Vanderbilt Summer Science Academy

Streamlined structural pipeline for Clostridium difficile proteins differentially expressed in the absence of TcdA/TcdB toxins

Clostridium difficile infections in the colon cause severe gastritis and diarrhea, with pathology stemming from two secreted toxic proteins, TcdA and TcdB. With C. difficile's already high transmission and infection rates on the rise in the United States, hospitals are desperate for more effective treatments. Preliminary data show that C. difficile TcdA/TcdB knockout strains still produce proteins that cause toxicity in gut epithelial cells. Finding the structure of these proteins, in particular those with no known function or homologs, would allow for a deeper understanding of significant components that may play roles in C. difficile infections. Amino acid sequences of selected knockout-strain proteins were analyzed through bioinformatics tools to identify promising crystallization targets. Recombinant expression of these proteins was tested in E. coli.; those with good expression and solubility underwent large-scale purification. Once high-purity protein samples are obtained, broad crystallization screens will be conducted to test optimal crystallization conditions for subsequent x-ray crystallography analysis. By conducting large proteomic screens and purification, C. difficile targets of interest can quickly be chosen for crystallization later on. This pipeline method will help define structures of unknown proteins and characterize their roles within the infection pathways of C. difficile and other harmful bacteria, possibly leading to future drug targets.