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Background

Clostridium difficile infection (CDI) is the leading recognized cause of nosocomial infectious diarrhea in the developed world. The importance of CDI amongst patients with inflammatory bowel disease (IBD) is increasingly being recognized. Recent studies on CDI in IBD patients have demonstrated a concerning trend towards increased rates of infection, morbidity, mortality and healthcare costs. Guidelines now promote testing for C. difficile in IBD patients experiencing a relapse of colitis. The major virulent factors of C. difficile are two large protein exotoxins - A and B, which initiate a marked intestinal inflammatory response. In this study we discovered that toxins secreted by Clostridium difficile contain significant amounts of bacterial DNA, which was further confirmed (by sequencing) to be of C. difficile origin. It is known that toll-like receptor 9 (TLR-9) recognizes bacterial DNA containing CpG dinucleotides and triggers an inflammatory response. In this study, we aim to examine the role of toxins-associated DNA in the inflammatory response in CDI.

Methods

HT-29 cells, THP-1 cells, wild type (WT) and TLR9 knock-out (TLR9KO) mouse macrophage lines were stimulated with C. difficile toxins A or its derivatives treated with DNAse I digestion or by heat inactivation. ODN TTAGGG was used to antagonize TLR9 receptor. Toxin cytotoxicity was measured by standardized cell rounding assay. IL-8 or KC(IL-8 homologue) release was measured by ELISA.

Results

Both C. difficile toxin A and B contain DNA from C. difficile as determined by gene sequencing. In vitro pull-down assays demonstrated that both toxin A and B have strong affinity for DNA. HT-29 intestinal epithelial cells, THP-1 monocytes and macrophages each secreted high levels of IL-8 or KC in response to toxin A (10nM) stimulation. IL-8 production was attenuated when cells were treated with the TLR9 antagonist ODN TTAGGG. Heat inactivation of toxin A demolished its cytotoxicity (i.e. no cell rounding in HT-29 cells), but IL-8 production was still induced although at lower levels than by untreated toxins. Digesting toxin A with DNAse I also reduced IL-8 production in all tested cell lines. Furthermore, compared to wild type control, TLR9 knock-out macrophages produced significantly lower amount of KC in response to toxin A.

Conclusion(s)

We have made the novel observation that C. difficile toxins contain genomic DNA that is bound with high affinity. C. difficile toxin A- associated DNA augments toxin-induced inflammation by activating TLR9 signaling pathways. Antagonizing or knocking out the TLR9 receptor results in reduced toxin A induced inflammatory cytokine production. Our data suggest that C. difficile DNA bound to exotoxins A and/or B and signaling via TLR 9 may be a significant contributing virulence factor in C. difficile infection.

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Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.
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