Assuntos
Ictiose Lamelar/metabolismo , Transtornos do Metabolismo dos Lipídeos/metabolismo , Animais , Modelos Animais de Doenças , Epiderme/embriologia , Epiderme/metabolismo , Humanos , Ictiose Lamelar/embriologia , Metabolismo dos Lipídeos , Transtornos do Metabolismo dos Lipídeos/embriologia , Camundongos Nus , Regulação para Cima/fisiologiaAssuntos
Proliferação de Células/fisiologia , Epiderme/patologia , Dermatopatias/patologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Diferenciação Celular/fisiologia , Técnicas de Inativação de Genes , Queratina-6/metabolismo , Queratinócitos/patologia , Queratinócitos/fisiologia , Camundongos , Camundongos Endogâmicos , PPAR gama/metabolismo , PPAR beta/metabolismo , Transdução de Sinais/fisiologia , Tretinoína/antagonistas & inibidores , Tretinoína/fisiologia , Proteína Supressora de Tumor p53/deficiência , Regulação para CimaRESUMO
Cytolethal distending toxin (CDT) from the diarrheagenic bacterium Campylobacter jejuni was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4N DNA content, indicative of cells in G2 or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G2. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease by C. jejuni and other diarrheagenic bacteria that produce CDT.
Assuntos
Toxinas Bacterianas/toxicidade , Campylobacter jejuni/patogenicidade , Fase G2/efeitos dos fármacos , Proteína Quinase CDC2/fisiologia , Células CACO-2 , Cafeína/farmacologia , Células HeLa , HumanosRESUMO
Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,11-6, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species.
Assuntos
Toxinas Bacterianas/genética , Campylobacter jejuni/patogenicidade , Genes Bacterianos , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/biossíntese , Sequência de Bases , Bovinos , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Campylobacter jejuni is a microaerobic bacterium that produces an acute, self-limiting, watery or bloody diarrhea in humans. Little is known about how C. jejuni causes disease or even what specific capabilities it requires for survival in vivo. The enzyme, superoxide dismutase (SOD), which catalyzes the breakdown of superoxide radicals to hydrogen peroxide and dioxygen is one of the bacterial cell's major defense mechanisms against oxidative damage. A PCR-based search for sod genes in C. jejuni 81-176 revealed that this bacterium contained at least one sod gene. We cloned and sequenced a sod gene from 81-176 and determined that its predicted protein product was most similar to that of FeSODs (sodB genes). Transcriptional analysis indicated that this gene is monocistronic and may be transcribed from a sigma 70-like promoter. Nondenaturing polyacrylamide gels stained to reveal SOD activities, accompanied by inhibition studies, demonstrated that C. jejuni produces five electrophoretically distinct bands of SOD activity, all of which appeared to be FeSODs. Analysis of an 81-176 sodB strain revealed that all of these FeSOD activities may be products of the one sodB gene that we cloned. The expression and enzymatic activity of the respective sodB and FeSOD produced by both C. jejuni and Helicobacter pylori were examined in Escherichia coli. Both genes were expressed in E. coli, and the proteins produced were enzymatically active. Finally, the ability of the 81-176 sodB strain to survive INT407 cell invasion was found to be significantly decreased (12-fold) compared with that of the parent, suggesting a potential role for SodB in C. jejuni intracellular survival.
Assuntos
Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidade , Intestinos/microbiologia , Superóxido Dismutase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Campylobacter jejuni/enzimologia , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Humanos , Intestinos/citologia , Ferro , Dados de Sequência Molecular , Mutagênese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/metabolismo , Virulência/genéticaRESUMO
A limited number of Escherichia coli isolates which produce an apparently novel toxin, termed cytolethal distending toxin (CDT), have been reported. The toxic activity produced by these strains causes certain cultured cell lines to become slowly distended and then disintegrate. DNA was isolated from the CDT-producing E. coli strain, 9142-88, and cloned into a cosmid vector. Plasmid DNA from a toxin-positive transductant was further subcloned until a plasmid with a 4-kb insert which still encoded the toxin activity was obtained. Nucleotide sequencing of a portion of this insert revealed the presence of three adjacent open reading frames. Further subcloning and deletion analysis suggested that the products of all three open reading frames may be required for toxin activity. Minicell experiments identified the products of all three open reading frames. The three proteins had predicted sizes of 27,753,29,531, and 19,938 Da, and all three appeared to have strong consensus leader sequences. None of the three predicted proteins had significant homology to known proteins.