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). The toxin proteins play a key role in the virulence of Bacillus anthracis. New results from studies of these proteins and their role in anthrax pathogenesis include the following. (1) The ability to measure changes in gene expression in Bacillus anthracis was enhanced by showing that RNA transcripts can be detected on DNA microarrays using a unique monoclonal antibody that recognizes DNA/RNA hybrids. This antibody detection scheme was shown to be especially useful in analysis of small regulatory RNAs. (2) Several important bacterial protein toxins act by modifying the unique diphthamide residue present on the protein synthesis elongation factor-2. The biosynthesis and normal role of diphthamide are not fully understood. A gene involved in diphthamide biosynthesis, dph3, was knocked out in mice to understand the gene?s function, and it was found that embryos lacking dph3 do not develop normally and die prior to birth. This suggests that diphthamide does have a normal role beyond its use as a target of bacterial toxins, but further work will be needed to find the exact role. (3) A number of advances were made in understanding how anthrax toxin binds and enters into cells. Working with researchers at Stanford University, it was shown that the cell surface protein LRP6 interacts with the previously identified toxin receptors, TEM8 and CMG2,to cause toxin uptake into cells. LRP6 therefore can be considered a co-receptor, one that offers a new target for therapeutics that might block toxin action. In separate studies with researchers in Switzerland, it was shown that the receptors are modified by attachment of a fatty acid, palmitic acid, and that this modification helps retain receptors on the surface where they can bind PA. In a third study of toxin internalization, it was shown that a fusion protein containing the N-terminal portion of LF and the enzyme beta-lactamase provides a very sensitive fluorescence-based assay for toxin internalization. This assay is suitable for high-throughput screening to identify drugs that block toxin uptake and action. (4) Collaborative work continued with several groups developing 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 LID, DIR, NIAID in which chimpanzee monoclonal antibodies to PA and LF were developed and tested. In another example, we worked with researchers in NICHD to show that a cyclodextran-based drug protected against anthrax toxin action in rats. (5) Knowledge of bacterial toxin structure and function was used to design cytotoxins specific for cancer cells. We extended our prior studies on treating mouse tumors to show that the anthrax toxin fusion proteins would work not only when injected adjacent to the tumor, but also when administered systemically, as would be preferred if these drugs were to be developed for human use.
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