Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria

Abstract

Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms.

Fig. 1

Some efflux pump substrates.

Fig. 2. Ligand-binding sites in the Binding Protomer of AcrB

A. The “deep” binding site for minocycline and doxorubicin found by Murakami and coworkers (Murakami et al., 2006). The orientation of the trimeric protein is shown in the inset, in which each protomer is shown in different color (Access, mauve; Binding, sand; and Extrusion, green). The binding pocket is shown as an orange-colored surface, with the bound minocycline molecule in stick representation. The site consists of two subpockets, the narrow “Groove” and the much wider “Cave.” The amino acid residues shown in the surface representation were specified earlier (Takatsuka et al., 2010). Parts of the protein close to the viewer have been clipped away to show the site more clearly. B. The two plausible binding sites of AcrB. This shows the “deep”, “ultimate” binding site shown in A (as orange surface), as well as the putative site on the surface of the periplasmic domain, at the entrance of the large cleft (as green surface). The residues used to generate the latter surface are residues 566, 664, 666, 668, 673, 676, 717, and 828, identified by Husain and Nikaido (2010). To get this view, the model was rotated by 70° as shown at the bottom. No clipping was applied here. C. A possible path for the substrate, from the surface site (green surface) to the deep site (orange surface), with the tunnel shown in gray. This is a view from the top, and the presumed direction of the substrate flow is shown by an arrowhead. The tunnel was detected by the program Caver (www.caver.cz), and all figures were generated by using Pymol (www.pymol.org).

Fig. 3. Efflux and hydrolysis of cefamandole

Efflux (V_e) and periplasmic hydrolysis rate (V_h) of cefamandole in E. coli strain HN1160, at different external concentrations (C_o) of the drug. From (Nagano & Nikaido, 2009).