Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli

Abstract

To understand better the mechanisms of resistance-nodulation-division (RND)-type multidrug efflux pumps, we examined the Escherichia coli AcrD pump, whose typical substrates, aminoglycosides, are not expected to diffuse spontaneously across the lipid bilayer. The hexahistidine-tagged AcrD protein was purified and reconstituted into unilamellar proteoliposomes. Its activity was measured by the proton flux accompanying substrate transport. When the interior of the proteoliposomes was acidified, the addition of aminoglycosides to the external medium stimulated proton efflux and the intravesicular accumulation of radiolabeled gentamicin, suggesting that aminoglycosides can be captured and transported from the external medium in this system (corresponding to cytosol). This activity required the presence of AcrA within the proteoliposomes. Interestingly, the increase in proton efflux also occurred when aminoglycosides were present only in the intravesicular space. This result suggested that AcrD can also capture aminoglycosides from the periplasm to extrude them into the medium in intact cells, acting as a "periplasmic vacuum cleaner."

FIG. 1.

Overexpression and purification of AcrD (A) and AcrA (B). Protein samples were loaded onto an SDS-7.5% polyacrylamide gel and stained with Coomassie brilliant blue. Lanes 1, crude extract; lanes 2, column flowthrough fractions; lanes 3, AcrD-6His after cobalt resin batch purification (A) and AcrA-6His after purification on an Ni²⁺ column (B). The rightmost and leftmost lanes in panels A and B, respectively, show molecular mass markers, with molecular masses shown in kilodaltons. (C) Western blot analysis of AcrD reconstituted into proteoliposomes. Here AcrD proteoliposomes were loaded onto an SDS-7.5% polyacrylamide gel and analyzed by immunoblotting with polyclonal anti-AcrD antibodies (left lane). The right lane shows molecular mass markers as for panels A and B.

FIG. 2.

Effects of aminoglycoside addition to the extravesicular space on proton efflux from AcrD-containing proteoliposomes. AcrD proteoliposomes were reconstituted in a buffer containing 0.2 M KCl and then were diluted in the same buffer containing 0.2 M NaCl instead. ΔpH was generated by the addition of 10 μM valinomycin. Decreased fluorescence of the pyranine corresponds to the interior-acid ΔpH, which dissipated slowly in the absence of drugs. Aminoglycosides were added to the extravesicular space at a concentration of 70 μM. (A) Curve 1, AcrD proteoliposomes alone; curve 2, gentamicin was added to AcrD proteoliposomes; curve 3, gentamicin was added to AcrD proteoliposomes containing AcrA plus Mg²⁺. (B) A single batch of AcrD proteoliposomes with entrapped AcrA and Mg²⁺ was used. Curve 1, without added substrates, as a control; curve 2, anti-AcrD antibodies and gentamicin added to the external medium; curve 3, gentamicin alone added to the external medium. (C) A single batch of AcrD proteoliposomes containing AcrA and Mg²⁺ was used. Curve 1, no-substrate control; curve 2, amikacin (70 μM) added externally; curve 3, gentamicin (70 μM) added externally; curve 4, tobramycin (70 μM) added externally. AU, arbitrary units. Insets in this and subsequent figures show schematically the experimental setup. S, substrate. Black squares, AcrA; grey structures, AcrD.

FIG. 3.

Accumulation of [³H]gentamicin in AcrD-containing proteoliposomes. AcrD proteoliposomes were made in KCl buffer so that AcrA and Mg²⁺ were entrapped within. These were diluted into NaCl buffer containing 10 μM [³H]gentamicin as described in Materials and Methods (▪). The results shown are averages of five independent experiments. Liposomes without AcrD and AcrA served as a control (▴). At time zero 10 μM valinomycin was added to produce a proton motive force.

FIG. 4.

Effect of the presence of aminoglycosides in the intravesicular space on proton efflux from AcrD proteoliposomes. Experiments were carried out as for Fig. 2, using proteoliposome preparations made side by side on the same day, except that the substrate was entrapped within one batch of vesicles. Curve 1, AcrD vesicles with entrapped AcrA and Mg²⁺, as the control; curve 2, AcrD vesicles with entrapped AcrA, Mg²⁺, and gentamicin (70 μM).

FIG. 5.

Effect of streptomycin on proton efflux from AcrD proteoliposomes. Experiments were carried out as for Fig. 2 and 4. Two kinds of proteoliposomes used were made side by side on the same day. Curve 1, AcrD proteoliposomes containing AcrA and Mg²⁺ (but no substrate) as the control; curve 2, streptomycin (70 μM) was added to the extravesicular space of AcrD proteoliposomes containing AcrA and Mg²⁺; curve 3, AcrD proteoliposomes containing AcrA, Mg²⁺, and 70 μM streptomycin. AU, arbitrary units.