Fosfomycin Permeation through the Outer Membrane Porin OmpF

Abstract

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.

Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

“…34,35 Such simulations have been tremendously useful to understand the transport mechanisms of solutes through nanopores, but remained computationally very exhaustive. 26,36 In this context, we employ our recently developed temperature accelerated Brownian dynamics (TABD) approach 27,37 to investigate the passage of uoroquinolones and especially noroxacin through bacterial outer membrane porins from E. coli (OmpF and OmpC) and E. aerogenes (Omp35 and Omp36). 38 Quinolones belong to the commonly prescribed classes of antimicrobials in clinics.…”

Large‐Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca**

“…In a second set of electrophysiological measurements, we characterised the concentration‐driven relative permeation by using the so‐called reversal potential measurement technique [30, 31] (see Section 2 in the SI for details on Material and Methods). Starting with two compartments having 0.1 m m potassium sulfate separated by a ΔCymA channel embedded in a DPhPC bilayer, a concentration gradient of 0.25 m m Ptm sulfate was introduced on the cis side.…”

“…shorter poly-arginine molecules through hetero-oligomeric channels and through the general diffusion channel OmpF. [29] In asecond set of electrophysiological measurements,we characterised the concentration-driven relative permeation by using the so-called reversal potential measurement technique [30,31] (see Section 2inthe SI for details on Material and Methods). Starting with two compartments having 0.1 mm potassium sulfate separated by a DCymA channel embedded in aDPhPC bilayer,aconcentration gradient of 0.25 mm Ptm sulfate was introduced on the cis side.Inprinciple,the largesize Ptm molecules are expected to have al ower electrophoretic mobility compared to that of the sulfate counterions.…”

Permeation eines 5.1‐kDa‐Peptides durch einen Proteinkanal: Molekulare Basis der Translokation von Protamin durch CymA aus Klebsiella Oxytoca**

“…In Figure S7 of the SI, a linear fit of the rates at lower voltages is shown on a semi-log scale, suggesting a permeation rate of 1-2 Ptm molecules per second for a 1 µM gradient at 0 V, where the permeation time for individual events is estimated from a logarithmic plot as 20-25 ms. Astonishingly, this flux rate agrees nicely with that estimated from the initial rates obtained by the ensemble fluorescence assays in vesicles (see above). In a second set of electrophysiological measurements, we characterized the concentration-driven relative permeation by using the so-called reversal potential measurement technique [31,32] (see Section 2 in the SI for details on Material and Methods). Starting with two compartments having 0.1 mM potassium sulfate separated by a ΔCymA channel embedded in a DPhPC bilayer, a concentration gradient of 0.25 mM Ptm sulphate was introduced on the cis side.…”

“…Studien wurde ein ähnlicher Tr end fürd ie Permeation kürzerer Polyargininmoleküle durch hetero-oligomere Kanäle und durch den allgemeinen Diffusionskanal OmpF beobachtet. [28] In einer zweiten Serie elektrophysiologischer Messungen haben wir die konzentrationsbedingte relative Permeation mithilfe der so genannten Umkehrpotential-Messtechnik charakterisiert [29,30] (Einzelheiten in Material und Methoden, siehe Abschnitt 2d er SI). Ausgehend von zwei Kompartimenten mit 0,1 mm Kaliumsulfat, die durch einen in eine DPhPC-Doppelschicht eingebetteten DCymA-Kanal getrennt waren, wurde auf der cis-Seite ein Konzentrationsgradient von 0,25 mm Ptm-Sulfat angelegt.…”