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Newcastle University Project – Monisha Pathania


Antibiotic permeation in Outer Membrane Proteins of Vibrio Cholerae

Fellow: Monisha Pathania

Supervisor: Bert van den Berg (Newcastle University)

Co-supervisors: Matteo Ceccarelli (University of Cagliari), Ulrich Kleinekathöfer (Jacobs University)


The main aim of the project at Newcastle University is to study and understand the antibiotic permeation in the OmpF/C-like outer membrane proteins OmpU and OmpT of Vibrio cholera.  For this, we aim to solve the OmpU/T X-ray crystal structures along with structural studies on other porins of Gram-negative bacteria for comparison purposes, like Enterobacter cloacae (OmpE35 and OmpE36) and Klebsiella pneumoniae (OmpK36). In addition to solving structures we want to test uptake of different antibiotics via these outer membrane proteins via antibiotic permeation assays. (eg. liposome swelling experiments and disk diffusion assays) and study the interaction of OmpU and OmpT with antibiotics by single-channel electrophysiology. The results will be used to obtain co-crystal structures of selected antibiotics with OmpU and/or OmpT. Together the project should give new insights into how antibiotics enter V. cholerae and other pathogenic vibrios.

We have expressed and purified OmpU and OmpT from V. cholerae as well as OmpK36 from Klebsiella pneumoniae and OmpE35/36 from Enterobacter cloacae. All proteins are available in milligram quantities. We have solved the X-ray crystal structures of OmpU, OmpT, OmpK36 and OmpE36 to high resolution. The structure of OmpE36 has been shown here below.

Structure of OmpE36- The crystal of OmpE36 diffracted to ~ 1.5 Å resolution with two trimers in the asymmetric unit. Figure shows arrangement of three β-barrel monomers arranged into a trimer, with long flexible loops facing the extracellular side. . Each monomer is a 16-stranded β-barrel with longer loops on the extracellular side and short turns on the periplasmic side. As seen in other porins, the Loop L3 of OmpE36 departs from the regular β-barrel fold and enters into the pore, with the residues of the loop constricting the pore. Loop L2 latches into the groove of adjacent monomer, forming electrostatic interactions, and thus stabilizes the trimer.

LPS attached to OmpE36 protein- In the structure, we found bound lipopolysaccharide (LPS) molecules present for each monomer of the OmpE36 trimer. One OmpE36 monomer has two bound LPS molecules. So far only one other structure of an OMP (the TonB dependent transporter FhuA) has been reported with bound LPS.

Cartoon of OmpE36 trimer shown with four bound LPS molecules                                                   Rainbow cartoon of OmpE36.monomer (periplasmic view) 





Calcium ion bridging between LPS molecule and OmpE36 protein residues