immunity are discussed.

Animals↗
PubMed1997

Protection against murine listeriosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the naturally somatic antigen superoxide dismutase.

A recombinant (r)-Salmonella typhimurium aroA vaccine strain was constructed which secretes the naturally somatic protein of Listeria monocytogenes, superoxide dismutase (SOD), by the HlyB/HlyD/TolC export machinery. Vaccine efficacy of the SOD-bearing carrier strain was compared with that of the p60-secreting construct, S. typhimurium p60s (J. Hess, I. Gentschev, D. Miko, M. Welzel, C. Ladel, W. Goebel, and S. H. E. Kaufmann, Proc. Natl. Acad. Sci. USA 93:1458-1463, 1996). Vaccination of mice with both constructs induced protection against a lethal challenge with the intracellular pathogen, L. monocytogenes. While the somatic listerial antigen, SOD, is immunologically uncharacterized, the naturally secreted protein of L. monocytogenes, p60, is known to be highly immunogenic. Our data emphasize the high vaccine potential of r-Salmonella constructs secreting antigens of somatic or secreted origin. Moreover, they suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated Salmonella spp. as the carrier is capable of potentiating the immune response against foreign proteins independent from their immunogenicity in and display by the natural host.

Animals↗