Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis via lipid rafts and pass through several endocytic vesicle populations, finally allowing LF and EF escape to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Collaborative studies have examined the interaction of LF with peptide substrates and clarified structural details of the catalytic mechanism. Inhibitors of furin and of the LF protease activity were shown to be effective in protecting cells and, in some cases, animals from toxin action. To understand how the toxin contributes to pathogenesis in animals, we extended to rats the prior studies in mice, and showed that LF causes a circulatory shock response, but one that clearly differs from that induced by gram-negative bacteria and endotoxin. Comparisons among inbred mouse strains showed that the sensitivity of mouse macrophages from certain strains to LF cannot explain the large differences in susceptibility of mice to the toxin. In studies of Bacillus anthracis, we compared regulation of gene expression in the B. anthracis to that in Bacillus cereus to explain why these bacteria differ in hemolytic phenotype. The PlcR regulon in B. anthracis was shown to lack several components present in B. cereus. A novel fusion of the PlcR and PapR proteins was shown to constitute an active transcriptional regulator able to bind to the specific PlcR DNA target sequence. Collaborative efforts related to anthrax vaccine development included studies that mapped an epitope in the receptor binding region of PA that is recognized by a neutralizing monoclonal antibody. Antibodies of higher affinity to this epitope were obtained by phage display of randomly mutated antibody libraries. A conjugate of polyglutamate to PA was shown to induce antibodies to both PA and to the bacterial capsular material, so that both anti-toxin and anti-bacterial antibodies were provided by a single immunogen. Knowledge of anthrax toxin structure and function was used to design cytotoxins specific for cancer cells. Specifically, we created diphtheria-toxin fusion proteins dependent on cell-surface plasminogen activator.
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