Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli Keiji Nagano1 and Hiroshi Nikaido2 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Communicated by Alexander N. Glazer, University of California System, Oakland, CA, February 13, 2009 (received for review November 26, 2008) Multidrug efflux transporters, especially those that belong to the With intact cells, it is difficult to determine the substrate resistance-nodulation-division (RND) family, often show very concentration in the periplasm, a location where most of the broad substrate specificity and play a major role both in the substrates are thought to be captured (1, 8–10). When ␤-lactams intrinsic antibiotic resistance and, with increased levels of expres- are used as substrates, however, their periplasmic concentrations sion, in the elevated resistance of Gram-negative bacteria. How- can be calculated from their hydrolysis rate by intact cells and ever, it has not been possible to determine the kinetic behavior of from the kinetic constants of periplasmic ␤-lactamases (11). these important pumps so far. This is partly because these pumps Using this approach, we determined the kinetic behavior of form a tripartite complex traversing both the cytoplasmic and several cephalosporins in AcrB-catalyzed efflux and indeed outer membranes, with an outer membrane channel and a obtained indications of positive cooperativity in the ligand– periplasmic adaptor protein, and it is uncertain if the behavior of pump interaction. an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli Results containing the intact multiprotein complex AcrB-AcrA-TolC, and Principle of the Assay. When a ␤-lactam is added to the external measure the kinetic constants for various cephalosporins, by as- medium at a given concentration Co (␮M), it diffuses sponta- sessing the periplasmic concentration of the drug from their rate of neously across the outer membrane into the periplasm (Fig. 1). ␤ hydrolysis by periplasmic -lactamase and the rate of efflux as the The influx Vin is given by Fick’s first law of diffusion difference between the influx rate and the hydrolysis rate. Nitro- ϭ ͑ Ϫ ͒ MICROBIOLOGY cefin efflux showed a Km of about 5 ␮M with little sign of Vin P * A * Co Cp cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, how- where P, A, and Cp denote the permeability coefficient (cm/s) of 2 ever, kinetics showed strong positive cooperativity, which is con- the outer membrane, the surface area of bacterial cells (cm /mg ␮ sistent with the rotating catalysis model of this trimeric pump. For dry weight), and the periplasmic drug concentration ( Mor 3 2 the very hydrophilic cefazolin there was little sign of efflux. nmol/cm ). We used 132 cm /mg (12) for A. We measure the rate of hydrolysis by the periplasmic ␤-lactamase (Vh) spectropho- cephalosporin ͉ drug efflux pump ͉ multidrug resistance tometrically with suspensions of intact cells. We then solve the Michaelis-Menten equation for hydrolysis for Cp, using the values of Km (determined earlier) and Vmax (determined by using ultidrug efflux pumps of the resistance-nodulation- cell extracts). Mdivision (RND) family in Gram-negative bacteria can With nitrocefin, P was determined by coupling influx with the pump out a surprisingly wide range of antimicrobial compounds enzymatic hydrolysis in periplasm (11), with the active efflux (1, 2). For example, AcrB of Escherichia coli, which has been inactivated by deenergization of the cytoplasmic membrane with studied most extensively as a prototype of similar pumps, exports a proton conductor, 40 ␮M carbonyl cyanide m-chlorophenyl- a number of dyes, detergents, chloramphenicol, tetracyclines, hydrazone (CCCP). When this was done in the presence of, e.g., macrolides, novobiocin, fluoroquinolones, and organic solvents. ␮ ␤ 25 M of nitrocefin, the rate of hydrolysis increased severalfold Importantly, it also pumps out -lactams, some of which cannot upon the addition of CCCP, giving us qualitative confirmation easily cross the cytoplasmic membrane and thus stay in the that proton-motive-force-dependent pump(s) were pumping out periplasm (3). However, in the 13 years since their discovery (4, much of the nitrocefin from the periplasm in nonpoisoned cells. 5), no success was achieved in the determination of kinetic The influx rate, Vin, can then be calculated from the values of behavior of these pumps, although the affinity of some substrates P, A, and C , as described above. In our model, the rate of efflux, could be assessed on a relative scale by their activity as com- p V , is the difference between the (calculated) V and the petitive inhibitors in a proteoliposome reconstitution assay (6). e in (measured) V (Fig. 1). Recently an attempt to measure the binding of dyes to purified h One possible complication is that some of the periplasmic AcrB was made by using fluorescence polarization (7); however, cephalosporin may cross the cytoplasmic membrane [V (dashed it is unclear if the experiments measured binding to active sites c arrow) in Fig. 1]. Such diffusion is known to occur with relatively or to generally hydrophobic pockets in the protein. lipophilic cephalosporins such as cephalothin (3, 13). The first- The knowledge of kinetic constants is essential in our attempts order rate constant for this influx into cytosol can be calculated to understand the contribution of the pumps to drug resistance (14) from the half-equilibration time determined earlier (13). in a quantitative manner. Furthermore, recent crystallographic Ϫ For cephalothin, this is 2.9 ϫ 10 9 cm3/s/mg cells. When C ϭ 10 studies of AcrB (8–10) suggest that each protomer in this p trimeric transporter undergoes a succession of conformational changes that are dependent on the conformations of the neigh- Author contributions: H.N. designed research; K.N. performed research; K.N. and H.N. bors, that is, a functionally rotating mechanism. This mechanism analyzed data; and H.N. wrote the paper. predicts that positive cooperativity may exist in the export of The authors declare no conflict of interest. drugs by AcrB. In this study we measured the kinetic constants 1Present address: Department of Microbiology, Aichi-gakuin University School of Dentistry, of AcrB. We used intact cells, because AcrB, which exists as a Nagoya 464-8650, Japan. tripartite complex with the periplasmic protein AcrA and the 2To whom correspondence should be addressed. E-mail: [email protected]. outer membrane channel TolC (1, 2), might be altered in its This article contains supporting information online at www.pnas.org/cgi/content/full/ behavior when it is separated from these physiological partners. 0901695106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901695106 PNAS Early Edition ͉ 1of5 Downloaded by guest on September 30, 2021 Co 0.04 V 0.03 0.05 in V (mM) Cp Cp e 0.04 β- Tol C- Cp V lactamase , Cp h AcrA- OM e 0.03 V , V h h 0.02 Ve Cp AcrB V 0.02 0.01 Ve 0 04080120Co (nmol/mg/s); e , V IM h 0.01 V V c Vh Fig. 1. Principle of the assay. A ␤-lactam compound is added to the external medium bathing the intact cells of E. coli at a concentration, Co. At steady state, the drug crosses the outer membrane at the net rate Vin, which, 0 Ϫ 0 50 100 150 200 according to Fick’s first law of diffusion, is equal to P * A *(Co Cp) (see text). Co This influx is balanced by the sum of 3 processes: (i) hydrolysis by the periplas- (µM) ␤ mic -lactamase at the rate Vh,(ii) periplasmic capture followed by efflux into Fig. 2. Changes of various processes at different external concentrations of the external medium through AcrAB-TolC complex at the rate Ve, and (iii) slow nitrocefin. Results from one typical experiment with strain HN1157 are shown. diffusion across the cytoplasmic membrane into cytosol at the rate Vc. The Inset shows similar results from an experiment with HN1159 (a ⌬acrAB derivative of HN1157), where efflux is undetectable at least within the range of C shown. Ϫ5 p ␮M, Vc will then be 2.9 ϫ 10 nmol/s/mg. This is about 1,000 times smaller than the rates we are concerned with here, Ve, Vin, and Vh (see below), and we neglected Vc in our analysis. rate Vh and the efflux rate Ve, it was not a simple function of Co. In practice, however, this approach demands certain condi- Examination of Vh and Ve (obtained by subtracting Vh from Vin) ␤ tions. For example, the Km of the -lactamase must be reason- showed that the values of Ve had a shape suggesting some ably high, so that Vh becomes a sensitive indicator of Cp, in the saturation, but those of Vh did not in this concentration range of range of the concentrations we are interested in. Ideally, Ve, Vh, Cp, as expected from the large value (340 ␮M) of Km. Efflux and Vin should all be of the same order of magnitude so that high represented by Ve was dominant in the low concentration range precision can be attained in our calculation. The Vh values must for Co, but the enzymatic hydrolysis represented by Vh became be reasonably high to allow for precise measurement of hydrolysis. more important at high values of Co. As an important control, a similar analysis was carried out by Optimization of Assay Conditions for Nitrocefin. In our initial stud- using the isogenic strain HN1159, where the main efflux genes ␤ ies, we used nitrocefin (15) as the -lactam substrate, because the acrAB were absent. As expected, at least up to 40 ␮M Cp, the changes in absorption occur at 486 nm, where the light scattering efflux rate Ve was insignificant (Fig.