Structural characterization of the cell wall binding domains of Clostridium difficile toxins A and B; evidence that Ca2+ plays a role in toxin A cell surface association.
Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.
Demarest
Stephen J
SJ
Department of Protein Therapeutics, Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA. stephen.demarest@biogenidec.com
Salbato
Jared
J
Elia
Marikka
M
Zhong
Jingping
J
Morrow
Theresa
T
Holland
Trevin
T
Kline
Katie
K
Woodnutt
Gary
G
Kimmel
Bruce E
BE
Hansen
Geneviève
G
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D003001:Cloning, Molecular; D016360:Clostridioides difficile; D006224:Cricetinae; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D002118:Calcium