Krish Patel (VI), Keira Troy (VI), Mia Cuiffo (VI), Ting Ting Luo (V), Hannah Diao (V), Aanvi Trivedi (V), Leah Holmes (IV), Riya Prabhu (IV)
Project Summary
The pathogen Shigella is a leading cause of bacterial foodborne illness worldwide. The virulence cascade of shigellosis requires a transcription factor in the AraC superfamily, VirF. Other members of this superfamily, such as ToxT, which is involved in the virulence cascade of Vibrio cholerae, shows inhibition of DNA binding via attaching a small fatty acid to a hydrophobic pocket. We wish to determine if VirF can be inhibited in a similar manner, and therefore we are working to express, purify, and solve the crystal structure of VirF. We also wish to use biochemical property testing to assess for homologous function to ToxT. Such will help reveal the mechanism of transcriptional regulation in S. dysenteriae and could lead to development of novel treatments for Shigella related illnesses.
Project Goals
- Successfully lyse cells and maintain as much soluble protein as possible
- Purify a high concentration (1 mg/mL) of VirF protein in high quantities (200mmL)
- Locate a lab which we could send purified protein to for crystallization
- Run several biochemical property assays, including:
- Microscale Thermophoresis Assays (MST) for Homodimerization
- ESMAs for fatty acid binding
- Once obtaining the 3D crystallized structure, use programs like PyMol and AlphaFold to compare structures of VirF to ToxT or HilD
Works Cited
Lowden, M. J., Skorupski, K., Pellegrini, M., Chiorazzo, M. G., Taylor, R. K., & Kull, F. J. (n.d.). Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proceedings of the National Academy of Sciences of the United States of America. https://doi.org/10.1073/pnas.0915021107
Di Martino, M. L., Falconi, M., Micheli, G., Colonna, B., & Prosseda, G. (2016). The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle. Frontiers in molecular biosciences, 3, 61. https://doi.org/10.3389/fmolb.2016.00061
Shigella infection. (n.d.).
Joe D. Joiner, Wieland Steinchen, Nick Mozer, Thales Kronenberger, Gert Bange, Antti Poso, Samuel Wagner, Marcus D. Hartmann, HilE represses the activity of the Salmonella virulence regulator HilD via a mechanism distinct from that of intestinal long-chain fatty acids, Journal of Biological Chemistry, Volume 299, Issue 12, 2023, 105387, ISSN 0021-9258, https://doi.org/10.1016/j.jbc.2023.105387. (https://www.sciencedirect.com/science/article/pii/S0021925823024158)
Faculty Advisor
Dr. D'Ausilio
