Efflux Pumps of the Resistance–Nodulation–Division Family: A Perspective of their Structure, Function, and Regulation in Gram‐Negative Bacteria

With the initial discovery of penicillin and the ensuing mass production of antibiotics in the 1940s, infectious bacteria quickly adapted and developed resistance to the deleterious molecules. In fact, a report published in 1947 found that of 100 staphylococcus infections tested, 38 were classified as highly resistant to penicillin (1). The initial resistance was primarily associated with individual enzymes inactivating specific antibiotics, such as β-lactamases on penicillin. As novel antibiotics were implemented to combat resistant pathogens, selective pressure led to fundamentally new methods of drug resistance. Currently, there are roughly three major mechanisms utilized by bacteria to evade the toxic effects of biocidal agents. These mechanisms include enzymes that modify the drug, alteration of the antibacterial target, and reduced drug uptake due to the presence of efflux pumps or a decrease in porin expression. Enzymatic modification involves two classes of enzymes, including those that degrade specific antibiotics (2) and enzymes that chemically modify the antibacterial compound (3), resulting in inhibition of drug function. The second mechanism employed by bacteria is alteration of the drug target. Nearly all relevant fluoroquinolone resistance has been attributed to target alteration, whereby specific mutations inhibit the drugs interaction to DNA gyrase and topoisomerase IV (4). Although highly effective, these mechanisms are limited by inhibiting only specific classes of antibiotics. A more critical issue is the broad spectrum of antibiotic resistance associated with multidrug efflux pumps. Ubiquitous in most living cells, multidrug efflux transporters have gained recognition as the major contributor to drug resistance observed in many pathogenic microorganisms (5, 6). These transporters are capable of capturing and exporting toxic compounds before they reach their cellular target, thereby decreasing the effectiveness of the drug (6–8). The first drug transporter TetA, identified in 1980 (9), specifically conferred tetracycline resistance on its host. Following this breakthrough, ensuing discoveries identified protein families capable of eliminating various structurally unrelated toxic compounds. Based on sequence and functional similarities, there are currently five families of multidrug-resistant (MDR) efflux pumps, including primary transporters of the ATP-binding cassette (ABC) family (10), and secondary transporters in the resistance-nodulation-division (RND) (11), multidrug and toxic compound extrusion (MATE) (12, 13), major facilitator (MF) (14–16), and small multidrug resistance (SMR) families (17). Of these protein families, RND transporters are considered the primary contributor to multidrug resistance associated with gram-negative bacteria (11, 18, 19). Transporters in the RND family are energized through the proton-motive force (PFM), with translocation of protons occurring in the transmembrane (TM) domain. These pumps generally function as a tripartite efflux complex in conjunction with a membrane fusion protein and an outer membrane channel to export substrates completely out of the bacterial cell (11). Strict regulation of these proteins is maintained at the transcription level through repressors and activators that respond to a similar library of compounds to initiate protein expression. Recently, tremendous strides have been made toward understanding the mechanisms that govern the RND transporter function. These works have focused on both the transporters and the regulatory networks that control their expression. In this chapter we focus on the tripartite RND transporters that are the primary cause of biocidal resistance identified in the three gram-negative bacterial species. These tripartite systems include the AcrAB-TolC and CusABC efflux complexes of Escherichia coli, the CmeABC pump of Campylobacter jejuni, and the MtrCDE transporter of Neisseria gonorrhea. Further, we discuss the regulation of these efflux pumps, especially for the structural aspects of the E. coli AcrR and C. jejuni CmeR transcriptional regulators. With a detailed understanding of the nature of these protein machineries, it will be possible to generate novel therapeutics capable of inhibiting the function of these efflux transporters, thus making futile antibiotics viable once again.