University of Birmingham
Multidrug efflux pumps
Du, Dijun; Wang-Kan, Xuan; Neuberger, Arthur; van Veen, Hendrik W.; Pos, Klaas M.;
Piddock, Laura J.V.; Luisi, Ben F.
DOI:
10.1038/s41579-018-0048-6
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Citation for published version (Harvard):
Du, D, Wang-Kan, X, Neuberger, A, van Veen, HW, Pos, KM, Piddock, LJV & Luisi, BF 2018, 'Multidrug efflux
pumps: structure, function and regulation', Nature Reviews Microbiology, vol. 16, no. 9, pp. 523-539.
https://doi.org/10.1038/s41579-018-0048-6
Link to publication on Research at Birmingham portal
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Checked for eligibility 14/09/2018
Dijun Du, Xuan Wang-Kan, Arthur Neuberger, Hendrik W. van Veen, Klaas M. Pos, Laura J. V. Piddock & Ben F. Luisi Multidrug efflux
pumps: structure, function and regulation Nature Reviews Microbiologyvolume 16, pages 523–539 (2018)
https://doi.org/10.1038/s41579-018-0048-6
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Multi-drug efflux pumps: structure, function and regulation
Dijun Du
1
, Xuan Wang-Kan
2
, Arthur Neuberger
1,3
, Hendrik W. van Veen
3
, Klaas M. Pos
4
, Laura
Piddock
2
and Ben F. Luisi
1
1. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2
1GA, UK
2. Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham
B15 2TT, UK
3. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2
1PD, UK
4. Institute of Biochemistry, Goethe Universität Frankfurt, Max-von-Laue-Straße 9, D-60438,
Frankfurt, Germany
Email: B.F. Luisi bfl20@cam.ac.uk
Summary
Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout
the world and threaten to become untreatable. The origins of resistance are numerous and
complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the
intrinsic activity of efflux pumps. In this Review, we describe recent advances that have
increased our understanding of the structures and molecular mechanisms of multidrug efflux
pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in
the drug extrusion process, but also in virulence and the adaptive responses that contribute to
antimicrobial resistance during infection. The emerging picture of the structure, function and
regulation of efflux pumps suggests opportunities for countering their activities.
[H1] Introduction
Bacterial antibiotic resistance has numerous origins, and several general mechanisms of adaptive
responses have been described that give rise to resistant behavior in bacterial populations
1
.
Resistant phenotypes can arise from the boost of intrinsic efflux activity through the
overexpression, asymmetric accumulation during division or mutation of genes encoding energy-
dependent transporters
2-4
. Depending on the antibiotic or toxin challenge, efflux can be the
fastest acting and most effective resistance mechanism in the bacterial repertoire of stress
responses. Exposure to antibiotics and other drugs often triggers very complex bacterial reactions
that involve changes in the level of expression of numerous transporter genes, as seen by
phenotypic profiling of Escherichia coli
5
. These transporters provide several antibiotic efflux
pathways that can work in a cooperative manner or provide redundant functionality
6,7
.
Currently, six families of bacterial drug efflux pumps have been identified that contribute to the
efflux pathways. One of these, the ATP-binding cassette family (ABC) directly utilizes ATP as
the energy source to drive transport. The other five groups are secondary-active transporters that
are powered by electrochemical energy captured in transmembrane ion gradients; they are the
major facilitator superfamily (MFS), the multidrug and toxin extrusion (MATE) family, the
small multidrug resistance (SMR) family, the resistance-nodulation-cell division (RND)
superfamily and the proteobacterial antimicrobial compound efflux (PACE) family
8,9
. Most of
the efflux families have an early origin and have been sustained during the course of evolution,
as seen for example in the ubiquity of the MFS, MATE and RND families or superfamilies
among all domains of life. The efflux machinery is well-tuned to cope with hazardous
compounds and harmful metabolic waste products of immensely diverse chemical character.
Currently, about 80% of all severe bacterial infections observed clinically are attributed to multi-
drug resistant Gram-negative species
10,11
. These bacteria are characterized by a cell envelope
comprising two membranes that function as a barrier to the entry of drugs and other compounds.
Tripartite efflux pumps span this envelope to drive the efflux of compounds across this barrier
(Figure 1). Not all transporters form such assemblies, but the pumps can cooperate as part of a
system that moves the efflux substrates first into the periplasm and then outward through a
tripartite machine
6,7
.
Advances in structural analyses of the different classes of transporters have recently provided
unprecedented insight into their detailed function. Investigations of pump expression patterns
reveal complex regulatory networks at the transcriptional and post-transcriptional level with
connections to many cellular processes, including central metabolism. Accumulating evidence
suggest that efflux pumps have much broader functional roles during infection, beyond transport
of noxious compounds (Box 1). For example, pumps can contribute to bacterial pathogenicity
through transport of proteinaceous toxins and other virulence factors, as well as having roles in
cell-to-cell communication and formation of protective biofilms. In addition, efflux pumps play a
part in lipid transport (in Mycobacteria) and possibly persistence in the presence of antibiotics
12
.
In this Review, we summarize our current understanding of the structures and molecular
mechanisms of multidrug efflux pumps in bacteria. We discuss their regulation by two
component systems as well as by transcription and post-transcription factors and explore efflux-
mediated resistance to antibiotics.
[H1] Molecular mechanisms of multidrug efflux
[H2] ATP-binding cassette transporters.
The ABC transporters are functionally diverse and mediate ATP-dependent import or export of
solutes. Some function as modulators of ion channels
13
. The structures of ABC transporters
reveal transmembrane domains (TMDs) that contain substrate-binding pockets and nucleotide-
binding domains (NBDs) that bind and hydrolyse ATP to drive the transport cycle
14-19
.
The ABC exporters can be divided into homo- and heterodimeric groups, with the latter being
particularly relevant for intrinsic and acquired antibiotic resistance in the Gram-positive bacteria
(for example, PatAB, LmrCD, BmrCD, EfrCD). Although most homodimeric ABC exporters are
thought to have two equivalent nucleotide-binding sites, heterodimeric ABC exporters contain a
degenerate binding site that does not support ATP hydrolysis
20
. ATP binding at the degenerate
site establishes additional contacts across the NBD-NBD dimer interface and prevents the NBD
dimers from fully separating, a molecular feature that might distinguish the hetero- and
homodimeric ABC transporters. For the heterodimeric ABC exporter BmrCD, the nucleotide-
binding domains have been shown to be non-equivalent, conferring an intrinsic asymmetry in the
transporter
21
.
The current structural and functional data for both importers and exporters support an
‘alternating access’ mechanism, whereby the conformation switches states between inward-open,
occluded and outward-open to translocate substrates across the membrane bilayer (Figure 2A).
The conformational changes are linked with NBD dimerization and dissociation mediated by
ATP binding and hydrolysis
22-24
.
The bacterial homodimeric MsbA uses its ATPase activity to move the lipopolysaccharide
precursor lipid A from the cytoplasmic leaflet of the inner membrane to the periplasmic leaflet.
This catalytic action confers the enzyme with the descriptive title of ‘flippase’. The structures of
MsbA in defined functional states have been elucidated by cryo-electron microscopy (cryo-EM)
and enabled the visualization of the transport process
25
. In the absence of ATP, the
lipopolysaccharide can be identified in the transmembrane region of MsbA near the periplasm. In
the presence of a transition state analogue that mimics ATP hydrolysis (ADP-vanadate), MsbA
assumes a closed state conformation, but when ADP is present, an inward-facing conformation is
observed. These structures can be considered as snapshots of the transport process and are
consistent with the alternative access model. The bacterial ABC transporter McjD exports
antibacterial peptides, and structural and spectroscopic data support a mechanism whereby a
binding cavity is transiently opened in the outward-facing conformation causing release of the
peptide
26
. In addition to the conformational changes in ABC exporters facilitated by ATP
binding and hydrolysis, a role of electrochemical ion gradients in transport has been indicated by
studies of the bacterial homodimeric ABC exporters LmrA
27
and MsbA
28
. By analogy with the
chemiosmotic coupling of secondary-active transporters, the coupling to electrochemical ion
gradients most likely imposes directionality on steps in the transport cycle of ABC transporters
that are not regulated by nucleotide. Thus, although bacterial ABC multidrug exporters share
proteins motifs and common features, the detailed structural differences might translate into a
diversity in molecular mechanisms
29
.
[H2] Tripartite assemblies involving ABC transporters
In E. coli and other Gram-negative bacteria, the ABC exporter MacB contributes to drug
resistance and virulence. In those organisms, MacB forms a tripartite assembly with the outer
membrane protein TolC and the periplasmic partner protein MacA. These pumps drive not only
the efflux of macrolide antibiotics, but also the transport of outer membrane lipopeptides,
protoporphyrin, polypeptide virulence factors and lipopolysaccharides. The transport processes
are coupled to ATP hydrolysis by MacB.
Cryo-EM structures have been obtained for the MacAB-TolC assembly, which consists of a
TolC homotrimer, six protomers of MacA and a MacB homodimer (Figure 1)
30
. The 3:6:2
stoichiometry of the assembly is consistent with results from biophysical experiments
31
, and the
dimer seen in the crystal structure of MacB homologues
32,33
. The assembly may also accept
some transport substrates from the periplasm. For example, the precursor of one of its substrates,
the heat-stable enterotoxin II
34
, is transported by the Sec machinery into the periplasm, where it
undergoes maturation and might subsequently enter the MacAB–TolC pump through an opening
observed in the cryo-EM structure
30,33
. For substrates that gain access from the periplasmic side,
MacB possibly uses an ‘outward-only’ transport mechanism proposed for some ABC
transporters, whereby the substrate-binding pocket remains in an outward-facing state. The
transporters may intercept their substrates from in an outward-facing binding pocket, then
undergo a conformational change that is coupled to ATP hydrolysis that decreases affinity for the
ligand so that it is displaced into the exterior compartment
35,36
.
[H2] Transporters of the major facilitator superfamily.
The MFS group is found in all domains of life and is the largest and most diverse family of
transporters. The group encompasses ‘uniporters’ (that move substrates across the lipid bilayer
without any coupling ions), ‘symporters’ (that couple transport with ion-transport in the same
direction as substrate) and ‘antiporters’ (that facilitate the movement of ion and substrates in
opposite directions). Most members of this superfamily function as single, monomeric units.
They range from 400–600 amino acid residues in length and possess 12 or 14 transmembrane
helices (TMH) organized as two domains, each of which is composed of bundles of six helices
(referred to as the MFS fold)
37
. The available structural data support an ‘alternating access’
mechanism for the MFS proteins, whereby the two domains undergo a conformational switch
between inward-open and outward-open states during a transport cycle, similar to that described
above for ABC transporters. Data for the lactose permease symporter LacY show that the
conformational switch for alternating access is triggered by induced fit from ligand binding, and
that the electrochemical proton gradients controls the rate of transport
38
. The key element of the
model is that the binding of proton and substrate are ordered, and that a ternary complex is
formed (Figure 2b). For various MFS transporters, the sequence of binding (that is, proton
binding followed by substrate binding, or substrate binding followed by proton binding) is
different.
Multidrug efflux is conferred by members of the drug:H
+
antiporters-1 (DHA1) and DHA2
families. The crystal structures of DHA1 proteins from E. coli have been determined in outward-
open (YajR), occluded (EmrD) and inward-open (MdfA) conformations
39-41
. The structural
gallery of three different conformational states resembles closely the states captured for the
symporters lactose permease LacY and XylE
38,42,43
. The common structural architecture of the
DHA1 group comprises amino-terminal- and carboxy-terminal domains that are linked by a long
cytoplasmic amphipathic helix-containing loop. In the substrate-bound structures of MdfA, drugs
and inhibitors bind in a central cavity (formed by TMH1, TMH4, TMH7 and TMH10) (Figure
4c). DHA2 family homologues, such as YbgG, have similar domain organization to DHA1
proteins, except that the domains are connected by two V-shaped TMHs
44
. A motif (termed
