Fig 1.
Major fluxes into bacteria, including passive diffusion across the inner and outer membranes, facilitated diffusion through porins and channels (blue), and active efflux by multidrug transporters, such as AcrAB-TolC of E. coli (red).
Labeled are the four main compartments considered in the kinetic scheme including the outside (O), the outer membrane barrier (M), the periplasm (P), and internal space (I). The kinetic scheme summarizes these processes (see text for details).
Fig 2.
Characteristic regimes of drug accumulation in bacteria.
(a). Steady state (ss) drug levels in the periplasm plotted against the equilibrium (eq) drug concentration (Xp), which would be observed in the absence of active fluxes. Note that Xp is proportional to the external drug concentration, Xp = k1O/k2. Solutions to Eq 2 are shown for cells without active drug efflux (V = 0), or with the indicated values of the barrier constant B. Asymptotic behavior of the plots (Eq 11) is shown with dashed lines together with the underlying equations. The variables are defined in Eq 2; α = (1-B)/(1+B). Note the existence of two regimes, of efficient (B>1) and inefficient (B<1) efflux. (b). The relationship between the steady state and equilibrium drug concentrations in the periplasm of cells with active drug efflux (KE > 1) but no outer membrane barrier (B = 0). The transition between regimes of efficient and inefficient efflux occurs when the periplasmic drug concentration reaches the transporter’s Michaelis constant Km. (c). The initial rate of drug accumulation in the cytoplasm as a function of its external concentration. Dashed lines mark the asymptotes to the plots.
Fig 3.
Accumulation of Hoechst 33342 (HT) in Escherichia coli.
(a). Time courses of Hoechst accumulation (±SD; n = 4) in WT and WT-Pore cells grown without and with 0.1% arabinose to induce the expression of the pore. The data were fit to a burst- single exponential decay, I = A1 + A2∙(1-exp(-k2t)), where A1 describes the fast initial step, and the slow step reflects subsequent rise in fluorescence (see Methods for details). Arrow marks the moment when cells were mixed with 4 μM Hoechst (b). Fluorescence microscopy analysis of Hoechst uptake by WT and WT-Pore cells grown with 0.1% arabinose, following incubation with 1 μM Hoechst for 1 min. (c). Initial rates of Hoechst accumulation in WT and WT-Pore cells grown with 0.1% arabinose during the slow phase shown in panel a and S4 Fig. The data were fit to Eq 5. (d). Steady state concentrations of Hoechst in the cytoplasm fit to Eq 4. (e). Steady state concentrations of Hoechst in the periplasm.
Fig 4.
Contributions of active drug efflux and the outer membrane barrier to drug susceptibility of a bacterium.
(a) Effects of the inactivation of drug efflux are modeled for bacteria with efficient (B > 1) and inefficient (B < 1) efflux. Drug concentrations are measured in the units of Km, KE equals 10 for all compounds. Depending on the intracellular inhibition constant of a compound, KI, inactivation of efflux can reduce its observed inhibitory concentration 3-, 7- or 37-fold, respectively, in the case of inefficient (B < 1, KI > Km), partially efficient (B < 1, KI < Km) or efficient (B > 1, KI < Km) efflux. (b). Growth inhibition of bacteria with defects in multidrug efflux or membrane permeability by Hoechst. The IC50 values (in μM) are shown in parenthesis. (c). The ratio of IC50s of azithromycin (AZI), ciprofloxacin (CIP) and Hoechst (HT) for TolC+ and ΔTolC cells measured in the absence (no Pore) or presence (+Pore) of overproduced FhuA* (±SD; n ≥ 2) plotted on a logarithmic scale.