Structural and functional analyses of bacterial protein toxins were performed to discover how toxins contribute to bacterial pathogenesis. Studies focussed on identifying the routes of internalization and mechanisms of intracellular action of anthrax toxin proteins. A. Additional evidence was obtained to support the hypothesis that the obligatory cleavage of anthrax toxin protective antigen (PA) is caused by the eukaryotic protease furin. Thus, the relative potencies of a set of protease inhibitors in blocking cleavage of PA bound on cellular receptors matched their inhibitor action on purified furin. PA mutants altered at the cleavage site were digested by purified furin at rates proportional to their toxicity for cells. B. A fusion protein method was used to show that residues 1-254 of the anthrax toxin lethal factor (LF) are sufficient to cause binding to PA and internalization. C. Initial work toward using components of anthrax toxin as cell-type specific therapeutic agents was accomplished by placing an amino acid sequence recognized by HIV protease at the cleavage site of PA. It is expected that this mutant PA will target the cytotoxic LF fusion proteins specifically to HIV-infected cells. D. Characterization of the mechanism of toxin binding and uptake was advanced by isolation of CHO cell mutants lacking PA receptor. To clone and identify the receptor, a cDNA library was transfected into these mutants, and screening was begun for colonies which have regained receptor. Replicate plating and fluorescent cell sorting methods were developed to detect PA binding to its receptor. E. Collaborative work on the structure of PA was continued with the successful crystallization of several cysteine-substituted PA mutant proteins, and the collection of a complete set of X-ray diffraction data. F. Analysis of the control of anthrax toxin synthesis was advanced substantially by cloning and sequencing a positive regulatory gene needed for expression of all three toxin proteins. This gene does not have apparent sequence homology to known regulatory genes and may be of a novel regulatory type.
