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Jurgen Wehland - One of the best experts on this subject based on the ideXlab platform.
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purification of the inlb gene product of listeria monocytogenes and demonstration of its biological activity
Infection and Immunity, 1998Co-Authors: Simone Muller, A Lingnau, Eugen Domann, Trinad Chakraborty, Torsten Hain, Philippos Pashalidis, Jurgen WehlandAbstract:Entry of Listeria monocytogenes into nonphagocytic Cells requires the inlAB gene products. InlA and InlB are bacterial Cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial Cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as Listeria innocua or an L. monocytogenes inlB2 mutant, where it promoted entry of these strains into Vero Cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa Cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into Tissue Culture Cell lines in greater detail.
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expression of the listeria monocytogenes egd inla and inlb genes whose products mediate bacterial entry into Tissue Culture Cell lines by prfa dependent and independent mechanisms
Infection and Immunity, 1995Co-Authors: A Lingnau, Eugen Domann, Martina Hudel, M Bock, Thomas Nichterlein, Jurgen Wehland, Trinad ChakrabortyAbstract:Internalization of Listeria monocytogenes into nonphagocytic Cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and -independent mechanisms. Tissue Culture invasion assays employing various epithelial Cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.
Trinad Chakraborty - One of the best experts on this subject based on the ideXlab platform.
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purification of the inlb gene product of listeria monocytogenes and demonstration of its biological activity
Infection and Immunity, 1998Co-Authors: Simone Muller, A Lingnau, Eugen Domann, Trinad Chakraborty, Torsten Hain, Philippos Pashalidis, Jurgen WehlandAbstract:Entry of Listeria monocytogenes into nonphagocytic Cells requires the inlAB gene products. InlA and InlB are bacterial Cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial Cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as Listeria innocua or an L. monocytogenes inlB2 mutant, where it promoted entry of these strains into Vero Cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa Cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into Tissue Culture Cell lines in greater detail.
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expression of the listeria monocytogenes egd inla and inlb genes whose products mediate bacterial entry into Tissue Culture Cell lines by prfa dependent and independent mechanisms
Infection and Immunity, 1995Co-Authors: A Lingnau, Eugen Domann, Martina Hudel, M Bock, Thomas Nichterlein, Jurgen Wehland, Trinad ChakrabortyAbstract:Internalization of Listeria monocytogenes into nonphagocytic Cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and -independent mechanisms. Tissue Culture invasion assays employing various epithelial Cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.
A Lingnau - One of the best experts on this subject based on the ideXlab platform.
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purification of the inlb gene product of listeria monocytogenes and demonstration of its biological activity
Infection and Immunity, 1998Co-Authors: Simone Muller, A Lingnau, Eugen Domann, Trinad Chakraborty, Torsten Hain, Philippos Pashalidis, Jurgen WehlandAbstract:Entry of Listeria monocytogenes into nonphagocytic Cells requires the inlAB gene products. InlA and InlB are bacterial Cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial Cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as Listeria innocua or an L. monocytogenes inlB2 mutant, where it promoted entry of these strains into Vero Cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa Cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into Tissue Culture Cell lines in greater detail.
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expression of the listeria monocytogenes egd inla and inlb genes whose products mediate bacterial entry into Tissue Culture Cell lines by prfa dependent and independent mechanisms
Infection and Immunity, 1995Co-Authors: A Lingnau, Eugen Domann, Martina Hudel, M Bock, Thomas Nichterlein, Jurgen Wehland, Trinad ChakrabortyAbstract:Internalization of Listeria monocytogenes into nonphagocytic Cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and -independent mechanisms. Tissue Culture invasion assays employing various epithelial Cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.
Paul Britton - One of the best experts on this subject based on the ideXlab platform.
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infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes
Mbio, 2013Co-Authors: Helena J Maier, Philippa C Hawes, Eleanor M Cottam, Judith Mantell, Paul Verkade, Paul Monaghan, Thomas Wileman, Paul BrittonAbstract:ABSTRACT Replication of positive-sense RNA viruses is associated with the rearrangement of Cellular membranes. Previous work on the infection of Tissue Culture Cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian Cell line but also in primary avian Cells and in epithelial Cells of ex vivo tracheal organ Cultures. In all Cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected Cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes. IMPORTANCE All positive-sense single-stranded RNA viruses induce rearranged Cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from Cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected Cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.
Eugen Domann - One of the best experts on this subject based on the ideXlab platform.
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purification of the inlb gene product of listeria monocytogenes and demonstration of its biological activity
Infection and Immunity, 1998Co-Authors: Simone Muller, A Lingnau, Eugen Domann, Trinad Chakraborty, Torsten Hain, Philippos Pashalidis, Jurgen WehlandAbstract:Entry of Listeria monocytogenes into nonphagocytic Cells requires the inlAB gene products. InlA and InlB are bacterial Cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating agents including mutanolysin, thiol reagents, sodium chloride, and detergents like Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate did not release substantial amounts of InlB from the bacterial Cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or other protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purification. A panel of monoclonal antibodies against listerial Tris-Cl extracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enabled purification of the InlB protein to homogeneity with relatively high yields. When added externally, purified InlB associated with the surface of noninvasive bacteria such as Listeria innocua or an L. monocytogenes inlB2 mutant, where it promoted entry of these strains into Vero Cells >300- and 17-fold, respectively. This effect was even more dramatic for HeLa Cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified native, invasion-competent InlB will allow analysis of the molecular basis of InlB-mediated entry into Tissue Culture Cell lines in greater detail.
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expression of the listeria monocytogenes egd inla and inlb genes whose products mediate bacterial entry into Tissue Culture Cell lines by prfa dependent and independent mechanisms
Infection and Immunity, 1995Co-Authors: A Lingnau, Eugen Domann, Martina Hudel, M Bock, Thomas Nichterlein, Jurgen Wehland, Trinad ChakrabortyAbstract:Internalization of Listeria monocytogenes into nonphagocytic Cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and -independent mechanisms. Tissue Culture invasion assays employing various epithelial Cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.