Vanderbilt Summer Science Academy

Summer Research Description: Clostridium difficile is an opportunistic bacterium that causes pseudomembranous colitis and nosocomial diarrhea in infected hosts. A common nosocomial infection, C. difficile presents an enormous burden on hospitals – where there is a dense immunocompromised population – with 450,000 infections and 29,300 related deaths in 2011. C. difficile secretes an exotoxin, Toxin B (TcdB), which contains a glucosyltransferase domain (GTD) that inactivates host GTPases in endothelial cells, leading to the disruption of downstream signaling pathways and cytoskeleton regulation, causing cell death. However, their exact interactions are unknown due to the low binding affinity between the two proteins. We plan to incorporate photoactive unnatural amino acids (p-azido-L-phenylalanine and p-benzoyl-L-phenylalanine) into various positions on the GTPase Rac1 using amber codon suppression. Exposing the modified proteins to UV light will crosslink Rac1 to GTD. If successful, a future aim of this project will be to analyze the complex using x-ray crystallography and mass spectrometry. With a crystal structure, we hope to identify the binding site between GTD and Rac1, increasing our understanding of the TcdB mechanism. Additionally, this crosslinking method could prove a powerful tool to investigate other parts of the TcdB mechanism, allowing researchers to probe the exact host protein interactions throughout the toxin’s infection timeline.