title: 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.
abstract: 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.
authors.affiliation: Department of Protein Therapeutics, Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA. stephen.demarest@biogenidec.com
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
authors.forename: Jared
authors.initials: J
authors.lastname: Salbato
authors.forename: Marikka
authors.initials: M
authors.lastname: Elia
authors.forename: Jingping
authors.initials: J
authors.lastname: Zhong
authors.forename: Theresa
authors.initials: T
authors.lastname: Morrow
authors.forename: Trevin
authors.initials: T
authors.lastname: Holland
authors.forename: Katie
authors.initials: K
authors.lastname: Kline
authors.forename: Gary
authors.initials: G
authors.lastname: Woodnutt
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Kimmel
authors.forename: Geneviève
authors.initials: G
authors.lastname: Hansen
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D002118:Calcium