Werner Goebel - Academia.edu (original) (raw)

Papers by Werner Goebel

Molecular Microbiology, 2002

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of t... more A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5a as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene. P. Loewen (University of Manitoba, Winnipeg, Manitoba, Canada). The multipurpose-plasmid vector pTZ18R was described previously (32). Media and reagents. Listeria strains and B. thuringiensis were grown overnight at 37°C and 160 rpm in brain heart infusion broth (Difco Laboratories, Detroit, Mich.); E. coli was grown in Luria-Bertani (LB) broth. When needed, ampicillin was added to a final concentration of 100 mg/liter.

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanov... more The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence

Fems Microbiology Letters, 1997

Infection experiments have shown that the avirulent species Listeria seeligeri invaded the entero... more 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

Trends in Microbiology, 1997

Infection and Immunity

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to m... more 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 sprea...

A recombinant (r)-Salmonella typhimurium aroA vaccine strain was constructed which secretes the n... more 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.

The Journal of infectious diseases, Jan 13, 2015

Although the T-cell subset differentiation pathway has been characterized extensively from the v... more Although the T-cell subset differentiation pathway has been characterized extensively from the view of host gene regulation, the effects of genes of the pathogen on T-cell subset differentiation during infection have yet to be elucidated. Especially, the bacterial genes that are responsible for this shift have not yet been determined. Utilizing a single-gene-mutation Listeria panel, we investigated genes involved in the host-pathogen interaction that are required for the initiation of T-cell subset differentiation in the early phase of pathogen infection. We demonstrate that the induction of T helper types 1 and 2 (Th1 and Th2) subsets are separate phenomena and are mediated by distinct Listeria genes. We identified several candidate Listeria genes that appear to be involved in the host-Listeria interaction. Among them, arpJ is the strongest candidate gene for inhibiting Th2 subset induction. Furthermore, the analysis utilizing arpJ-deficient Listeria monocytogenes (Lm) revealed ...

Applied and environmental microbiology, 1999

The iap gene encodes the protein p60, which is common to all Listeria species. A previous compari... more 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...

Frontiers in Cellular and Infection Microbiology, 2014

Infection and immunity, 1992

Listeria monocytogenes is a rapidly growing, gram-positive, food-borne human and animal pathogen ... more Listeria monocytogenes is a rapidly growing, gram-positive, food-borne human and animal pathogen responsible for serious infections in immunocompromised individuals and pregnant women (17). The murine model of listeriosis has received enormous attention over the years because of the utility of L. monocytogenes as a model pathogen to study cell-mediated immunity. In fact, much of our current understanding of cell-mediated immunity, such as the concept of the activated macrophage, has its roots in the study of murine listeriosis . The beauty of the murine model is that it provides a highly reproducible system for the quantitation of L. monocytogenes virulence. However, until relatively recently almost nothing was known about the cell biology of intracellular growth or bacterial determinants of pathogenicity.

Gene Therapy, 2003

Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approac... more 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

Advances in Experimental Medicine and Biology, 2002

Escherichia coli is by far the most common causative agent of urinary tract infections. The abili... more Escherichia coli is by far the most common causative agent of urinary tract infections. The ability of uropathogenic strains to produce certain virulence factors distinguishes them from commensal and intestinal pathogenic strains. Pathogenicity factors, such as fimbriae, ferric uptake ...

Chemotherapy, 2007

The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif®, the only attenu... more The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif®, the only attenuated live oral vaccine against typhoid fever. In comparison with antibiotics, the ‘magic bullets’ which Paul Ehrlich was striving for to treat infectious diseases, this vaccine should be viewed as a ‘magic shield’, because rather than treating typhoid fever after the infection has started, immunisation with

International Journal of Medical Microbiology, 2001

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for eit... more 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 α-hemolysin secretion system (type I) 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 and dendritic cells in vitro and can be also used in animal models.

Molecular and General Genetics, 2000

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella p... more 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 (Pmip) and the sod (superoxide dismutase) promoter (Psod) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong

Molecular and General Genetics, 1979

The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication ori... more The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK 103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication

PLoS ONE, 2010

Background: In the environment as well as in the vertebrate intestine, Listeriae have access to c... more Background: In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems.

Molecular and General Genetics, 1985

The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstrea... more The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS

A cloned cytolytic determinant fromthegenome ofBacillus cereusGP-4hasbeencharacterized atthe mole... more A cloned cytolytic determinant fromthegenome ofBacillus cereusGP-4hasbeencharacterized atthe molecular level. Nucleotide sequencedetermination revealed thepresenceoftwoopenreading frames. Both openreading frames werefoundbydeletion andcomplementation analysis tobenecessaryforexpression ofthe hemolytic phenotype byBacillus subtilis andEscherichia coli hosts. The5'openreading frame was foundtobe nearly identical toa recently reported phospholipase C genederived froma mutantB.cereusstrain which overexpressestherespective protein, anditconferred a lecithinase-positive phenotype totheB.subtilis host. The3'open reading frameencoded a sphingomyelinase. Thetwotandemly encoded

Molecular Microbiology, 2002

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of t... more A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5a as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene. P. Loewen (University of Manitoba, Winnipeg, Manitoba, Canada). The multipurpose-plasmid vector pTZ18R was described previously (32). Media and reagents. Listeria strains and B. thuringiensis were grown overnight at 37°C and 160 rpm in brain heart infusion broth (Difco Laboratories, Detroit, Mich.); E. coli was grown in Luria-Bertani (LB) broth. When needed, ampicillin was added to a final concentration of 100 mg/liter.

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanov... more The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence

Fems Microbiology Letters, 1997

Infection experiments have shown that the avirulent species Listeria seeligeri invaded the entero... more 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

Trends in Microbiology, 1997

Infection and Immunity

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to m... more 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 sprea...

A recombinant (r)-Salmonella typhimurium aroA vaccine strain was constructed which secretes the n... more 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.

The Journal of infectious diseases, Jan 13, 2015

Although the T-cell subset differentiation pathway has been characterized extensively from the v... more Although the T-cell subset differentiation pathway has been characterized extensively from the view of host gene regulation, the effects of genes of the pathogen on T-cell subset differentiation during infection have yet to be elucidated. Especially, the bacterial genes that are responsible for this shift have not yet been determined. Utilizing a single-gene-mutation Listeria panel, we investigated genes involved in the host-pathogen interaction that are required for the initiation of T-cell subset differentiation in the early phase of pathogen infection. We demonstrate that the induction of T helper types 1 and 2 (Th1 and Th2) subsets are separate phenomena and are mediated by distinct Listeria genes. We identified several candidate Listeria genes that appear to be involved in the host-Listeria interaction. Among them, arpJ is the strongest candidate gene for inhibiting Th2 subset induction. Furthermore, the analysis utilizing arpJ-deficient Listeria monocytogenes (Lm) revealed ...

Applied and environmental microbiology, 1999

The iap gene encodes the protein p60, which is common to all Listeria species. A previous compari... more 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...

Frontiers in Cellular and Infection Microbiology, 2014

Infection and immunity, 1992

Listeria monocytogenes is a rapidly growing, gram-positive, food-borne human and animal pathogen ... more Listeria monocytogenes is a rapidly growing, gram-positive, food-borne human and animal pathogen responsible for serious infections in immunocompromised individuals and pregnant women (17). The murine model of listeriosis has received enormous attention over the years because of the utility of L. monocytogenes as a model pathogen to study cell-mediated immunity. In fact, much of our current understanding of cell-mediated immunity, such as the concept of the activated macrophage, has its roots in the study of murine listeriosis . The beauty of the murine model is that it provides a highly reproducible system for the quantitation of L. monocytogenes virulence. However, until relatively recently almost nothing was known about the cell biology of intracellular growth or bacterial determinants of pathogenicity.

Gene Therapy, 2003

Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approac... more 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

Advances in Experimental Medicine and Biology, 2002

Escherichia coli is by far the most common causative agent of urinary tract infections. The abili... more Escherichia coli is by far the most common causative agent of urinary tract infections. The ability of uropathogenic strains to produce certain virulence factors distinguishes them from commensal and intestinal pathogenic strains. Pathogenicity factors, such as fimbriae, ferric uptake ...

Chemotherapy, 2007

The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif®, the only attenu... more The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif®, the only attenuated live oral vaccine against typhoid fever. In comparison with antibiotics, the ‘magic bullets’ which Paul Ehrlich was striving for to treat infectious diseases, this vaccine should be viewed as a ‘magic shield’, because rather than treating typhoid fever after the infection has started, immunisation with

International Journal of Medical Microbiology, 2001

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for eit... more 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 α-hemolysin secretion system (type I) 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 and dendritic cells in vitro and can be also used in animal models.

Molecular and General Genetics, 2000

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella p... more 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 (Pmip) and the sod (superoxide dismutase) promoter (Psod) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong

Molecular and General Genetics, 1979

The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication ori... more The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK 103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication

PLoS ONE, 2010

Background: In the environment as well as in the vertebrate intestine, Listeriae have access to c... more Background: In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems.

Molecular and General Genetics, 1985

The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstrea... more The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS

A cloned cytolytic determinant fromthegenome ofBacillus cereusGP-4hasbeencharacterized atthe mole... more A cloned cytolytic determinant fromthegenome ofBacillus cereusGP-4hasbeencharacterized atthe molecular level. Nucleotide sequencedetermination revealed thepresenceoftwoopenreading frames. Both openreading frames werefoundbydeletion andcomplementation analysis tobenecessaryforexpression ofthe hemolytic phenotype byBacillus subtilis andEscherichia coli hosts. The5'openreading frame was foundtobe nearly identical toa recently reported phospholipase C genederived froma mutantB.cereusstrain which overexpressestherespective protein, anditconferred a lecithinase-positive phenotype totheB.subtilis host. The3'open reading frameencoded a sphingomyelinase. Thetwotandemly encoded