In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.

Adenylate cyclase (AC) toxin fromBordetella pertussis delivers its catalytic domain to the interior of target cells where it converts host ATP to cAMP in a process referred to as intoxication. This toxin also hemolyzes sheep erythrocytes by a mechanism presumed to include pore formation and osmotic lysis. Intoxication and hemolysis appear at strikingly different toxin concentrations and evolve over different time scales, suggesting that different molecular processes may be involved. The present study was designed to test the hypothesis that intoxication and hemolysis occur by distinct mechanisms. Although the hemolytic activity of AC toxin has a lag of >1 h, intoxication starts immediately. Because of this difference, we sought a surrogate or precursor lesion that leads to hemolysis, and potassium efflux has been observed from erythrocytes treated with other pore-forming toxins. AC toxin elicits an increase in K⁺efflux from sheep erythrocytes and Jurkat cells, a human T-cell leukemia line, that begins within minutes of toxin addition. The toxin concentration dependence along with the analysis of the time course suggest that toxin monomers are sufficient to elicit release of K⁺ and to deliver the catalytic domain to the cell interior. Hemolysis, on the other hand, is a highly cooperative event that likely requires a subsequent oligomerization of these individual units. Although induction of K⁺ efflux shares some structural and environmental requirements with both intoxication and hemolysis, it can occur under conditions in which intoxication is reduced or prevented. The data presented here suggest that the transmembrane pathway by which K⁺ is released is separate and distinct from the structure required for intoxication but may be related to, or a precursor of, that which is ultimately responsible for hemolysis.

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.

A previous study showed that opsonization with human immune serum could either promote or antagonize phagocytosis ofBordetella pertussisby human neutrophils depending on whether the bacteria expressed adenylate cyclase toxin. Opsonization of the wild-type strain inhibited phagocytosis relative to unopsonized controls. In contrast, mutants lacking adenylate cyclase toxin were efficiently phagocytosed when opsonized with human immune serum. In this study, we examined opsonization in the presence or absence of monoclonal antibodies to adenylate cyclase toxin. Addition of neutralizing monoclonal antibodies to adenylate cyclase toxin converted a serum that previously inhibited both attachment and phagocytosis of the wild-type strain to one that increased both attachment and phagocytosis compared to the no-serum control. Monoclonal antibodies that recognize the adenylate cyclase toxin but fail to neutralize activity were without effect. These results suggest that adenylate cyclase toxin inhibits both Fc receptor-mediated attachment and phagocytosis ofB. pertussisby neutrophils.