The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced byBordeteUapertussis, is believed to be due to the ADP-ribosyltransferase activity of the Si subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymaticaily deficient form of the Si subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant Si subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant Si displayed greatly diminished cytopathicity.These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.In an effort to create a fully efficacious yet less reactogenic pertussis vaccine, we have focused on the recombinant production of PTX subunit proteins, detoxification of the Si moiety by site-specific mutagenic inactivation of enzyme activity, and the in vitro assembly of a genetic "holotoxoid." For these purposes, the individual subunit polypeptides were expressed at high levels in Escherichia coli (36), and a region of the recombinant Si subunit (Val8-Pro'5) was identified as both necessary for enzyme activity and critical for the formation of its neutralizing, protective epitope (37). Selective site-directed mutagenesis of the subcloned S1 cistronic element permitted us to derive and analog polypeptide (Arg9 -k Lys) retaining the protective epitope, but with strikingly reduced ADP-ribosyltransferase activity (38). We wished to assess the biological activity of this genetically deactivated S1 mutant subunit and evaluate its potential to participate in the production of a recombinant holotoxoid. To this end, we have assembled holotoxin molecules in vitro that are composed of native PTX B oligomer and recombinant SI subunits of either native or mutant sequence and evaluated their relative abilities to elicit cytotoxic responses in cultured cells.Pertussis toxin (PTX) is both a major virulence factor of Bordetella pertussis (1), the etiologic agent of whooping cough (2), and an important antigenic element in vaccines for immunoprotection against disease (3-7). Certain clinical manifestations of infection (1,8,9) and minor reactions to vaccination (10,11) are attributable to the effects of this complex exotoxin. The origin of the extremely rare adverse neurologic events temporally related to pertussis immunization remains controversial (11-15). Chemic...

An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.

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