The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model

Abstract

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B

“…The second model is the murine cecum injection model as described previously [35]. A minor survival advantage (not statistically significant) was observed for mice receiving TcdB and DLD-4 compared with TcdB alone (Fig 6B).…”

“…Mouse survival was monitored for 4 days until the termination of the experiments, and data were analyzed by Kaplan–Meier survival analysis with log-rank test of significance. The cecum injection experiment was carried out as described previously by Zhang and colleagues [35]. Mice were anesthetized with intramuscular injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg).…”

Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection

“…We analyzed the toxicity of these TcdB1 mutants in comparison with the WT toxin by directly inject them into the mouse cecum 24 , 37 . This method has the advantage of controlling precisely the amount of toxins and incubation time, in order to capture any differences among these toxins.…”

Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells

“…To further examine the relevance of sGAG-TcdA interactions for TcdA-induced pathogenesis in vivo, we utilized a mouse cecum injection model previously established to assess pathogenesis of TcdA and TcdB 44 . Briefly, TcdA or TcdA pre-mixed with inhibitors was injected into the cecum.…”