1. A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp.
- Author
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McNichol BA, Rasmussen SB, Meysick KC, and O'Brien AD
- Subjects
- Animals, Bacterial Toxins genetics, Bacterial Toxins metabolism, Bordetella enzymology, Catalytic Domain genetics, Escherichia coli enzymology, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Humans, Lysine chemistry, Lysine genetics, Mice, Mutagenesis, Site-Directed, Phenotype, Protein Structure, Tertiary, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Recombinant Fusion Proteins toxicity, Swiss 3T3 Cells, Threonine chemistry, Threonine genetics, Transglutaminases genetics, Transglutaminases metabolism, Virulence Factors, Bordetella genetics, Virulence Factors, Bordetella metabolism, rhoA GTP-Binding Protein chemistry, rhoA GTP-Binding Protein drug effects, Amino Acid Substitution, Bacterial Toxins toxicity, Cell Nucleus drug effects, Escherichia coli Proteins toxicity, Transglutaminases toxicity, Virulence Factors, Bordetella toxicity
- Abstract
Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e. deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1 and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared with the parent toxins. Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity.
- Published
- 2006
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