| dc.description.abstract | Clostridium difficile is a gram-positive, anaerobic, spore-forming bacteria that causes severe diarrhea, abdominal pain, and pseudomembranous colitis (1). Clostridium difficile infection (CDI) can be fatal. The estimated annual cost for CDI management is around $6.1 billion in the U.S.. When treating CDI using broad-spectrum antibiotics, the rate for CDI recurrence increases significantly. Thus, developing new treatments for CDI is essential. TcdB is the major virulence factor secreted by Clostridium difficile, and it is responsible for most of the symptoms of CDI. It is critical to understand its mechanism of entry into intestine epithelial cells and subepithelial layers to devise methods to block as many of the uptake steps as possible. In this dissertation, I have studied the interaction of TcdB with its two receptors, frizzled-2 (FZD2) and chondroitin sulfate proteoglycan 4 (CSPG4), using cryo-EM and biochemical assays. TcdB utilizes hydrophobic interactions to bind FZD2 and CSPG4, and CSPG4 also has electric charge interactions with TcdB. Three snapshots of TcdB binding to CSPG4 were obtained, demonstrating that TcdB binds in an equilibrium among different states. I also analyzed TcdB binding at acidic pH and found that there is a large conformational change in the overall structure, primarily in the hydrophobic region of the delivery domain. Both of the receptors dissociated with TcdB when pH decreased, with CSPG4 dissociation at a larger rate, allowing TcdB to float near the endosome membrane for the following pore formation and translocation. By revealing detailed receptor binding mechanism as well as intermediate states of TcdB when the decrease of the pH triggers the conformational changes, this work extensively expand a structural view of TcdB uptake mechanism and provides strong reference to resolve homolog toxin invasion mechanism. To develop a potential treatment for CDI, a library of Designable Ankyrin Repeat Proteins (DARPins) was generated to screen for TcdB neutralizer. A dimeric DARPin, DLD-4, that is composed of two monomeric DARPins, U3 and 1.4E, is selected because it has the best neutralization ability as well as high binding affinity to TcdB. My cryo-EM structure of TcdB-DLD4 complex demonstrates that U3 interacts at the FZD2 receptor binding surface and 1.4E grips on the CSPG4 interaction region. Thus, DLD-4 is able to neutralize the TcdB by competing against its receptors with much higher binding affinity and block TcdB entry at the first step. | en |
