To reach their site of action, it is essential for antibiotic molecules to cross the bacterial outer membrane. The progress of enhanced sampling techniques in molecular dynamics simulations enables us to understand these translocations at an atomic level. To this end, calculations of free energy surfaces for these permeation processes are of key importance. Herein, we investigate the translocation of a variety of anionic solutes through the outer membrane pore OprO of the Gram-negative bacterium Pseudomonas aeruginosa using the metadynamics and umbrella sampling techniques at the all-atom level. Free energy calculations have been performed employing these two distinct methods in order to illustrate the difference in computed free energies, if any. The investigated solutes range from a single atomic chloride ion over a multiatomic monophosphate ion to a more bulky fosmidomycin antibiotic. The role of complexity of the permeating solutes in estimating accurate free energy profiles is demonstrated by performing extensive convergence analysis. For simple monatomic ions, good agreement between the well-tempered metadynamics and the umbrella sampling approaches is achieved, while for the permeation of the monophosphate ion differences start to appear. In the case of larger molecules such as fosmidomycin it is a tough challenge to achieve converged free energy profiles. This issue is mainly due to neglecting orthogonal degrees of freedom during the free energy calculations. Nevertheless, the freely driven metadynamics approach leads to clearly advantageous results. Additionally, atomistic insights of the translocation mechanisms of all three solutes are discussed.
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.
To reach their target site inside Gram-negative bacteria, almost all antibiotics need to cross the outer membrane. Computational modeling of such processes can be numerically demanding due to the size of the systems and especially due to the timescales involved. Recently, a hybrid Brownian and molecular dynamics approach, i.e., Brownian dynamics including explicit atoms (BRODEA), has been developed and evaluated for studying the transport of monoatomic ions through membrane channels. Later on, this numerically efficient scheme has been applied to determine the free energy surfaces of the ciprofloxacin and enrofloxacin translocation through the porin OmpC using temperature-accelerated simulations. To improve the usability and accuracy of the approach, schemes to approximate the position-dependent diffusion constant of the molecule while traversing the pore had to be established. To this end, we have studied the translocation of the charged phosphonic acid antibiotic fosfomycin through the porin OmpF from Escherichia coli devising and benchmarking several diffusion models. To test the efficiency and sensitivity of these models, the effect of OmpF mutations on the permeation of fosfomycin was analyzed. Permeation events have been recorded over millisecond-long biased and unbiased simulations, from which thermodynamics and kinetics quantities of the translocation processes were determined. As a result, the use of the BRODEA approach, together with the appropriate diffusion model, was seen to accurately reproduce the findings observed in electrophysiology experiments and all-atom molecular dynamics simulations. These results suggest that the BRODEA approach can become a valuable tool for screening numerous compounds to evaluate their outer membrane permeability, a property important in the development of new antibiotics.
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen responsible for many nosocomial infections. It is quite resistant to various antibiotics, caused by the absence of general diffusion pores in the outer membrane. Instead, it contains many substrate-specific channels. Among them are the two phosphate- and pyrophosphate-specific porins OprP and OprO. Phosphonic acid antibiotics such as fosfomycin and fosmidomycin seem to be good candidates for using these channels to enter P. aeruginosa bacteria. Here, we investigated the permeation of fosfomycin through OprP and OprO using electrophysiology and molecular dynamics (MD) simulations. The results were compared to those of the fosmidomycin translocation, for which additional MD simulations were performed. In the electrophysiological approach, we noticed a higher binding affinity of fosfomycin than of fosmidomycin to OprP and OprO. In MD simulations, the ladder of arginine residues and the cluster of lysine residues play an important role in the permeation of fosfomycin through the OprP and OprO channels. Molecular details on the permeation of fosfomycin through OprP and OprO channels were derived from MD simulations and compared to those of fosmidomycin translocation. In summary, this study demonstrates that the selectivity of membrane channels can be employed to improve the permeation of antibiotics into Gram-negative bacteria and especially into resistant P. aeruginosa strains.
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