In recent years, several exotoxin systems involved in bacterial pathogenesis--specifically, those exotoxins which translocate across target eukaryotic cell membranes into the cytoplasm, and which modify specific intracellular targets--have been well-characterized. These exotoxins, which are major determinants of pathogenicity in bacterial infections, offer potential systems (i) for understanding the receptor binding, membrane translocation, and enzymatic activation mechanisms shared by bacterial exotoxins from diverse sources; (ii) for developing immunizations against specific toxins; (iii) for constructing targeted cytotoxic agents. Despite the potential contribution of protein crystallography to understanding these systems, little three-dimensional structural information is available for members of this class of toxins. The recent 3.0 angstroms structure from this laboratory of Pseudomonas aeruginosa exotoxin A, a major determinant of lethality in P. aeruginosa infections, is currently the only available molecular model of a bacterial exotoxin. Structural work on exotoxin A will be extended. To produce a precise molecular model, data will be collected to approximately 2.0-2.2 angstroms resolution and the current model refined. Structural and biochemical experiments will be effected to define (i) the receptor binding domain, (ii) the membrane translocation mechanism, and (iii) the enzymatic mechanism of exotoxin A. In addition, crystallographic work on the three-protein exotoxin system of Bacillus anthracis, the pathogen responsible for anthrax disease, has been initiated. Two proteins of the system--the protective antigen and the lethal factor--have been crystallized. The protective antigen crystals diffract to approximately 3.0 angstroms resolution; an effort to determine the structure of the molecule is underway. Suitability of crystal forms of lethal factor for high resolution x-ray crystallograhic work is being examined. The exotoxin A portion of this proposal is a continuation of work accomplished under """"""""Three-Dimensional Structure of Pseudomonas Toxin""""""""; it is discussed in that context. The current proposal is directed toward mechanisms of exotoxins in general, including but not confined to exotoxin A.
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