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."Multidrug efflux pumps play an important role in the intrinsic resistance of gram-negative bacteria to a number of antimicrobial agents, and their overproduction causes increased resistance to a broad range of agents, a major concern in view of the occurrence of multidrug-resistant bacterial pathogens in the clinical setting (17). Among the five known families of multidrug transporters, the resistance-nodulation-division (RND) family (26) tends to play major roles in the intrinsic resistance of gram-negative bacteria (17). Some of them also can extrude a very large range of compounds (29). The Escherichia coli genome contains several genes coding for RND transporters (20,25). Among them, the best-studied transporter is AcrB, which provides the major mechanism for multiple drug resistance in E. coli (16,17,29) and whose crystal structure was elucidated by Murakami and coworkers (15). AcrB forms a multiprotein complex (31, 32) with AcrA, a periplasmic protein of the membrane fusion protein family (5), and the complex is likely to include an outer membrane channel protein, TolC (8). This tripartite construction allows the transporter to pump out drugs directly into the medium, rather than into the periplasm, making the complex much more efficient in producing resistance (16,17,29).Earlier studies using intact cells suggested that the AcrABTolC complex and its homolog MexAB-OprM of Pseudomonas aeruginosa may capture their substrates from the periplasm or the outer leaflet of the cytoplasmic membrane (which is in equilibrium with the periplasm), because carbenicillin, which does not diffuse across the cytoplasmic membrane, is pumped out by this complex (13, 18). Furthermore, the crystallographic structure of the AcrB trimer (15) contains "vestibules," which connect the outer leaflet of the cytoplasmic membrane outside with the central cavity within the trimer, which was recently shown to be t...

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

“…A similar suggestion has been made for the CzcA [54] and AcrD [18] efflux pumps. However, the major path for ion efflux should be carried out through the periplasm [55].…”

“…These seven transporters can be categorized into two distinct subfamilies, the hydrophobic and amphiphilic efflux RND (HAE-RND) and heavy-metal efflux RND (HME-RND) families [4,5]. Six of these transporters, such as AcrB [6][7][8][9][10][11][12][13][14][15][16], AcrD [7,17,18], AcrF [7,19,20], MdtB [7,21,22], MdtC [7,21,22] and YhiV [7,23,24], are multidrug efflux pumps, which belong to the HAE-RND protein family [4]. In addition to these multidrug efflux pumps, E. coli contains one HME-RND efflux transporter, CusA, which specifically recognizes and confers resistance to Ag(I) and Cu(I) ions [25,26].…”

Multidrug resistant Acinetobacter baumannii--the role of AdeABC (RND family) efflux pump in resistance to antibiotics.

“…The efflux transporter (AdeB) captures its substrates either from within the phospholipid bilayer of the inner membrane or the cytoplasm [4] and then transport them into the extracellular medium via OMP (AdeC) [28]. The periplasmatic protein AdeA mediates in the cooperation between AdeB and AdeC components.…”

In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

“…However, much care must be taken when manipulating this molecule in solution because its fluorescence and lipophilicity are highly dependent on the pH of the medium 13 . Therefore, in vitro studies on efflux pumps most often rely on a secondary reporter probe, specifically, the pH variations inside the liposome followed by the use of a pH-dependent fluorescent probe such as pyranine 9,[14][15][16][17] . Lately, we have optimized a functional test where the activity of MexB was indirectly monitored by measuring the pH inside proteoliposomes in which MexB was reconstituted in the presence or in the absence of MexA 16,17 .…”