The Binary Toxin CDT and Toll-like Receptor 2 Contribute to the Pathogenesis of Clostridioides difficile Infection

Clostridioides difficile, the causative agent of the most common hospital-acquired gastrointestinal infection, has been labeled an urgent health threat by the CDC. Despite readily available antibiotic therapy, C. difficile is responsible for approximately 500,000 infections and 29,000 deaths in the US each year. Increases in the rates of C. difficile infection (CDI) in recent decades have been linked to the emergence of epidemic, hypervirulent strains. While typical strains of C. difficile express only the primary virulence factors Toxin A and Toxin B, these newer hypervirulent strains express an additional third binary toxin called C. difficile transferase, or CDT. These CDT-expressing strains have been linked to increased disease severity, and while expression of CDT has been shown to enhance virulence during infection, there is still much that is unknown regarding the mechanisms responsible.
Previous work from our lab has demonstrated that CDT is recognized by TLR2 to induce downstream inflammation. To further investigate the impact of TLR2 signaling on the immune response during infection, we infected both wild-type and TLR2-deficient mice with a CDT-expressing strain of C. difficile and compared the immune gene expression between the groups. We found that the presence of TLR2 signaling during CDI had a broad impact on immune gene expression, with several immune pathways upregulated in the wild-type mice as compared to those lacking TLR2. Next, we explored the interaction between CDT and TLR2, and using an NF-κB reporter cell line, determined that CDT was recognized by the TLR2/6 heterodimer to induce a downstream NF-κB response. The binding component (CDTb) alone was sufficient to induce this response, and was also capable of inducing IL-1β transcript expression in bone marrow-derived dendritic cells. To study the contribution of CDTb to pathogenesis during in vivo infection, we developed novel isolates of C. difficile mutated to express the separate components of the binary toxin and tested them using both a mouse and hamster model of CDI. Infection of hamsters with an isolate of C. difficile expressing CDTb without CDTa resulted in more severe disease as compared to those infected with an isolate deficient in both components, indicating that CDTb enhances virulence during infection. Interestingly, CDTb expression in the absence of CDTa did not affect virulence in the mouse model, highlighting the impact that selection of an animal model can have on in vivo infection studies.
Overall, we found that CDTb alone interacts with the TLR2/6 heterodimer to induce downstream inflammation and that CDTb expression enhances virulence during infection. It is vital to understand how virulence factors like CDT can affect and influence the host response during infection, as this knowledge can be used to reveal novel targets for therapeutic treatment.