title: Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.
abstract: TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Lianfa
authors.initials: L
authors.lastname: Shi
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Yongrong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Gregorio
authors.initials: G
authors.lastname: Perez-Cordon
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Tuxiong
authors.initials: T
authors.lastname: Huang
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Ramsey
authors.affiliation: Texas Children's Microbiome Center, Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Numan
authors.initials: N
authors.lastname: Oezguen
authors.affiliation: Texas Children's Microbiome Center, Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Tor C
authors.initials: TC
authors.lastname: Savidge
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA hfeng@umaryland.edu.
authors.forename: Hanping
authors.initials: H
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000201:enzymology / Q000472:pathogenicity*; D057056:Cysteine Proteases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000276:immunology; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology*; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D051379:Mice; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000276:immunology*; D014709:Vero Cells; D037521:Virulence Factors / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015853:Cysteine Proteinase Inhibitors; D007143:Immunoglobulin Heavy Chains; D061905:Single-Domain Antibodies; D037521:Virulence Factors; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D057056:Cysteine Proteases