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Characterization of a Mutant Diphtheria Toxin that is Defective in Binding to Cell Membrane Receptors on Vero Cells

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Technical Report

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Uniformed Services University Of The Health Sciences Bethesda United States

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Mutants of corynebacteriophage Beta were isolated that coded for the production of mutant forms of diphtheria toxin cross-reacting materials, CRMs that were reduced in cytotoxicity due to defects in the fragment B. A sensitive cytotoxicity assay was used to screen the supernatants of cultures of Corynebacterium diphtheriae that were infected with the mutagenized Beta phages. Several nontoxinogenic or hypotoxinogenic mutant phages were isolated by this procedure, plaque-purified, and used to prepare lysogenic derivatives of C. diphtheriae C7-. A preliminary characterization of these lysogenic strains identified a new class of CRMS which were antigenically and enzymatically Indistinguishable from wild-type toxin but were considerably less cytotoxic for Vero cells. Of these mutanttoxins, CRM9 was the most reduced in cytotoxicity and was selected for purification and further characterization. Purified CRM9 was approximately 1,000-fold less toxic than diphtheria toxin in Vero cell cytotoxicity tests. The inhibition of protein synthesis in Vero cells by CRM9 and wild-type toxin proceeded at the same rate when equally toxic doses of each protein were compared. CRM9 and diphtheria toxin had identical electrophoretic mobilities in SDS-polyacrylamide gels, were antigenically indistinguishable in competitive-binding radioimmunoassays and in immunodiffusion tests, and had comparable NADEF2 ADPR-transferase activities after activation by trypsin treatment. 125 I-labeled CRM9 did not bind specifically to membrane receptors on Vero cells under conditions where specific binding of 125 I-labeled diphtheria toxin could be demonstrated. Competition between CRM9 and diphtheria toxin for cell membrane receptors could .not be demonstrated directly in Vero cell cytotoxicity assays however, CRM197 afforded complete protection to Vero cells from intoxication by both CRM9 and diphtheria toxin.

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