Cholera toxin (CT) and heat-labile enterotoxin (LT) from enterotoxigenic E. coli are two protein toxins, which cause severe diarrhea. These diseases are responsible for almost a million deaths each year. Both toxins possess a high degree of sequence similarity, and share a similar 3-dimensional structure, but they differ with respect to binding specificity. While the two toxins also share the same primary receptor, the GM1 ganglioside, they differ with respect to binding to secondary receptors in the intestine, even though the binding sites differ by only a few amino acid residues. Which of the amino acid residues are responsible for the more promiscuous binding specificity by this toxin?
Detailed knowledge of the toxins, including their three-dimensional structure, is essential. Recently, a protocol for production of fully labeled (production of protein with 98% 13C, 98% 15N and 98% 2H) LBT (human LT B-subunit) was established and the complete backbone was successfully assigned. These results are the basis for further interaction studies between hLTB and different ligands.
hLTB is delivered to the intestinal cells while being bound to outer membrane vesicles, whereas cholera toxin is secreted in soluble form. Binding of LPS, present in the outer bacterial membrane, would anchor the toxin to the bacterial surface. Although hLTB is known to bind to LPS, the molecular details of this interaction are not yet understood. NMR studies in combination with crystallization and biochemical experiments will allow insights into the underlying mechanism and may serve as basis for future drug or vaccine development.
Aim: The Master student will be focusing on the molecular characterization of toxin delivery to the intestinal cells, by analyzing the underlying molecular interactions of the toxins with their ligands. The aim is to get a greater understanding of these molecular interactions, and how they can be exploited to lower the extent and seriousness of bacterial infection.
With guidance from two supervising PhD students, the Master’s student will express recombinant toxin, purify it by protein purification methods, investigate binding to ligands by NMR and attempt to crystallize the protein with ligands. Details of the project will be discussed with the student. The group currently consists of 4 PhD students and 3 Postdocs.
Methods: Standard DNA techniques (PCR, mutagenesis), protein expression and purification, binding studies, crystallization, NMR and data analysis.
Selected publications:
1) F. Vasile, J. J. Reina, D. Potenza, J. E. Heggelund, A. Mackenzie, U. Krengel & A. Bernardi (2014). Comprehensive analysis of blood group antigen binding to classical and El Tor cholera toxin B-pentamers by NMR. Glycobiology. 24(8), s 766- 778
2) Røntgenkjemiker løser koleragåte, Forskningsmagasinet Apollon (2012), 3, 26-29.
3) J. E. Heggelund, E. Haugen, B. Lygren, A. Mackenzie, Å. Holmner, F. Vasile, J. J. Reina, A. Bernardi & U. Krengel (2012) Both El Tor and classical cholera toxin bind blood group determinants, Biochem. Biophys. Res. Comm. 418, 731-735.
4) Å. Holmner, A. Mackenzie & U. Krengel (2010) Molecular basis of cholera blood-group dependence and implications for a world characterized by climate change, FEBS Letters, 584, 2548-2555.
5) B. Mudrak, D. L. Rodriguez, M. J. Kuehn. Residues of heat-labile enterotoxin involved in bacterial cell surface binding. J Bacteriol. 2009 May; 191(9):2917-25.
6) Å. Holmner, M. Lebens, S. Teneberg, J. Ångström, M. Ökvist & U. Krengel (2004). Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 Å crystal structure reveals the details, Structure 12 (9), 1655-1667. Erratum in: Structure (2007) 15(2), 253.
7) Å. Holmner, G. Askarieh, M. Ökvist & U. Krengel (2007) Blood group antigen recognition by Escherichia coli heat-labile enterotoxin, J. Mol. Biol., 371, 754-764.
8) Å. Holmner, A. Mackenzie, M. Ökvist, L. Jansson, M. Lebens, S. Teneberg & U. Krengel (2011) Crystal structures exploring the origins of the broader specificity of Escherichia coli heat-labile enterotoxin compared to cholera toxin, J. Mol. Biol., 406, 387-402.
9) J.E. Heggelund, V. A. Bjørnestad & U. Krengel (2015). Vibrio cholerae and Escherichia coli heat-labile enterotoxins and beyond. Elsevier. ISBN 978-0-12-800188-2. 1182 s.