title: Clostridium difficile flagellin stimulates toll-like receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5.
abstract: OBJECTIVE
  Clostridium difficile is an important pathogen in nosocomial infections. Although C. difficile toxins are considered to be major virulence factors, pathogenesis of C. difficile associated diseases remains to be determined. In this study, we investigated whether C. difficile flagellin is involved in the pathogenesis of C. difficile-associated diseases.
  
  
  METHODS
  C. difficile flagellin was extracted from bacterial body by using a combination of ultracentrifugation and low speed centrifugation. Extracted C. difficile flagellin was added to HEK293T cells transiently transfected with pUNO-mcs (empty vector) or pUNO-hTLR5, and NF-kappaB activation was compared by a dual-luciferase assay. The amount of C. difficile flagellin-induced inflammatory mediators such as interleukin-8 and CCL20 was measured by ELISA assay in the culture media of intestinal epithelial cell lines, HT29 cells and Caco-2 cells. Flagellin induced phosphorylation of p38 mitogen-activated protein kinase was examined by Western blotting analysis in Caco-2 cells. The amount of C. difficile flagellin-induced inflammatory mediators in the presence, or absence of C. difficile toxin B was also measured by ELISA assay.
  
  
  RESULTS
  C. difficile flagellin induced activation of NF-kappaB in HEK293T cells via toll-like receptor 5. C. difficile flagellin also induced activation of p38 mitogen-activated protein kinase, and promoted the production of interleukin-8 and CCL20 in intestinal epithelial cells via toll-like receptor 5. Pretreatment with toxin B enhanced flagellin-induced cytokine productions.
  
  
  CONCLUSIONS
  Our results indicate that toxin B promotes flagellin-induced activation of intestinal epithelial cells, and that C. difficile flagellin may play a role in the occurrence of C. difficile-associated diseases.
authors.affiliation: Department of Infectious Diseases, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Tokyo 173-8606, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Yoshino
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Kitazawa
authors.forename: Mahoko
authors.initials: M
authors.lastname: Ikeda
authors.forename: Keita
authors.initials: K
authors.lastname: Tatsuno
authors.forename: Shintaro
authors.initials: S
authors.lastname: Yanagimoto
authors.forename: Shu
authors.initials: S
authors.lastname: Okugawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yotsuyanagi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ota
mesh_terms: D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D018938:Caco-2 Cells; D054418:Chemokine CCL20 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D016360:Clostridioides difficile; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism; D005408:Flagellin / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C108355:CCL20 protein, human; C508606:CXCL8 protein, human; D054418:Chemokine CCL20; D016209:Interleukin-8; D016328:NF-kappa B; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; C031815:toxB protein, Clostridium difficile; D005408:Flagellin; D048051:p38 Mitogen-Activated Protein Kinases