RESUMO
Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP. To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B. pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin. Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin. A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B. pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication. B. pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria. Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria. These data establish that delivery of this toxin by intact B. pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin.
Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/metabolismo , Animais , Antibacterianos/farmacologia , Anticorpos Monoclonais/metabolismo , Aderência Bacteriana , Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/efeitos da radiação , Antígeno CD11b/metabolismo , Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Linhagem Celular , AMP Cíclico/metabolismo , Gentamicinas/farmacologia , Ionóforos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , CamundongosRESUMO
Adenylate cyclase (AC) toxin from Bordetella pertussis intoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K(+) and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K(+) efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 micrograms/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37 degrees C; however, the total binding at 0 degrees C is less than that at 37 degrees C. In human erythrocytes, AC toxin does not cause an increase in K(+) efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K(+) efflux, and hemolysis produced by AC toxin.
Assuntos
Toxina Adenilato Ciclase , Adenilil Ciclases/metabolismo , Bordetella pertussis/enzimologia , Eritrócitos/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Citometria de Fluxo/métodos , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/metabolismo , Ovinos , TemperaturaRESUMO
Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.
Assuntos
Toxina Adenilato Ciclase , Escherichia coli/metabolismo , Hemólise , Ácidos Palmíticos/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Virulência de Bordetella/metabolismo , Fatores de Virulência de Bordetella/toxicidade , Acilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bordetella pertussis/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Eritrócitos/efeitos dos fármacos , Lisina , Espectrometria de Massas , Dados de Sequência Molecular , Ácido Palmítico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Ovinos , Fatores de Virulência de Bordetella/biossínteseRESUMO
Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin which is able to bind to target cells but unable to elicit cAMP accumulation. In that AC toxin from BPDE386 is able to function normally when injected artificially to an intracellular site, we conclude that the disruption of cyaC produces a defect in insertion and transmembrane delivery of the catalytic domain.
Assuntos
Toxina Adenilato Ciclase , Adenilil Ciclases/metabolismo , Bordetella pertussis/enzimologia , Bordetella pertussis/genética , Genes Bacterianos , Genes Reguladores , Fatores de Virulência de Bordetella/genética , Adenilil Ciclases/genética , Adenilil Ciclases/isolamento & purificação , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Células Cultivadas , AMP Cíclico/metabolismo , Eletrofisiologia/métodos , Coração/efeitos dos fármacos , Coração/fisiologia , Hemólise , Humanos , Técnicas In Vitro , Modelos Biológicos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Rana catesbeiana , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Espectrometria de Fluorescência , Linfócitos T Auxiliares-Indutores , Fatores de Virulência de Bordetella/isolamento & purificação , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Adenylate cyclase (AC) toxin from Bordetella pertussis enters cells to cause supraphysiologic increases in cAMP. AC toxin is also hemolytic. Substitution of Lys-58 with a methionine residue by site-directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme-deficient AC toxin molecule. This mutant toxin molecule exhibits 1000-fold reduction in enzymatic activity relative to wild-type and has no toxin activity in J774 cells. The enzyme-deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme-deficient organisms were used to infect infant mice. The hemolytic, enzyme-deficient mutant organisms are reduced in virulence relative to wild-type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.
Assuntos
Toxina Adenilato Ciclase , Bordetella pertussis/patogenicidade , Hemólise , Fatores de Virulência de Bordetella/farmacologia , Animais , Animais Recém-Nascidos , Bordetella pertussis/enzimologia , Bordetella pertussis/genética , AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Cinética , Pulmão/microbiologia , Camundongos , Mutagênese Sítio-Dirigida , Mapeamento por Restrição , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/metabolismo , Coqueluche/microbiologia , Coqueluche/mortalidadeRESUMO
Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.
Assuntos
Toxina Adenilato Ciclase , Adenilil Ciclases/isolamento & purificação , Bordetella pertussis/enzimologia , Fatores de Virulência de Bordetella/isolamento & purificação , Adenilil Ciclases/metabolismo , Anticorpos Monoclonais , Calmodulina , Cromatografia de Afinidade , Cinética , Peso Molecular , Fatores de Virulência de Bordetella/metabolismoRESUMO
Pertussis toxin (PT) has been shown to have a variety of effects on T lymphocyte function, and its activity has been used to suggest the involvement of a G protein in the early events of T lymphocyte activation. In this report, the effects of PT on T lymphocytes have been investigated in detail. PT at a concentration of 10 micrograms/ml rapidly stimulated early events that are normally induced by occupancy of the TCR complex in Jurkat cells and cloned, murine CTL including increased intracellular Ca2+ concentration, serine esterase release, and induction of Ag non-specific target cell lysis. However, 1-h treatment with this concentration of PT induced a state that was refractory to further receptor stimulation in Jurkat cells but not cloned CTL although substrate membrane proteins were modified to a similar extent in both cell lines. The functional effects of PT were mimicked by the B oligomer of PT which did not, however, catalyze ADP-ribosylation of membrane proteins. In addition, overnight exposure of Jurkat cells to a lower concentration of PT also modified substrate membrane proteins but did not inhibit receptor stimulation. These findings indicate that PT catalyzed ADP-ribosylation of a G protein does not account for the actions of the toxin on T lymphocytes. Finally, direct stimulation of increased intracellular Ca2+ concentration by PT and the B oligomer only occurred in T lymphocytes expressing CD3. This suggests that the mitogenic effect of PT holotoxin is mediated by the interaction of the B oligomer with CD3 and that this may account for many of the effects of PT holotoxin both in vivo and in vitro.