TY - JOUR A1 - Heisig, Martin A1 - Frentzen, Alexa A1 - Bergmann, Birgit A1 - Gentschev, Katharina Ivaylo A1 - Hotz, Christian A1 - Schoen, Christoph A1 - Stritzker, Jochen A1 - Fensterle, Joachim A1 - Rapp, Ulf R. A1 - Goebel, Werner T1 - Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines N2 - Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization. KW - Listeria monocytogenes Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68705 ER - TY - JOUR A1 - Kreft, Jürgen A1 - Goebel, Werner T1 - Complex Co1E1 DNA in Escherichia coli and Proteus mirabilis N2 - Incubation of the colicinogenic Escherichia coli strain JC 411 (ColE1) at elevated temperatures (47-49°) leads to the accumulation of catenated molecules and replicative intermediates of this plasmid. Mature supercoiled OolE1 DNA molecules synthesized under these conditions have an increased number of tertiary turns as shown by electron microscopy. The monomeric tightly supercoiled molecules possess a slightly slower sedimentation rate and a higher binding capacity for ethidium bromide than supercoiJed monomers synthesized at lower temperatures. Recombination deficient mutants of E. coli recA, recB and recC, which carry the ColE1 plasmid, form about the same amount of catenated molecules at the elevated temperature as a rec+ strain. In addition, we have observed by electron microscopy a small percentage (.--.5% of the circular DNA molecules) of minicircular DNA molecules in all preparations of JC 411 (CoIE1). They are homogenous in size, with a molecular weight of 1.4 X 106 daltons. Addition of chloramphenicol to a culture of Proteus mirabilis (ColE1) leads to an increased amount of higher multiple circular oligomers and to a stimulated accumulation of catenated OolE1 DNA molecules of varying sizes. ColE1 DNA synthesis is more thermosensitive than chromosomal DNA replication in P. mirabili8. Plasmid replication stops completely at temperatures above 43°C. Y1 - 1974 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47044 ER - TY - JOUR A1 - Kreft, Jürgen A1 - Bernhard, K. A1 - Goebel, Werner T1 - Recombinant plasmids capable to replication in B. subtilis and E. coli N2 - The plasmid pBC16 (4.25 kbases), ongtnally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBCl6 (pBCI6-1), 2,7 kb) which has lost an EcoRI fragment of pBCI6 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS], a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et aI., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBSl61 (8.2 kb) and the smaller plasmid pBS162 (2. I kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindlII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-l is generated, which can be used as a HindlII and EcoRI cloning vector in Bacillus suhtilis. Hybrid plasmids consisting of the E. coli plasmids pBR322, p WL 7 or pACl84 and different HindlII fragments of pBSI61 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindlII fragment of pBS161 can replicate in E. coli and B. sublilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. suhtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. suhtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli. Y1 - 1978 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47000 ER - TY - JOUR A1 - Kreft, Jürgen A1 - Funke, D. A1 - Schlesinger, R. A1 - Lottspeich, F. A1 - Goebel, Werner T1 - Purification and characterization of cytolysins from Listeria monocytogenes serovar 4b and Listeria ivanovii N2 - Several exoproteins from Listeria monocytogenes serovar 4b (NCTC 10527) and Listeria ivanovii (ATCC) 19119, SLCC 2379), respectively, have been purified to homogeneity by thiol-disulfide exchange chromatography and gel filtration. Both strains produce a haemolytic/cytolytic protein of Mr 58 kDa, which has all the properties of a SH-activated cytolysin, the prototype of which is streptolysin 0 (SLO), and this protein has therefore heen termed Iisteriolysin 0 (LLO). In addition a protein of Mr 24 kDa from culture supernatants of L. ivanovii co-purified withLLO. The N-terminal aminoacid sequences of both proteins from L. ivanovii have been determined. By mutagenesis with transposons of Gram-positive origin (Tn916 and TnI545), which have been introduced via conjugation into L. ivanovii, several phenotypic mutants (altered haemolysis on sheep blood agar or lecithinase-negative) were obtained. Results on the properties of these muntants will he presented. Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47036 ER - TY - JOUR A1 - Kreft, Jürgen A1 - Haas, Albert A1 - Goebel, Werner T1 - Isolation and characterization of genes coding for proteins involved in the cytolysis by Listeria ivanovii N2 - We established a library of chromosomal DNA of Listeria ivanovii in the pTZ19R plasmid system, using Escherichia coli DH5alpha as the host. One recombinant clone reacted strongly with a polyclonal antiserum raised against the listeriolysin 0 and a second exoprotein (24kDa) of L. ivanovii, which is most probably also involved in cytolytic processes. The recombinant E. coli clone may contain part of the listeriolysin 0 gene of L. ivanovii. Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46991 ER - TY - JOUR A1 - Goetz, Andreas A1 - Eylert, Eva A1 - Eisenreich, Wolfgang A1 - Goebel, Werner T1 - Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells N2 - Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens. KW - Metabolismus KW - Carbon Metabolism Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68555 ER - TY - JOUR A1 - Lampidis, Robert A1 - Gross, Roy A1 - Sokolovic, Zeljka A1 - Goebel, Werner A1 - Kreft, Jürgen T1 - The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators N2 - No abstract available KW - Biologie Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60503 ER - TY - JOUR A1 - Brehm, Klaus A1 - Haas, Albert A1 - Goebel, Werner A1 - Kreft, Jürgen T1 - A gene encoding a superoxide dismutase of the facultative intracellular bacterium Listeria monocytogenes N2 - A gene (Imsod) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listeria monocytogenes, was cloned by functional complementation of an SOD-deficient Escherichia coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganese-containing SOD's from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODs formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeria. KW - Biologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60515 ER - TY - JOUR A1 - Haas, Albert A1 - Brehm, Klaus A1 - Kreft, Jürgen A1 - Goebel, Werner T1 - Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases N2 - A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the grain-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DHSa as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately SO times that of the combined E. coli catalases. The nucleutide 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 tbe L. seeligeri catalase gene. KW - Biologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60536 ER - TY - JOUR A1 - Kreft, Jürgen A1 - Burger, Klaus J. A1 - Goebel, Werner T1 - Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis N2 - Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by σ55 of B. subtilis RNA polymerase. KW - Biologie Y1 - 1983 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60600 ER -