The Mechanism Of Intestinal Toxicity Of Clostridium Difficile Toxin

Clostridium difficile is a gram-positive,spore-forming anaerobic bacillus.It is widly found in nature and feces of humans and animals.C.diff is normal flora in humans.C.diff infection is caused by C.diff accumulation in the intestinal tract of humans and animals due to abuse antibiotics,destrorying the the balance of the intestinal flora.In recent years,the incidence of C.diffe-associated diarrhea and intestinal inflammatory disease has increased significantly in the world,causing lengthy hospitalization,substantial morbidity and mortality.In further CDI is the emergence of hypervirulent strains that are resistant to antibiotics.Previous studies showed that CDI was a toxin-mediated disease,the cause of the disease is mainly due to C.diff secretion two exotoxins(TcdA and tcdB).TcdA and Tcd B are multi-domain proteins,it was found that Clostridium difficile toxin function domain include: glucosyltransferase domain(GTD),cysteine proteinase domain(CPD),transmembrane domain(TMD)and receptor binding domain(RBD).And TcdB is generally more potent(~1000 fold)than Tcd A.Experiments in animal models have clearly demonstrated that TcdA is an enterotoxin.TcdB was initially reported to exhibit no enterotoxic activity in animal models.However,the pathological mechanism of C.diffe toxin TcdA and TcdB in CDI is still not very clear.This paper focuses on studying the relationship between the pathological mechanism and structure function.For the study of Clostridium difficile toxin study,we focus on: 1.research on C.diff mutant toxin intestinal;2.the pathogenesis of C.diff toxin research.In the first part,we mutated two mutant toxins based on C.diff toxin B: injection the TcdB-D97 and TcdB-L543A(LA)to mouse ileal loop,and then carried on the quantitative analysis on intestinal fluid and detected the inflammatory factor secretion,neutrophils and intestinal epithelial tissue damage in the tissue,experimental results showed that: the two mutant toxins have intestinal toxicity.At the same time,we carried out the autocleavage of two toxins,glucosyltransferase activity of toxins,as well as the detection of GTD in the cytosolic faction or cells cytoplasm.We found that the GTD of LA and TcdB-D97 could not normally enter the cytoplasm.The results showed that the infection of C.diff may be due to the C.diff toxin GTD which could not enter into the cytoplasm,so that caused inflammatory reaction.In the second part,we introduced a kind of Rac1 glucosylation inhibitor TUDCA,which was mixed with TcdB to induce severe intestinal fluid and inflammatory responses in mice.The results showed that C.diff toxin could enter the cytoplasm,causing the phosphorylation of Rac1 on the surface of the cell membrane.Animal experiments with mutant toxin aTcdB-D97 demonstrated that Rac1 phosphorylation on the surface of the cell membrane was one of the necessary conditions for the C.diff infection.At the same time,we also found that the Tcd B transmembrane domain 1756-1780 these 25 amino acids and TUDCA can be combined with each other,preventing the TcdB’s GTD from entering into the cytoplasm.The Rac1 inhibitor experiment also proved that Rac1 phosphorylation is also a necessary condition for the induction of enteritis in C.diff toxin.High mobility group protein Box 1(HMGB1)is a member of the HMGB family.HMGB1 is a non histone chromosomal protein,and is mainly in the nucleus.HMGB1 is as an important inflammatory factor,mainly by macrophages,mononuclear cells and the secretion of pituitary cells.In the extracellular HMGB1 cell surface receptor binding and the activation of inflammatory reaction and immune response through signal transduction pathway mediated by mouse ileum.This paper focuses on the study the role of HMGB1 in TcdA-induced acute enteritis in rats based on ileal loop model.We used HMGB1 inhibitors glycyrrhizine which can counteract with HMGB1 and inhibit the activity of HMGB1.The results found that the mice intestinal swelling symptoms improved significantly,Tcd A-induced acute enteritis was also significantly improved.We can get the following conclusions: HMGB1 is involved in the inflammatory factor Clostridium botulinum toxin induced acute inflammatory bowel disease response.To sum up,a variety of molecular biology,pathological methods and animal experiments to study the structure function of Tcd B,and to explore the role of Tcd B in the pathogenesis of disease.In our research,we found that: 1.mix with TcdB and TUDCA have intestinal toxicity in the ileal loop in mice;2.Intestinal toxicity depend on TcdB’s GTD enter into the cytoplasmic or not;3.Cell membrane surface of Rac1 phosphorylation is necessary conditions for C.diff toxin induced intestinal inflammation;4.GTD is not intestinal toxicity determinants;5.TcdB transmembrane region(1756-1780)25 amino acids can be combined with TUDCA and prevent GTD from entering the cytoplasm.In this study,the pathogenic mechanism of TcdB was studied through the study of the relationship between the structure and the intestinal toxicity of TcdB.These results changed the people’s some traditional understanding about TcdB,to fill the new knowledge in the field of C.diff,in order to further understand the principle of C.diff infection and treatment of the disease model.