Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity.
Toxins A and B of Clostridium difficile are large cytotoxic proteins that share several unusual structural features, including four conserved cysteines, a potential nucleotide binding site, a hydrophobic region, and a series of contiguous repeating units at the carboxyl terminus. In the following study, we developed a series of toxin B mutants with altered properties in each of these features and examined the effect of the mutation on cytotoxic activity. Altering conserved cysteines to serine resulted in a 90% reduction in activity, whereas altering a histidine residue located in the potential nucleotide binding site to glutamine resulted in a 99% reduction. Removing the repeating units lowered the activity by 90% whereas removing the repeating units plus a conserved cysteine located just upstream of the units reduced the activity by more than five logs, resulting in an inactive toxin. Deleting the internal hydrophobic region had a similar effect. Our findings demonstrate that these conserved features appear to be important for expression of cytotoxic activity.
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
L A
LA
Barroso
J S
JS
Moncrief
D M
DM
Lyerly
T D
TD
Wilkins
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity*; D016466:CHO Cells; D016360:Clostridioides difficile / Q000737:chemistry* / Q000235:genetics; D006224:Cricetinae; D003545:Cysteine / Q000502:physiology; D004768:Enterotoxins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006639:Histidine / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017384:Sequence Deletion / Q000502:physiology*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D006639:Histidine; D003545:Cysteine