Areas of Interest
Human diseases caused by enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli remain a major problem worldwide, especially for children in developing countries. Unlike laboratory strains, these bacteria are surrounded with a high molecular weight group 4 capsule polysaccahride. We collaborate with Prof. Ilan Rosenshine (Hebrew University Faculty of Medicine), whose lab recently demonstrated that EHEC group 4 capsule is important for bacterial virulence. Together, we identified the g4c operon encoding seven proteins, each essential for group 4 secretion (Peleg et al., 2005). Our long-term goal is a detailed molecular understanding of how reversible tyrosine phosphorylation regulates capsule biosynthesis.
Applying biochemical and structural techniques, we are focusing on two enzymes: a transmembrane protein tyrosine kinase (Etk) and a cytoplasmic low molecular weight tyrosine phosphatase (Etp). The Etk catalytic domain is structurally unrelated to eukaryotic kinases but is most similar to some bacterial ATPases. It autophosphorylates the seven tyrosines found at the C-terminus of other Etk molecules, while Etp dephosphorylates Etk. The catalytic rates of both enzymes appear to be important for proper G4C secretion. We are studying kinetics of both Etk and Etp mutants to understand the effect of the mutations on polysaccharide secretion , as well as on the activity of each other. Another project investigates why a particular Etp mutant, which is fully active in vitro and in vivo, prevents formation of the capsule. The lab is also expressing new constructs of Etk in order to obtain crystals of enzyme-substrate complexes for structure determination. Another study involves studying the size of capsule polysaccharide molecules and how they are attached to the bacteria's outer membrane.
Honors & Awards
1993 Pew Scholars Program in Biomedical Sciences
2002 Fulbright Scholar Research Award
Nadler, C., Koby, S., Peleg, A., Johnson, A. C., Suddala, K. C., Sathiyamoorthy, K., Smith, B. E., Saper, M. A. & Rosenshine, I. (2012). Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli. PLoS ONE 7, e37984. PMID: 22675501
Rojviriya, C., Pratumrat, T., Saper, M. A. & Yuvaniyama, J. (2011). Improved X-ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration. Acta Crystallogr Sect F Struct Biol Cryst Commun 67, 1570–1574. PMID: 22139169
Sathiyamoorthy, K., Mills, E., Franzmann, T. M., Rosenshine, I. & Saper, M. A. (2011). The Crystal Structure of Escherichia coli Group 4 Capsule Protein GfcC Reveals a Domain Organization Resembling That of Wza. Biochemistry 50, 5465–5476. PMID: 21449614
Vijayalakshmi, J., Akerley, B. J. & Saper, M. A. (2008). Structure of YraM, a protein essential for growth of Haemophilus influenzae. Proteins 73, 204–217. PMID: 18412262
Peleg, A., Shifrin, Y., Ilan, O., Nadler-Yona, C., Nov, S., Koby, S., Baruch, K., Altuvia, S., Elgrably-Weiss, M., Abe, C. M., Knutton, S., Saper, M. A. & Rosenshine, I. (2005). Identification of an Escherichia coli operon required for formation of the O-antigen capsule. J Bacteriol 187, 5259–5266. PMID: 16030220
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