title: Identification of an Essential Region for Translocation of Clostridium difficile Toxin B.
abstract: Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and translocation domain. Few studies have investigated the translocation domain and its mechanism of action. Recently, it was demonstrated that a segment of 97 amino acids (AA 1756-1852, designated D97) within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB. However, the mechanism by which D97 regulates the action of TcdB in host cells and the important amino acids within this region are unknown. In this study, we discovered that a smaller fragment, amino acids 1756-1780, located in the N-terminus of the D97 fragment, is essential for translocation of the effector glucosyltransferase domain into the host cytosol. A sequence of 25AA within D97 is predicted to form an alpha helical structure and is the critical part of D97. The deletion mutant TcdB∆1756-1780 showed similar glucosyltransferase and cysteine protease activity, cellular binding, and pore formation to wild type TcdB, but it failed to induce the glucosylation of Rho GTPase Rac1 of host cells. Moreover, we found that TcdB∆1756-1780 was rapidly degraded in the endosome of target cells, and therefore its intact glucosyltransferase domain was unable to translocate efficiently into host cytosol. Our finding provides an insight into the molecular mechanisms of action of TcdB in the intoxication of host cells.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. shuyichan@foxmail.com.
authors.forename: Shuyi
authors.initials: S
authors.lastname: Chen
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. yingzi224926@163.com.
authors.forename: Haiying
authors.initials: H
authors.lastname: Wang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. guhuawei1990@163.com.
authors.forename: Huawei
authors.initials: H
authors.lastname: Gu
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. chunlis@163.com.
authors.forename: Chunli
authors.initials: C
authors.lastname: Sun
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. lishan@scut.edu.cn.
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, USA. hfeng@umaryland.edu.
authors.forename: Hanping
authors.initials: H
authors.lastname: Feng
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. jufwang@scut.edu.cn.
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D004705:Endocytosis; D004761:Enterocolitis, Pseudomembranous / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D008807:Mice, Inbred BALB C; D009154:Mutation; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021381:Protein Transport; D059748:Proteolysis; D014709:Vero Cells; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D037521:Virulence Factors; C031815:toxB protein, Clostridium difficile
keywords: Clostridium difficile; conformational change; endocytosis; toxin; translocation