Research was conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure and mechanism of action of the toxin. We completed a study to address the question of whether vaccination can provide selective pressure for antigenic variation of PT and, if so, whether antigenic drift might result in decreased efficacy of acellular pertussis vaccines. We compared the sequence of the PT (ptx) genes of Japanese isolates of Bordetella pertussis obtained both before and after wide-spread use of acellular pertussis vaccines. The ptx regions from Tohama I (the vaccine strain), two clinical isolates obtained in 1979, and three clinical isolates obtained in the 1990's were sequenced. We found that the ptx regions of the clinical isolates were identical indicating that antigenic drift had not occurred, at least in the strains analyzed. In addition, we examined whether antigenic variation of PT could affect the ability of vaccine to protect against B. pertussis. In order to do this, we cloned the ptx genes from B. bronchiseptica, and exchanged these genes for those found in B. pertussis. We then examined the ability of sera from mice immunized with PT originating from B. pertussis to neutralize the bronchiseptica toxin. We found that the bronchiseptica toxin was neutralized as efficiently as pertussis toxin, indicating that the multiple amino acid differences between bronchiseptica toxin and pertussis toxin, most of which occur on the exposed surface of the protein, did not affect the antigenicity of the protein. These results suggest if evolutionary drift of pertussis toxin does occur, it likely occurs very slowly and many of the alterations that would be allowed, since they would not affect biological function of the protein, do not significantly affect the antigenicity of the protein. We are continuing our work on the analysis of the secretion of PT from B. pertussis. Previously, we identified nine ptl genes that are essential for the secretion of the toxin. These genes include proteins that belong to the Type IV family of transporters. In order to gain further insight into the mechanism of secretion, we studied the ability of individual components of the toxin to be secreted. In order to do this, we constructed mutants of B. pertussis that expressed only the S1 subunit of the toxin or only the B oligomer of the toxin. We found that the B. oligomer was not secreted in the absence of the S1 subunit. Likewise, in the absence of the B oligomer, the S1 subunit was not secreted by a Ptl-dependent mechanism. These data indicate that it is the holotoxin form of the protein that is released from the organism by the Ptl transport system. In order to further understand the biogenesis of PT, we initiated studies to localize the subunits of the toxin prior to secretion. We first localized the S1 subunit of the toxin in strains that produce only S1, S1+B oligomer, and S1 + B oligomer + Ptl proteins (wild-type). We found that the S1 subunit localized to the outer membrane of the bacterium. Thus, the outer membrane may serve as the site of assembly of the toxin. After the toxin assembles, the Ptl proteins may act using a piston-like action to push the toxin through the outer membrane and into the extracellular space. Such a model would be consistent with the pilus structures that are often seen with Type IV transporters.
