Permeability barriers of Gram-negative pathogens

Abstract

Most clinical antibiotics do not have efficacy against Gram-negative pathogens, mainly because these cells are protected by the permeability barrier comprising the two membranes with active efflux. The emergence of multidrug-resistant Gram-negative strains threatens the utility even of last resort therapeutic treatments. Significant efforts at different levels of resolution are currently focused on finding a solution to this nonpermeation problem and developing new approaches to the optimization of drug activities against multidrug-resistant pathogens. The exceptional efficiency of the Gram-negative permeability barrier is the result of a complex interplay between the two opposing fluxes of drugs across the two membranes. In this review, we describe the current state of understanding of the problem and the recent advances in theoretical and empirical approaches to characterization of drug permeation and active efflux in Gram-negative bacteria.

Keywords: Gram-negative; antibiotic resistance; bacteria; multidrug efflux; permeability barrier.

Figure 1.. Schematics and kinetic model of compound permeation in the context of the two membranes with active efflux.

Small molecules traverse the outer membrane via facilitated or passive diffusion and can be extruded from the periplasmic space by active transporters. The kinetic scheme explicitly considers four compartments, outside the cell (O), within the outer membrane (M), in the periplasm (P) and in the cytoplasm (I). Active efflux is approximated as a Michaelis-Menten process. The binding to the membrane is postulated saturable, with the maximal flux F. The degree of saturation is denoted as ϕ; k₁ through k₄ are microscopic rate constants.

Figure 2.. The energy profile of drug permeation across a lipid bilayer.

(A) Schematic structure of the asymmetric bilayer. (B) A simplified energy profile recognizes the Donnan equilibrium of the membrane, ΔG_d, and the free energy of partitioning between the membrane and the medium, ΔG_t. (C) In addition to the above, a more realistic energy profile needs to incorporate the excluded volume created by the lipid A modification, ΔG_A, and the attractive interaction at the lipid-water interface, ΔG_b.

Figure 3.. Bifurcation kinetics in simulation and live cells.

(A) The main regimes of compound accumulation in Gram-negative bacteria calculated for a model in Fig. 1. The steady state (SS) concentration of a drug in the periplasm is plotted against its equilibrium concentration (EQ), which takes into account the Donnan equilibrium. The black line is for compounds without active efflux, the three other lines are for compounds with the Efflux constant of 10 and the indicated values of the Barrier constant. At low drug concentration, the steady state is reduced compared to equilibrium by a factor of 1+K_E in all cases. At high concentrations, the result depends on the value of B. If B < 1, the steady state asymptotically approaches the equilibrium modified by a factor α = (1-B)/(1+B). If B > 1, the intracellular drug level cannot exceed its threshold. (B) Experimental observation of a bifurcation in accumulation of Hoechst 33342 (HT) in E. coli cells harboring and inducible pore. The expression level of the pore and the resulting B-values are indicated. Reproduced from.

Figure 4.. Rules of permeation and their hierarchy.

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