title: Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis.
abstract: 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.
authors.affiliation: Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Gray
authors.forename: G M
authors.initials: GM
authors.lastname: Donato
authors.forename: F R
authors.initials: FR
authors.lastname: Jones
authors.forename: T
authors.initials: T
authors.lastname: Kim
authors.forename: E L
authors.initials: EL
authors.lastname: Hewlett
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001422:Bacterial Adhesion; D001886:Bordetella pertussis / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D039481:CD11b Antigen / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D051379:Mice
chemical_list: D037361:Adenylate Cyclase Toxin; D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D039481:CD11b Antigen; D005839:Gentamicins; D007476:Ionophores; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000242:Cyclic AMP; D002118:Calcium