This program is aimed at understanding disease mechanisms for Bordetella pertussis and Bacillus anthracis. An understanding of the pathogenesis of the organisms and host immunity will lead to improved vaccines for pertussis and anthrax. Studies of the structure and functions of pertussis toxin. Research was conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure and mechanism of action of the toxin. We have focused much or 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. We have previously shown that, while the holotoxin is secreted from B. pertussis, the individual S1 subunit and B oligomer of PT are not secreted. Moreover, we localized the S1 subunit that remained within the cell to the outer membrane of the bacterium suggesting that the outer membrane may serve as the site of assembly of the toxin. In order to gain further insight into the biogenesis of the toxin and its secretion, we examined subassemblies of the toxin composed of either S1-S2, S1-S4, S2-S4, or S1-S2-S4. We were able to determine that certain of these subassemblies can exist at least transiently within the bacterium. We are currently determining whether any of the subassemblies composed of the S1 subunit and a portion of the B oligomer can be secreted from the bacterium. Such information would aid in our understanding of toxin biogenesis and might shed light on nature of the interactions that occur between the toxin and its transporter. We have also examined the relative levels of production of the PT subunits and the Ptl proteins by fusing a phoA gene to the ATG start codon of various ptx and ptl genes. The ptx and ptl genes are known to be part of the same operon. We found that the transcription/translation of genes decreased as the distance from the promoter increased. These results indicate that the Ptl proteins may be synthesised at lower levels than the PT subunits. This finding led to the hypothesis that the Ptl proteins may be rate limiting in secretion and, in fact, this appears to be the case since overexpression of certain Ptl proteins results in higher levels of secretion of the toxin. Studies on the Pathogenesis of B. anthracis and Protective Immunity. Anthrax is a deadly disease caused by the Gram-positive bacterium Bacillus anthracis. The organism infects humans and many other animals. The primary virulence factors are thought to be anthrax toxin and a glutamic acid capsule, both of which are encoded on large plasmids, although other important virulence factors will likely be discovered. The dissemination of anthrax spores into the air, which results in inhalation anthrax, is considered to be the most likely method by which this organism would be used as a bioterrorist agent. Inhalation anthrax, the most severe form of the disease, results in a systemic infection in which the organism spreads to the lymph nodes and then into the blood where it is able to replicate to very high levels. While the release of anthrax toxin from the bacteria is thought to play a major role in the morbidity and mortality associated with the disease, other bacterial factors likely contribute to the ability of the bacteria to escape from alveolar macrophages and to grow to extremely high levels in the bloodstream. Since bacterial surface proteins often are critical for the survival of bacterial pathogens within the harsh environment of the host, we are examining the role of certain surface proteins in the pathogenesis of B. anthracis. Specifically, we are focusing on proteins that are tethered to the outer surface of the bacterium by the enzyme sortase. Sortases are enzymes that are ubiquitous among gram-positive bacteria. Elimination of sortase activity results in the inability of the sortase substrates to be properly anchored to the bacterial cell surface. Since the substrates for sortases can be critical virulence factors, disruption of sortase activity often results in attenuation of virulence. In order to determine whether the sortase substrates of B. anthracis play a role in virulence, we are creating mutations in each of the three putative sortases of B. anthracis. We will then examine the virulence of the resulting mutants in in vitro and in vivo models of infection and disease. This project incorporates FY2002 projects 1Z01BJ003001-18 and 1Z01BJ003014-03.
