Clostridium difficile flagellin stimulates toll-like receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5

• Published in: Life sciences (1973) Vol. 92; no. 3; pp. 211 - 217
• Main Authors: Yoshino, Yusuke, Kitazawa, Takatoshi, Ikeda, Mahoko, Tatsuno, Keita, Yanagimoto, Shintaro, Okugawa, Shu, Yotsuyanagi, Hiroshi, Ota, Yasuo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 27.02.2013
• Subjects: Bacterial Proteins - pharmacology; Bacterial Toxins - pharmacology; Caco-2 Cells; chemokine CCL20; Chemokine CCL20 - biosynthesis; Chemokine CCL20 - genetics; Chemokine CCL20 - immunology; Clostridium difficile; culture media; enzyme-linked immunosorbent assay; epithelial cells; Epithelial Cells - immunology; Epithelial Cells - metabolism; Flagellin; Flagellin - pharmacology; HEK293 Cells; human cell lines; Humans; interleukin-8; Interleukin-8 - biosynthesis; Interleukin-8 - genetics; Interleukin-8 - immunology; intestinal mucosa; Intestinal Mucosa - immunology; Intestinal Mucosa - metabolism; mitogen-activated protein kinase; NF-kappa B - genetics; NF-kappa B - immunology; NF-kappa B - metabolism; p38 Mitogen-Activated Protein Kinases - genetics; p38 Mitogen-Activated Protein Kinases - immunology; p38 Mitogen-Activated Protein Kinases - metabolism; pathogenesis; pathogens; phosphorylation; Toll-like receptor 5; Toll-Like Receptor 5 - genetics; Toll-Like Receptor 5 - immunology; Toll-Like Receptor 5 - metabolism; Toxin B; toxins; transcription factor NF-kappa B; Transfection; ultracentrifugation; virulence; Western blotting; Flagellin; Toll-like receptor 5; Toxin B; Clostridium difficile
• ISSN: 0024-3205; 1879-0631
• DOI: 10.1016/j.lfs.2012.11.017

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. 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. 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. 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.