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Christopher D. Bahl Alumnus, and current Postdoctoral Fellow with David Baker at the University of Washington, Seattle Ph.D., Biochemistry    Dartmouth Medical School. 2012 M.S., Biochemistry    University of Maine, Orono. 2006 B.S., Biochemistry, Molecular & Cellular Biology    University of Maine, Orono. 2005 email: cdbahl@uw.edu

Research Summary

Cif is likely a colonization factor during airway infection. Cif is secreted by P. aeruginosa in the lung, which is able to promote the removal of CFTR from the apical membrane of airway epithelial cells. This leads to a dehydrated airway-surface liquid, inhibition of mucociliary clearance, and promotion of bacterial colonization.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic infection in the lungs of cystic fibrosis (CF) patients. P. aeruginosa has been shown to secrete a soluble protein, termed Cif (CFTR inhibitory factor), which inhibits the endocytic recycling of cystic fibrosis transmembrane conductance regulator (CFTR). The result of Cif treatment on airway epithelial cells is a loss of CFTR mediated chloride efflux across the apical membranes. This effect is due to mislocalization of the transporter to endosomes, and by preventing trafficking back to the plasma membrane, CFTR is shunted to the lysosomes for degradation. Since the majority of CF patients are colonized by P. aeruginosa, Cif represents a potential roadblock for the effectiveness of CFTR targeted therapies in a clinical setting. Additionally, elucidation of the Cif mechanism may illuminate novel pathways regulating CFTR trafficking.

We have determined the structure of Cif by X-ray crystallography and demonstrated it to be an epoxide hydrolase (EH) with unique substrate selectivity. Mammalian EHs can function as regulators of blood pressure and inflammation, while bacterial EHs have mostly been characterized for their ability to detoxify xenobiotic compounds. Cif is the first example of an EH serving as a virulence factor. Based on structural comparison, it appears that the enzyme utilizes a catalytic triad of residues Asp129, Glu153 and His297, with accessory residues His177 and Tyr239 coordinating the epoxide oxygen during ring opening. Additionally, His207 sits along the substrate access tunnel to the active site. Cif is also the first example of an EH utilizing a His-Tyr pair to coordinate an epoxide substrate, rather than the canonical Tyr-Tyr pair. In the proposed enzyme mechanism, Asp129 nucleophilically attacks a carbon of the epoxide moiety of a substrate, forming an ester linked enzyme-acyl intermediate. The preference for which carbon is attacked varies depending upon the substrate. In the second step of the reaction, a water molecule is activated by the charge-relay His297-Glu153 pair, and undergoes nucleophilic attack on the Cγ of Asp129. This hydrolyzes the ester group, liberating the hydrolysis product as a vicinal diol.

Active site and epoxide hydrolase mechanism of Cif. The X-ray crystal structure of Cif is shown with a Cα trace in gray, and side chains of select residues playing a role in catalysis are displayed as sticks.

Mutation of any active site residue results in the disruption of enzymatic hydrolysis of epoxide compounds. Furthermore, these mutations completely abolish the CFTR inhibitory effect seen on airway epithelial cells. Thus, it appears that EH activity is strictly required for Cif mediated mislocalization of CFTR, suggesting that Cif likely acts on a physiological epoxide with a role in regulating CFTR trafficking. It is possible that this effect could be due to either the depletion of an epoxide target or the generation of a vicinal-diol hydrolysis product. Current work is aimed at elucidation of the endogenous human epoxide/diol pair that is responsible for mediating this effect.

For more information, feel free to visit the Cif Wikipedia page.

Publications

A.E. Ballok, C.D. Bahl, E.L. Dolben, A.K. Lindsay, J.D. St. Laurent, D.A. Hogan, D.R. Madden, G.A. O'Toole (2012) Epoxide-mediated CifR repression of cif gene expression utilizes two binding sites in Pseudomonas aeruginosa. J Bacteriol. 194, 5315-5324.

C.D. Bahl (2012) Searching For a Hijacked Epoxide: Structural, Mechanistic, and Functional Characterization of Cif, a Virulence Factor Secreted by Pseudomonas aeruginosa. (Ph.D. thesis) Dartmouth College, Hanover, NH

C.D. Bahl, D.R. Madden (2012) Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair. Protein Pept Lett. 19, 186-193.

C.D. Bahl, C. Morisseau, J.M. Bomberger, B.A. Stanton, B.D. Hammock, G.A. O'Toole, D.R. Madden (2010) Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor Cif reveals novel active-site features of an epoxide hydrolase virulence factor. J Bacteriol. 192, 1785-1795.

C.D. Bahl, D.P. MacEachran, G.A. O'Toole, D.R. Madden. (2010) Purification, crystallization and preliminary X-ray diffraction analysis of Cif, a virulence factor secreted by Pseudomonas aeruginosa. Acta Crystallogr. F66, 26-28.

C.D. Bahl (2006) Characterization of a Putative Palmitoyltransferase in Dictyostelium discoideum. (M.S. thesis) The University of Maine, Orono, ME

Structures

A full list of structures can be found at the Protein Data Bank

Published Poster Abstracts

C.D. Bahl, S.A. Gerber, D.R. Madden (2010) The Pseudomonas virulence factor Cif alters human ABC transporter trafficking and stability through epoxide hydrolase enzyme activity. A Special Symposium Celebrating the 40th Anniversary of the Protein Data Bank Abstract Book. Cold Spring Harbor Laboratory, New York.

C.D. Bahl, S.A. Gerber, D.R. Madden (2010) The Pseudomonas virulence factor Cif requires epoxide hydrolase activity to mediate CFTR degradation. Pediatr Pulmonol. 45, 349.

C.D. Bahl, C. Morisseau, J.M. Bomberger, D.P. MacEachran, G.A. O'Toole, B.A. Stanton, B.D. Hammock, D.R. Madden (2009) Epoxide hydrolase activity of CFTR inhibitory factor, a virulence factor on airway epithelial cells. Pediatr Pulmonol. 44, 318.

Invited Talks

Fellowships and Awards

Teaching Experience

Professional Memberships

Biophysical Society
   Student Spotlight in the June 2011 Newsletter

American Society for Biochemistry and Molecular Biology

Dartmouth Medical School • Department of Biochemistry • Dartmouth Lung Biology Center • Biophysics at Dartmouth
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