Whooping cough remains a worldwide problem. In developing nations an estimated 60 million cases occur annually, resulting in over half a million deaths. The disease is controlled in the United States by the use of the whole-cell pertussis vaccine, however, a high failure rate was noted in recent epidemics. Both whole cell and acellular-component vaccines give only short term protection from disease, and little protection from infection. Bordetella pertussis, the bacterium that causes whooping cough produces many toxins and virulence factors, the most important of which is pertussis toxin. Pertussis toxin, not growth of the bacteria in the respiratory tract is thought to cause the severe disease manifestations of whooping cough. In the previous granting period we discovered an operon of eight genes (called ptl for Pertussis Toxin Liberation) that promotes the secretion of assembled pertussis toxin across the outer membrane of B. pertussis. In this grant the applicant proposes to elucidate the molecular mechanism of pertussis toxin secretion using a genetic and biochemical approach. The applicant proposes to: 1. Examine the role of disulfide bond formation during pertussis toxin maturation. The application has shown that newly synthesized pertussis toxin subunits first appear covalently bound to other proteins via intermolecular disulfide bonds, suggesting that the subunits must be extracted from these complexes before assembly and secretion can occur. 2. Polyclonal antibodies, prepared from fusion proteins will be used to determine the cellular location of the Ptl proteins using immunofluorescent microscopy. 3. Mutants deficient in each Ptl protein will be generated to determine the phenotype. 4. Protein-protein interactions between the Ptl proteins and pertussis toxin will be characterized. This work has important implications in development of novel therapeutic strategies. Conventional antimicrobial therapy requires that the agent have a selective toxicity to the microorganism and not the human hosts, for example the bacterial cell wall has no human counterpart and is the target of penicillins and beta-lactam antibiotics. The applicant believes that observations could lead to development of a new class of antimicrobial agents that will be targeted against a subset of bacteria, in this case, Gram-negative bacteria which secrete toxic factors. The applicant has already shown that B. pertussis strains with mutations in the ptl pathway are reduced in virulence. It seems likely that a therapeutic, including a vaccine, directed against this pathway would also result in less severe disease.
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