The enzymatic domain of Clostridium difficile toxin A is located within its N-terminal region.
Clostridium difficile, an anaerobic pathogen encountered in human enteric disease, produces two major virulence factors, toxins A and B, which are members of a clostridial family of large cytotoxins. These are glucosyltransferases, which use a UDP-sugar as co-substrate to glucosylate and inactivate small GTPases of the Rho or Ras families, culminating in cytotoxicity. Clinically, toxin A is perhaps the most important family member, because it causes major tissue damage in the course of disease, leading to a potentially lethal, pseudomembranous colitis. The location of the enzymatic domain of toxin A and mechanistic details of its action are not yet known, so we wished to localize this domain using gene deletion constructions from the full-length gene and by monitoring glucosylation activity of encoded protein products. Toxin A deletions were obtained by successively truncating the C-terminal coding region. These were transformed into E. coli, cell lysates were prepared and they were assayed for their ability to glucosylate Rho A protein, using an in vitro enzymatic assay. We report that the UDP-glucose binding site, the catalytic site for glucose transfer and the Rho A interaction site occur within the first 659 N-terminal amino acids of toxin A, i.e., less than 25% of the length of holotoxin A. Localization of the enzymatic domain of toxin A to these 659 N-terminal amino acids should greatly simplify studies on mechanistic details of this clinically important toxin.
Faust
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Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 Fourth Street, Lubbock, Texas, 79430, USA.
Ye
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Song
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KP
D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016360:Clostridioides difficile / Q000737:chemistry / Q000201:enzymology*; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis
D001427:Bacterial Toxins; D004768:Enterotoxins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile