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Biomedical subjects

W Goebel

Publications and source records attributed to W Goebel.

At least 19 recordsLinked to original sources
PubMed2006

Overexpression of PrfA leads to growth inhibition of Listeria monocytogenes in glucose-containing culture media by interfering with glucose uptake.

Listeria monocytogenes strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when they are supplemented with glucose but not when they are supplemented with glucose-6-phosphate compared to the growth of isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake was observed in a PrfA*-overexpressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and an isogenic strain carrying multiple copies of prfA or prfA* on a plasmid. These analyses revealed that in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), there was less pronounced up-regulation of the expression of several phage and metabolic genes (group II) and there was strong down-regulation of several genes involved mainly in carbon and nitrogen metabolism in the PrfA*-overexpressing strain (group III). Among the latter genes are the nrgAB, gltAB, and glnRA operons (involved in nitrogen metabolism), the ilvB operon (involved in biosynthesis of the branched-chain amino acids), and genes for some ABC transporters. Most of the down-regulated genes have been shown previously to belong to a class of genes in Bacillus subtilis whose expression is negatively affected by impaired glucose uptake. Our results lead to the conclusion that excess PrfA (or PrfA*) interferes with a component(s) essential for phosphotransferase system-mediated glucose transport.

Base Sequence↗
PubMed2004

Listeria monocytogenes infection-dependent transfer of exogenously added DNA to fibroblast COS-1 cells.

The addition of double-stranded circular or linear DNA encoding EGFP (the Enhanced Green Fluorescent Protein) to a Listeria -containing infection medium resulted in up to 8.6% COS-1 cells expressing the reporter protein. The transfer of naked DNA into host cells upon infection by Listeria was found to be dependent on the ability of the bacteria to synthesize internalins and listeriolysin. Since no binding of DNA to the bacterial cells was detected, DNA uptake seems to be the consequence of the simultaneous entry of infection medium, and thus of naked DNA, via the phagosomes induced by the bacterium to facilitate its own entry into the host cells.

Animals↗
PubMed2003

Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery.

Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approach to express plasmid-encoded heterologous proteins (protein antigens, toxins or enzymes) in a large set of different cell types including phagocytic and nonphagocytic mammalian cells. Previously, we have described a Listeria monocytogenes-mediated DNA delivery system, which releases plasmid DNA directly into the cytosol of mammalian cells by partial self-destruction of the carrier bacteria. Here we report on a second generation of this phage lysin supported bactofection system, which is greatly improved with respect to plasmid stability, transfer efficacy and biosafety. In this case, DNA release is initiated by spontaneous bacterial lysis in the infected cells cytosol which is subsequently enhanced by the simultaneously released phage lysin produced by the intracellular carrier bacteria. Bacteria that are capable of cell-to-cell spread are found to be much more efficient in bactofection than their non spreading counterparts.

Animals↗
PubMed2002

Efficient expression of the alpha-haemolysin determinant in the uropathogenic Escherichia coli strain 536 requires the leuX-encoded tRNA(5)(Leu).

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two alpha-haemolysin determinants which are located on different pathogenicity islands (PAI I(536) and PAI II(536)). PAI II(536) is associated with the tRNA gene leuX. The leuX-encoded tRNA(5)(Leu) is required for the efficient expression of the hly determinants in strain 536. HlyA levels were reduced and secretion of the protein was delayed in the leuX-negative mutant strain 536Delta102. The lack of a functional tRNA(5)(Leu) resulted in a decrease in hly transcript levels in comparison to the wild-type strain. Analysis of several genes whose products are involved in the regulation of hly expression revealed that levels of RfaH and Hha, as well as the corresponding rfaH and hha transcripts, were higher in the leuX-negative background, whereas the expression of tolC and hns was not influenced by the leuX genotype. The analysis of hly transcript levels in hha deletion mutants of the E. coli strains 536 and 536Delta102 demonstrated that the increase in hha expression is partially responsible for the reduction in hly transcript levels in the leuX-negative background. These results demonstrate that the tRNA(5)(Leu) affects the expression of the alpha-haemolysin determinant at different levels in a regulatory cascade, and imply that, in addition to Hha, at least one further, as yet unidentified, regulatory factor must be involved in the regulation of hly transcription in the uropathogenic E. coli strain 536.

Bacterial Proteins↗
PubMed2001

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Adaptation, Physiological↗
PubMed2001

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.

Animals↗
PubMed2001

Gram-positive and Gram-negative bacteria as carrier systems for DNA vaccines.

Vaccination by intradermal or intramuscular injection of eukaryotic antigen expression vectors (so-called DNA vaccines) elicits strong cellular and humoral immune responses. A novel approach employs attenuated mutant strains of Gram-positive and Gram-negative intracellular bacteria as carriers for the delivery of DNA vaccines. This strategy allows the administration of the DNA vaccines via mucosal surfaces and a direct delivery of the plasmid DNA to professional antigen presenting cells (APC), such as macrophages and dendritic cells (DC). In this work, we have found that several Gram-negative bacteria are capable of delivering plasmid vectors to human DC. In addition, we tested the suitability of the Gram-positive bacterium Listeria monocytogenes as a vaccine carrier for the immunization of fish.

Animals↗
PubMed2001

Recombinant attenuated bacteria for the delivery of subunit vaccines.

Using attenuated intracellular bacteria as carriers, we have developed two different approaches for the delivery of subunit vaccines encoding heterologous antigens. The first system is based on the direct secretion of the heterologous antigens in Gram-negative bacteria via the hemolysin secretion system of Escherichia coli into either phagosome or cytosol of infected cells. The second approach is based on the transport of eukaryotic antigen expression vectors by intracellular bacteria like Listeria and Salmonella into the host cell and here, preferably, into the cytosolic compartment. After release of the plasmid DNA from the bacteria, the plasmid-encoded antigens can be expressed directly by the host cell. Finally, we combined both types of subunit vaccines in one live vector - we equipped Salmonella strains with a phagosomal escape function by utilization of the hemolysin secretion system and used this recombinant vaccine strain for the delivery of a eukaryotic antigen expression vector into the cytosol of macrophages.

Animals↗
PubMed2001

A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes.

We used a novel approach for the direct ex vivo identification and characterization of T cell epitopes based on the screening of peptide spot libraries with freshly isolated splenocytes in a sensitive enzyme-linked immunospot (ELISPOT) assay. This technique was applied for the analysis of splenocytes from Listeria monocytogenes-infected BALB/c and C57BL/6 mice. The screening of peptide spot libraries covering the whole listeriolysin O and p60 of L. monocytogenes confirmed all known CD4 and CD8 T cell epitopes of these proteins and additionally revealed six new H-2(d) and six new H-2(b)-restricted T cell epitopes. New epitopes were categorized into CD4 and CD8 T cell epitopes by ex vivo ELISPOT analysis with separated T cell populations. The quantitative analysis of cells reactive with these CD4 and CD8 T cell epitopes revealed the existence of dominant and subdominant CD4 and CD8 T cell populations during L. monocytogenes infection. As a consequence of these data we suggest that ELISPOT-based screening of peptide spot libraries could be a general approach for the rapid identification and characterization of pathogen-specific T cell populations during various infectious diseases.

Animals↗
PubMed2001

From evil to good: a cytolysin in vaccine development.

Current vaccination strategies mainly target antigens into the phagosomal, major histocompatibility complex class II antigen-processing pathway and thus lead predominantly to humoral immune responses. The elicitation of cytotoxic T-cell responses instead requires introduction of antigens into the cytosol of professional antigen-presenting cells (APCs). The intracellular bacterium Listeria monocytogenes gains access to the host cell cytosol by means of a cytolysin, listeriolysin O. Vaccine researchers have successfully employed listeriolysin in novel vaccination approaches to provide access to the cytosol of professional APCs for purified protein antigens, attenuated bacterial vaccine strains, DNA vaccines and liposome contents.

Animals↗
PubMed2001

Intracellular survival strategies of mutualistic and parasitic prokaryotes.

Endosymbiotic bacteria closely related to mammalian pathogens are widespread in invertebrates. Mutualistic and parasitic bacteria-host interactions on the various evolutionary levels apparently involve similar factors, indicating that relevant genetic information developed early in evolution. The detailed characterization of symbiotic interactions of bacteria with non-mammalian hosts should provide profound insights into the basic mechanisms of bacteria-host interactions and their evolution.

Animals↗
PubMed2001

Pathogenicity islands and virulence evolution in Listeria.

As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.

Amino Acid Sequence↗
PubMed2001

In vitro transcription of PrfA-dependent and -independent genes of Listeria monocytogenes.

In vitro transcription starting from the promoters of the Listeria monocytogenes genes hly, plcA, actA, mpl, prfA and iap has been studied. Whereas transcription from Phly, PplcA and PactA is strictly PrfA-dependent, that from Piap, PprfA1/2 and, unexpectedly, also from Pmpl is independent. Initiation of in vitro transcription at all tested promoters except PprfA requires high concentrations of ATP but not GTP. The nucleotides required in higher concentrations for efficient in vitro transcription are always included in the first three nucleotides of the corresponding transcript. RNA polymerase prepared from L. monocytogenes cultured either in rich culture medium (RNAP(BHI)), exposed to heat shock conditions (RNAP48) or conditioned in minimal essential medium (RNAP(MEM)) shows significant differences in the transcription efficiencies when transcription is initiated at these promoters. Transcription starting from the PrfA-dependent promoters PactA and Phly is enhanced with RNAP48 and RNAP(MEM) (in relation to Piap-mediated transcription), while transcription from the other promoters is reduced when compared with RNAP(BHI). These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded with the vegetative sigma factor 43, but may require a modified RNA polymerase, possibly loaded with an alternative sigma factor.

Bacterial Proteins↗
PubMed2001

No effect of vitamin D3 treatment on active calcium absorption across ruminal epithelium of sheep.

A significant contribution of the forestomachs in net calcium (Ca2+) absorption from the gastrointestinal tract has been postulated from in vivo and in vitro studies in different ruminant species. However, the potential role of vitamin D3 and its metabolites in controlling these mechanisms is still under discussion. It was therefore the aim of the present study to investigate the effectiveness of treatment with vitamin D3 in stimulating active Ca2+ absorption from sheep rumen. Four mature, non-lactating, non-pregnant sheep that had been treated 7 and 4 days before the Ca2+ flux rate measurements with intramuscular injections of 300000 IU of vitamin D3 each in aqueous solution were used. Two female and three male placebo-treated sheep served as controls. To characterize the effects of vitamin D3 application on plasma parameters the time courses of total calcium, inorganic phosphate, calcitriol and intact parathyroid hormone (iPTH) were recorded. In vitro studies of unidirectional Ca2+ flux rates across isolated, intact rumen wall epithelia were carried out by applying the Ussing-chamber technique. Western blot analysis and reverse transcriptase-polymerase chain reaction analysis (RT-PCR) were applied to identify vitamin D receptors (VDR) in ruminal and jejunal tissues. In addition, Western blot analysis for qualitative examination of epithelial calbindin D9k levels was carried out in these tissues. Total calcium and phosphate levels in plasma were not significantly affected treatment with vitamin whereas calcitriol concentrations significantly increased by about 130 and 63% after the first and second application, respectively. In contrast, iPTH tended to decrease by about 60% indicating regulatory effects of calcitriol on systemic Ca homeostasis. The Ca2+ flux rate measurements in Ussing-chambers revealed significant net Ca2+ absorption indicating the contribution of active mechanisms for Ca2+ transport in rumen epithelia. This, however, was not significantly affected by increased calcitriol concentrations in plasma. Western blot analysis on the basis of a human recombinant VDR protein and RT-PCR clearly indicated the presence of VDR in ruminal and jejunal epithelia, but, in contrast to jejunum, this was not reflected by respective amounts of calbindin-D9k in ruminal tissues. The results suggest the absence of classical calbindin-D9k-mediated mechanisms for active Ca2+ transport in sheep rumen.

Absorption↗
PubMed2001

Listeria pathogenesis and molecular virulence determinants.

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal individuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

Animals↗
PubMed2001

Antibodies against listerial protein 60 act as an opsonin for phagocytosis of Listeria monocytogenes by human dendritic cells.

Human-monocyte-derived dendritic cells (MoDC) are very efficient in the uptake of Listeria monocytogenes, a gram-positive bacterium which is an important pathogen in humans and animals causing systemic infections with symptoms such as septicemia and meningitis. In this work, we analyzed the influence of blood plasma on the internalization of L. monocytogenes into human MoDC and compared the uptake of L. monocytogenes with that of Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. While human plasma did not significantly influence the uptake of serovar Typhimurium and Y. enterocolitica by human MoDC, the efficiency of the uptake of L. monocytogenes by these phagocytes was strongly enhanced by human plasma. In plasma-free medium the internalization of L. monocytogenes was very low, whereas the addition of pooled human immunoglobulins resulted in the internalization of these bacteria to a degree comparable to the highly efficient uptake observed with human plasma. All human plasma tested contained antibodies against the 60-kDa extracellular protein of L. monocytogenes (p60), and anti-p60 antibodies were also found in the commercially available pooled immunoglobulins. Strikingly, in contrast to L. monocytogenes wild type, an iap deletion mutant (totally deficient in p60) showed only a minor difference in the uptake by human MoDC in the presence or the absence of human plasma. These results support the assumption that antibodies against the listerial p60 protein may play an important role in Fc-receptor-mediated uptake of L. monocytogenes by human MoDC via opsonization of the bacteria. This process may have a major impact in preventing systemic infection in L. monocytogenes in immunocompetent humans.

Antibodies, Bacterial↗
PubMed2001

Positive selection of mutations leading to loss or reduction of transcriptional activity of PrfA, the central regulator of Listeria monocytogenes virulence.

Transcription factor PrfA controls the expression of virulence genes essential for Listeria monocytogenes pathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerial iap gene, encoding the p60 protein, is lethal if overexpressed in Bacillus subtilis. A plasmid in which the iap gene is placed under the control of the PrfA-dependent hly promoter was constructed and introduced into B. subtilis. This strain was rapidly killed when expression of iap was induced by introduction of a second plasmid carrying prfA. Two classes of B. subtilis survivor mutants were identified: one carried mutations in iap, and the second carried mutations in prfA. Sequence analysis of the defective prfA genes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the beta-roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure. PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.

Amino Acid Sequence↗