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 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). New results from studies of these proteins include the following. (1) Studies of gene expression in the Bacillus anthracis were extended by developing a new method for inactivating genes and for making large genomic deletions. In a demonstration of the utility of the method, a region of the chromosome containing 30 genes was deleted, and the viability of the resulting strain showed that none of the genes are essential. (2) In continuation of studies on the susceptibility of mice to anthrax lethal toxin (PA+LF), we showed that adrenalectomy greatly sensitized mice. Surprisingly, feeding dexamethasone to partially restore adrenal function also greatly sensitized mice. These findings may have relevance to the types of supportive care that are appropriate for anthrax patients, although these types of studies need to be performed in actual infection models. (3) We also continued to collaborate in studies of vaccines and therapeutics for anthrax. We worked with groups developing inhibitors of the anthrax lethal factor protease activity, human and humanized antibodies that neutralize anthrax toxin, and new methods for producing and delivering candidate vaccine antigens derived from the toxin and the poly-glutamic acid capsule. In one example, we supported work in the NIH Clinical Center in which a human monoclonal antibody was shown to protect rats from slowly infused lethal toxin even when administered after toxin exposure was initiated. In another example, we worked with investigators in Florida who demonstrated high level production of PA in plant chloroplasts. (4) Knowledge of bacterial toxin structure and function was used to design cytotoxins specific for cancer cells. We reported a novel method for achieving higher tumor cell specificity by exploiting the oligomeric nature of the activated PA protein so as to restrict cytotoxic action to cells expressing two separate tumor-related proteases. In addition, with experienced cancer clinicians, we worked to develop diphtheria toxin fusion proteins that require activation by the tumor-enriched plasminogen activator protease.
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