Quantification of fluoroquinolone uptake through the outer membrane channel OmpF of Escherichia coli
Journal of the American Chemical Society
American Chemical Society
Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
This work was supported by a European Research Council (ERC) Grant (261101 Passmembrane) to UFK. JC acknowledges support from an Internal Graduate Studentship, Trinity College, Cambridge, and a Research Studentship from the Cambridge Philosophical Society. SP was supported by the Leverhulme Trust through an Early Career Fellowship. TM acknowledges support from the Konrad-Adenauer Foundation and the German National Merit Foundation. HB, YB and MW are part of the TRANSLOCATION consortium and have received support from the Innovative Medicines Joint Undertaking under grant agreement 115525, the European Union’s seventh framework program (FP7/2007-2013), and European Federation of Pharmaceutical Industries and Associates companies in-kind contribution.
This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.
Vol. 137, pp. 13836 - 13843
Place of publication