The structure and function of bacterial protein toxins is studied to determine how toxins contribute to bacterial pathogenesis. Mammalian cells are studied to identify the subcellular targets of the toxins. A. A collaborative project to determine the structure of anthrax toxin protective antigen (PA) by X-ray diffraction is nearing completion. Mutant PA proteins containing single cysteine residues yielded heavy metal derivatives that helped to solve the structure. Nearly all the amino acids have been located in the three-dimensional structure. The structure is being used as a guide for mutagenesis. B. Biochemical and genetic methods are used to identify the animal cell systems involved in toxin action. Mutations are induced in cultured cells to confer resistance to bacterial toxins and fusion proteins. Characterization of these mutants helps to identify toxin receptors, activating proteases, endocytic uptake mechanisms, intracellular trafficking, and cytosolic catalytic activities. C. Site-specific mutagenesis is used to identify functionally important amino acids in toxin proteins and in cytotoxic fusion proteins. Proteins are expressed in Bacillus sp., secreted to the culture medium, and purified by chromatography. Functional assays are performed in cultured cell lines. Non-toxic mutant proteins are evaluated for possible use in vaccines. D. Toxin fusion proteins are used as a general system for delivering heterologous polypeptides into the cytosol of animal cells. The fusion proteins have residues 1-254 of anthrax toxin lethal factor attached to catalytic domains from several different toxins. When combined with PA, these fusion proteins are highly toxic to mammalian cells because they are efficiently translocated to the cytosol. Fusions of polypeptides from transcriptional activators, regulatory substances, etc. are designed that will have a therapeutic action when delivered to cytosol of animal cells.
