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.
Barroso
L A
LA
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Moncrief
J S
JS
Lyerly
D M
DM
Wilkins
T D
TD
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