Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli

Abstract

Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic ␤-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a Km of about 5 M with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.cephalosporin ͉ drug efflux pump ͉ multidrug resistance M ultidrug eff lux pumps of the resistance-nodulationdivision (RND) family in Gram-negative bacteria can pump out a surprisingly wide range of antimicrobial compounds (1, 2). For example, AcrB of Escherichia coli, which has been studied most extensively as a prototype of similar pumps, exports a number of dyes, detergents, chloramphenicol, tetracyclines, macrolides, novobiocin, fluoroquinolones, and organic solvents. Importantly, it also pumps out ␤-lactams, some of which cannot easily cross the cytoplasmic membrane and thus stay in the periplasm (3). However, in the 13 years since their discovery (4, 5), no success was achieved in the determination of kinetic behavior of these pumps, although the affinity of some substrates could be assessed on a relative scale by their activity as competitive inhibitors in a proteoliposome reconstitution assay (6). Recently an attempt to measure the binding of dyes to purified AcrB was made by using fluorescence polarization (7); however, it is unclear if the experiments measured binding to active sites or to generally hydrophobic pockets in the protein.The knowledge of kinetic constants is essential in our attempts to understand the contribution of the pumps to drug resistance in a quantitative manner. Furthermore, recent crystallographic studies of AcrB (8-10) suggest that each protomer in this trimeric transporter undergoes a succession of conformational changes that are dependent on the conformations of the neighbors, that is, a functionally rotating mechanism. This m...

Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB ofEscherichia coli

“…S2C). Finally, all binding poses for cefazolin, for which we could not find any evidence for AcrB-mediated efflux in our quantitative efflux assay (28), were outside the binding pocket (Fig. S2C), confirming that prediction of binding at these locations has no functional significance.…”

“…We therefore examined the possible competition between the substrates by measuring the efflux of nitrocefin (a groove-binder; Fig. 3) quantitatively (28) in the presence and absence of potential competitors. Because the assay depends on the β-lactamase-catalyzed hydrolysis of nitrocefin, we could not use β-lactams as competitors.…”

“…However, computer docking has many serious limitations. We therefore sought to evaluate the reliability of the docking predictions by using two experimental approaches recently developed in our laboratory (25,28). The initial results, although limited in scope, indeed appear to suggest that the prediction by the computer docking may not be far from reality, at least in those cases examined experimentally.…”

Quinazoline derivatives are efficient chemosensitizers of antibiotic activity in Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa resistant strains

“…A recent paper presented differences in the affinity constant for some antibiotics [13]. Consequently, as the concentration of a determined compound is increased, competition for these internal binding sites favours the compound depending on the respective affinity constant ratio, thereby promoting intrabacterial retention of the antibiotic.…”

“…This can explain the divergence observed in the competitive capacity of the compounds with drugs of different classes (chloramphenicol, quinolones, ethidium bromide) for binding sites within the pump type (AcrB, MexB) as well as the level of expressed efflux pump activity in the tested strains. Kinetic studies have reported variation in the affinity constant of various -lactams for AcrB in E. coli cells [13]. This flexibility of the substrate site may be directly involved in the polyselectivity of the efflux pump; a site that can be modified by site-directed mutagenesis [15,16].…”