The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

82 Pages·2015·4.52 MB·English

crossmark
The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-
Negative Bacteria
Xian-ZhiLi,aPatrickPlésiat,bHiroshiNikaidoc
HumanSafetyDivision,VeterinaryDrugsDirectorate,HealthProductsandFoodBranch,HealthCanada,Ottawa,Ontario,Canadaa;LaboratoiredeBactériologie,Faculté
deMédecine-Pharmacie,CentreHospitalierRégionalUniversitaire,UniversitédeFranche-Comté,Besançon,Franceb;DepartmentofMolecularandCellBiology,University
ofCalifornia,Berkeley,California,USAc
SUMMARY..................................................................................................................................................338
INTRODUCTION............................................................................................................................................339
D
BIOCHEMISTRYANDGENETICSOFMULTIDRUGEFFLUXPUMPS........................................................................................339
o
ClassesofEffluxPumps...................................................................................................................................339 w
RNDTransporters ........................................................................................................................................340 n
AcrBofEscherichiacoli.................................................................................................................................340 lo
OtherRNDtransportersinE.coli.......................................................................................................................347 a
MFSTransporters.........................................................................................................................................347 d
e
ABCTransporters.........................................................................................................................................348 d
SMRTransporters.........................................................................................................................................348 f
MATETransporters.......................................................................................................................................349 ro
SYNERGYWITHTHEOUTERMEMBRANEBARRIER........................................................................................................349 m
PathwaysofDrugInfluxacrosstheOM..................................................................................................................349
h
DrugsTraversingtheOMMainlythroughPorinChannels...............................................................................................350 t
t
DrugsTraversingtheOMthroughtheLipidBilayerRegion..............................................................................................351 p
:
GAMMAPROTEOBACTERIA:ENTEROBACTERIACEAE.......................................................................................................351 //
E.coli......................................................................................................................................................351 c
m
Fluoroquinolones......................................................................................................................................352
r
(cid:2)-Lactams.............................................................................................................................................352 .a
Otherdrugs............................................................................................................................................353 s
Salmonellaspp. ..........................................................................................................................................353 m
Citrobacterspp............................................................................................................................................353 .o
EnterobacteraerogenesandEnterobactercloacae.........................................................................................................354 r
g
Klebsiellapneumoniae....................................................................................................................................354 /

Proteus,Providencia,andMorganellaspp.................................................................................................................356 o
n
Serratiamarcescens.......................................................................................................................................356
Shigellaflexneri............................................................................................................................................356 J
a
YersiniaenterocoliticaandYersiniapestis..................................................................................................................356 n
OTHERGAMMAPROTEOBACTERIA:VIBRIO,AEROMONAS,LEGIONELLA,ANDPASTEURELLACEAE.......................................................357 u
a
Vibriospp.................................................................................................................................................357 r
Aeromonasspp. ..........................................................................................................................................357 y

Legionellaspp. ...........................................................................................................................................357 4
,
Pasteurellaceae...........................................................................................................................................358 2
Pasteurellamultocida...................................................................................................................................358 0
Haemophilusinfluenzae................................................................................................................................358 1
9
GAMMAPROTEOBACTERIA:PSEUDOMONAS,ACINETOBACTER,ANDSTENOTROPHOMONAS............................................................358
b
Pseudomonasaeruginosa.................................................................................................................................358
y
OMpermeability.......................................................................................................................................358
g
RNDeffluxpumps. ....................................................................................................................................358 u
(continued) e
s
t
Published18March2015
CitationLiX-Z,PlésiatP,NikaidoH.18March2015.Thechallengeofefflux-
mediatedantibioticresistanceinGram-negativebacteria.ClinMicrobiolRev
doi:10.1128/CMR.00117-14.
AddresscorrespondencetoHiroshiNikaido,[email protected].
Copyright©2015,AmericanSocietyforMicrobiology.AllRightsReserved.
doi:10.1128/CMR.00117-14
April2015 Volume28 Number2 ClinicalMicrobiologyReviews cmr.asm.org 337
Lietal.
(i)MexAB-OprM.....................................................................................................................................358
(ii)MexXY-OprM(OprA)..............................................................................................................................359
(iii)MexCD-OprJ.....................................................................................................................................360
(iv)MexEF-OprN.....................................................................................................................................361
(v)OtherRNDpumps...............................................................................................................................361
Acinetobacterspp.........................................................................................................................................361
OMpermeability.......................................................................................................................................361
RNDeffluxpumps.....................................................................................................................................362
Non-RNDeffluxpumps................................................................................................................................362
Stenotrophomonasmaltophilia...........................................................................................................................363
ALPHAPROTEOBACTERIA:BRUCELLA,BARTONELLA,ANDRICKETTSIA....................................................................................363
Brucellaspp...............................................................................................................................................364
Bartonellaspp. ...........................................................................................................................................364
Rickettsiaspp. ............................................................................................................................................364
BETAPROTEOBACTERIA:ACHROMOBACTER,BURKHOLDERIA,ANDNEISSERIA...........................................................................364
Achromobacterspp.......................................................................................................................................364 D
Burkholderiaspp..........................................................................................................................................364 o
Neisseriaspp..............................................................................................................................................365 w
EPSILONPROTEOBACTERIA:CAMPYLOBACTERANDHELICOBACTER......................................................................................365 n
Campylobacterspp. ......................................................................................................................................366 lo
a
Helicobacterspp. .........................................................................................................................................366 d
BACTEROIDACEAEANDPREVOTELLACEAE.................................................................................................................367 e
DRUGEFFLUXGENESONPLASMIDS......................................................................................................................367 d

REGULATIONOFMUTLIDRUGEFFLUXPUMPS............................................................................................................369 fr
E.coliEffluxPumps.......................................................................................................................................369 o
m
AcrAB-TolC.............................................................................................................................................369

Regulationofotherpumps............................................................................................................................371 h
t
SalmonellaEffluxPumps.................................................................................................................................371 t
p
K.pneumoniaeEffluxPumps..............................................................................................................................371 :
/
P.aeruginosaEffluxPumps...............................................................................................................................372 /c
MexAB-OprM..........................................................................................................................................372 m
MexXY.................................................................................................................................................373 r
.
OtherMexpumps.....................................................................................................................................375 a
s
A.baumanniiEffluxPumps...............................................................................................................................375 m
S.maltophiliaEffluxPumps...............................................................................................................................376
.
NeisseriaEffluxPumps....................................................................................................................................376 o
r
C.jejuniEffluxPumps.....................................................................................................................................376 g
/
ROLEOFEFFLUXPUMPSINBIOFILMFORMATIONANDRESISTANCE....................................................................................376
o
INVOLVEMENTOFMUTLIDRUGEFFLUXPUMPSINOTHERFUNCTIONS.................................................................................377 n
BacterialStressResponses................................................................................................................................378 J
Fitness,Colonization,andVirulence......................................................................................................................378 a
n
Entericbacteria........................................................................................................................................378
u
P.aeruginosa...........................................................................................................................................379 a
Otherbacteria.........................................................................................................................................380 ry
MULTIDRUGEFFLUXPUMPSASACHALLENGEINDRUGDEVELOPMENT...............................................................................380
4
EFFLUXPUMPINHIBITORS.................................................................................................................................381 ,

PA(cid:2)N.....................................................................................................................................................381 2
0
NMP......................................................................................................................................................383 1
D13-9001.................................................................................................................................................384 9
MBX2391.................................................................................................................................................384 b
OtherCompoundsThatInhibitRNDPumps.............................................................................................................385 y

METHODOLOGICALCONSIDERATIONS...................................................................................................................386 g
CONCLUSIONS.............................................................................................................................................387 u
e
ACKNOWLEDGMENTS......................................................................................................................................387 s
ADDENDUMINPROOF.....................................................................................................................................387 t
REFERENCES................................................................................................................................................387
AUTHORBIOS..............................................................................................................................................418
SUMMARY and acquired multidrug resistance (MDR) but also are in-
The global emergence of multidrug-resistant Gram-negative volved in other functions, including the bacterial stress re-
bacteriaisagrowingthreattoantibiotictherapy.Thechromo- sponseandpathogenicity.Additionally,effluxpumpsinteract
somally encoded drug efflux mechanisms that are ubiquitous synergisticallywithotherresistancemechanisms(e.g.,withthe
inthesebacteriagreatlycontributetoantibioticresistanceand outer membrane permeability barrier) to increase resistance
present a major challenge for antibiotic development. Multi- levels. Since the discovery of RND pumps in the early 1990s,
drug pumps, particularly those represented by the clinically remarkablescientificandtechnologicaladvanceshaveallowed
relevantAcrAB-TolCandMexpumpsoftheresistance-nodu- foranin-depthunderstandingofthestructuralandbiochem-
lation-division(RND)superfamily,notonlymediateintrinsic icalbasis,substrateprofiles,molecularregulation,andinhibi-
338 cmr.asm.org ClinicalMicrobiologyReviews April2015 Volume28 Number2
DrugEffluxinGram-NegativeBacteria
tion of MDR pumps. However, the development of clinically (17–19)andisavailableintheTransporterClassificationDatabase
usefuleffluxpumpinhibitorsand/ornewantibioticsthatcan (http://www.tcdb.org/). Transporter genes in hundreds of se-
bypass pump effects continues to be a challenge. Plasmid- quencedbacterialgenomesareclassifiedinIanPaulsen’sdatabase
borne efflux pump genes (including those for RND pumps) (20)foreachofthesegenomes(http://www.membranetransport
have increasingly been identified. This article highlights the .org/). Among many families of transporters, several contain
recentprogressobtainedfororganismsofclinicalsignificance, prominentmembersofeffluxtransporters:especiallyimportant
togetherwithmethodologicalconsiderationsforthecharacter- in bacteria are the RND, MFS (major facilitator superfamily),
izationofMDRpumps. MATE(multidrugandtoxiccompoundextrusion),SMR(small
multidrug resistance), and ABC (ATP-binding cassette) super-
INTRODUCTION familiesorfamilies.ABCtransportersutilizeATPhydrolysisasthe
Antibioticresistancehasemergedasamajorthreattopublic energy source, but all others are dependent on proton motive
force and are thus secondary transporters or proton/drug anti-
healthinthiscentury,asevidentfromglobalsurveillancedata
porters.
(1).Indeed,withtheancientoriginandwidespreadpresenceof
The transporters also differ in their subcellular organization.
diverseresistancegenes(2,3),themodernevolutionofresistance D
TheRNDpumps,whichareallexportersofdrugsandtoxiccat-
hasledtotheglobalemergenceandspreadofalargenumberof o
ions,arelocatedintheinnermembrane(IM)(cytoplasmicmem- w
resistantbacteriathatpossesssophisticatedgenotypesandpheno- brane) but must interact with the periplasmic adaptor protein n
typesagainstantibiotics.Thisphenomenonisaconsequenceof lo
(alsocalledmembranefusionprotein)andtheoutermembrane a
thenaturalselectionprocessinmicroorganismsandpromotion
(OM)channel,thusproducingatripartitecomplexspanningthe d
byhumanactivitiesoverthepast70yearsoftheantibioticera(4, e
IM, the periplasm, and the OM (represented by E. coli AcrAB- d
5).In2013,theU.S.CentersforDiseaseControlandPrevention
(6)listedcurrentresistancethreats,ofwhichmultidrug-resistant TolCandP.aeruginosaMexAB-OprM)(seethemulticomponent fro
pumpdepictedinFig.1).SomemembersoftheABCsuperfamily m
Gram-negativebacteriaconstitutealargeproportion(e.g.,Enter-
(e.g.,MacB),theMATEfamily(e.g.,MdtK),andeventheMFS
obacteriaceae, Acinetobacter, and Pseudomonas). Of the various h
(e.g.,EmrB)(allfromE.coli)alsoareorganizedinthismanner. t
molecularandbiochemicalmechanismsofresistancetoantibiot- tp
ics,activeeffluxofantibioticsinbacteriaplaysanimportantrole Thetripartitetransportersexcretedrugsdirectlyintotheexternal :/
mediumsothatthereentryofdrugsrequirestheslowtraversalof /c
in both intrinsic and acquired multidrug resistance (MDR) of m
theOM,aneffectivepermeabilitybarrier(21,22).Forthisreason,
clinicalrelevance.Italsointerplayswithotherresistancemecha- r
nisms,suchasthemembranepermeabilitybarrier,enzymaticin- thesepumpsarefarmoreefficientincreatingdetectableresistance .a
toantibiotics(especiallyAcrB,aconstitutiveRNDtransporterof s
activation/modificationofdrugs,and/orantibiotictargetchang- m
E. coli [9]) (see Gammaproteobacteria: Enterobacteriaceae, be-
es/protection,insignificantlyincreasingthelevelsandprofilesof .o
resistance. low).Incontrast,thepumpsthatarenotorganizedinthismanner rg
andexistassingle-componentor“singlet”pumpsintheIM(Fig. /
Energy-dependentdrugeffluxwasdiscoveredinthe1970s,ini-
1),includingthevastmajorityofMFSandSMRpumps,areless o
tiallywithP-glycoproteininmammaliancells(7)andlaterwith n
effectiveinproducingadetectabledecreaseinsusceptibility,be-
Tet proteins in Escherichia coli isolates resistant to the specific J
causethedrugmoleculesareexcretedonlyintotheperiplasmand a
antibioticclasstetracyclines(8).Thesubsequentdiscoveryinthe n
canspontaneouslydiffusebackintothecytosol,sincemostanti- u
early1990sofMDRpumpsinE.coliandPseudomonasaeruginosa, a
bioticsarerelativelylipophilicmoleculesthatcancrossthephos-
representedbytheresistance-nodulation-division(RND)super- ry
pholipidbilayerregionoftheIM.However,RNDpumps,which
familyexporters(9–13),hasmadeanimportantcontributionto 4
arethoughttocaptureantibioticsmostlyfromtheperiplasm(23, ,
our understanding of resistance mechanisms (14). Since then, 2
24),cancollaboratewiththesingletpumpsandthusincreasetheir
withrapidtechnologicaladvancesinbiochemistryandmolecular 0
efficacy(25,26). 1
biology,therehavebeenever-growingidentificationandcharac- 9
Themostdetailedinformationonthecontributionofvarious
terizationofMDRpumpsinnumerousbacterialspeciesofpublic b
pumpstodrugsusceptibilityisavailableforE.coliK-12,andTable y
healthconcern(e.g.,intheESKAPE[Enterococcusfaecium,Staph-
1listsdataonknownandpredictedmultidrugpumpsidentifiedin g
ylococcusaureus,Klebsiellapneumoniae,Acinetobacterbaumannii, u
theTransporterClassificationDatabasementionedabove.Anob- e
P.aeruginosa,andEnterobacterspecies]pathogens),whichcom- s
pellinglydemonstratetheirpredominantroleinclinicalsettings vious way to detect the contribution of individual pumps is to t
measuretheMICsofdrugsindefectivemutants.Thiswasdonein
(15,16).Meanwhile,effortsofscientistsledtotheunderstanding
2001 by Sulavik and coworkers (27) and showed that the RND
ofnotonlythestructuralandfunctionalbasisofthesedrugtrans-
transporter AcrB (in cooperation with its periplasmic and OM
portersbutalsotheirregulationandinhibition.Inthisreview,we
partnersAcrAandTolC)playsatrulypredominantroleinraising
aim to provide a comprehensive and up-to-date description of
theMIClevelsinawild-typestrain.Thisalsocreatesaproblem
efflux-mediatedantibioticresistanceinGram-negativebacteria.
because deletion of other pumps rarely produces detectable
changes in MICs in the presence of the active AcrB-AcrA-TolC
BIOCHEMISTRYANDGENETICSOFMULTIDRUGEFFLUX
system.Asimilarproblemwasreportedinastudy(28)examining
PUMPS
theMICvaluesofnearly4,000deletionmutantsofallnonessential
ClassesofEffluxPumps E. coli genes (the “Keio collection” [29]). Thus, although that
Becausetherearesomanydifferenteffluxtransporters,theonly studyshowedthatthefunctionsofmanymetabolicgeneshavean
feasiblewayfortheirclassificationistousephylogeneticgrouping, unsuspectedinfluenceondrugsensitivity,intermsoftransporter
based on protein sequences. Such a classification for all trans- genes, it essentially identified the effect of only the acrAB-tolC
porter proteins has been established by Milton Saier’s group complexandnothingelse.Onepossibleexceptionisthedeletion
April2015 Volume28 Number2 ClinicalMicrobiologyReviews cmr.asm.org 339
Lietal.
D
o
w
n
lo
a
d
e
d

f
r
o
FIG1LocationofdrugeffluxpumpsandpathwaysofdruginfluxandeffluxacrosstheOMandIMinGram-negativebacteria.Theinfluxofdrugs(shownas m
pills)throughtheOMoccursinoneormoreofthefollowingthreepathways:porinchannels(e.g.,OmpFofE.coliandOprFofP.aeruginosa),specificprotein h
channels(e.g.,CarOofA.baumanniiandOprDofP.aeruginosaforcarbapenems),andtheLPS-containingasymmetriclipidbilayerregion.Aftertheirentryinto tt
theperiplasmicspace,thedrugmoleculescanfurtherpenetratetheIMviadiffusion.However,thesedrugscanbeextrudedoutofthecellbyeffluxtransporters, p
:
whichexistaseithersingle-componentpumps(“singlet”;e.g.,Tetpumps)ormulticomponentpumps(e.g.,AcrAB-TolCandMexAB-OprMtripartiteefflux //
c
systemsthateachtypicallycontainapump,anOMchannelprotein[OMP],andanaccessorymembranefusionprotein[MFP]).Whilethesingletpumpsmay m
takeupthedrugfromthecytosolandtheperiplasmandfunctionwithporinsorothertypesofproteinchannelstomaketheeffluxprocesseffective,the r
multicomponentexporterscapturetheirsubstratesfromtheperiplasmandtheIManddirectlypumpthemintothemedium.Thecompetitionbetweentheinflux .a
andeffluxprocessesultimatelydeterminesthesteadystateofdrugmoleculesinbacterialcells.WiththelipophilicdrugmoleculesthatcrosstheOMslowlyorthe s
m
hydrophilicdrugsthatpenetratetheA.baumannii/P.aeruginosalow-permeabilityporins(i.e.,“slowporins”),theeffluxmechanismbecomeveryeffective,thus
.
beingabletoyieldMDR.Incontrast,withthelesshydrophobicandsmallerdrugmoleculesthatcanrapidlypenetrate,forexample,E.coliporins,effluxisnot o
effectivetocounteractdruginflux,thushardlydecreasingtheconcentrationsofthedruginthecell. rg
/

o
n

J
oftheycdZgene,whichproducedhypersusceptibilitytotetracy- RNDTransporters a
n
clineandmaycodeforanexporter.However,thisconclusionis AcrBofEscherichiacoli.TheconstitutivelyexpressedpumpAcrB u
a
notsupportedbyastudyfromtheCarolGrossgroup,whoquan- ofEscherichiacoliplaysamajorroleinraisingtheMICsofmost r
y
titated the growth phenotype of the same set of mutants in the antibiotics,duemostlytothefactthatitexistsastheAcrB-AcrA- 4
presence of sub-MICs of various drugs (30). This approach is TolCtripartitecomplexsothattheexporteddrugmoleculesend ,
2
moresensitivethanthedeterminationofMICvaluesand,indeed,
upintheexternalmedium,notintheperiplasm,andthuscannot 0
aspresentedinTable1,showedthatthedeletionofpracticallyall 1
easilyreenterthecellsexceptbycrossingtheeffectiveOMperme- 9
knownandsuspectedpumpsproduceshypersusceptibilitytoat
abilitybarrier(24).Therefore,theeffectivenessofRNDpumpsis b
leastoneagenttested.Theseresults,however,mustbeinterpreted y
intimatelytiedtothestrengthoftheOMbarrier;permeabilizing
withcare,sincethisapproachisverysensitiveandcouldproduce g
theOMdestroystheeffectofRNDpump-mediatedeffluxalmost u
false-positiveresultsinspiteofeffortstoavoidthem(Table1). e
aseffectivelyastheinactivationofthepumpitself(42,43).Avail- s
Acompletelydifferentapproachistheplasmid-basedover- t
ablereviewsonRNDpumpsincludethoseemphasizingthestruc-
expression of putative efflux genes. This analysis by Nishino
tureandmechanism(24,44–49),computationalapproaches(50),
and Yamaguchi (31) indeed detected efflux activity in those
rolesinsolventtolerance(51),andfunctionsotherthandrugre-
genes whose activity was difficult to detect by deletion-based
sistance(34,52–54).
approaches. However, these data do not tell us whether the
AcrB has been studied most intensively as the prototype of
pumps are functioning in the wild-type or even mutant cells,
although the level of expression of most pumps can be in- RNDpumps.Ithasanextremelywidespecificity,includingprac-
creasedbyregulatorysignals(seeRegulationofMultidrugEf- ticallyalltypesofantibacterialagents(exceptaminoglycosides),
fluxPumps,below). detergents,microbicides,dyes(Table1),freefattyacids(55),and
Reviews describing various types of efflux pumps include evensimplesolvents(56).Inareconstitutionassay(57),AcrBwas
thosewrittenbyPoole(32,33),Piddock(34–36),Paulsenetal. showntoalsoextrudemodifiedphospholipids.Acommonprop-
(37),Saierandothers(38),VanBambekeandothers(39),and erty of these AcrB substrates is the presence of a hydrophobic
Higgins(40).AreviewbyAlekshunandLevy(41)isuseful,as domain (24, 58). Such a wide specificity appeared surprising at
italsoemphasizesthecontributionofnoneffluxmechanismsof first.However,aspointedoutbyNeyfakh(59),thismaybeex-
resistance. pected.Thus,whenatypicalsolubleenzymecapturesitshydro-
340 cmr.asm.org ClinicalMicrobiologyReviews April2015 Volume28 Number2
DrugEffluxinGram-NegativeBacteria
philicsubstratefromtheaqueousmedium,theprocessrequires externalspace,theactivitieswereratherweak,anditseemslikely
the removal of the substrate molecule, already stabilized by its thatAcrD(andpossiblyalsootherRNDpumps)atleastprefersto
numeroushydrogen-bondinginteractionswiththesurrounding captureitssubstratesfromtheperiplasm.Interestingly,theaddi-
water.Stablebindinginthebindingsiteoftheenzymetherefore tionofAcrAwasnecessaryforthefunctionofAcrD.Becausethe
mustinvolveprecise,stronginteractionswiththeresiduesinthe assaydoesnotrequirethejuxtapositionofvesicles,AcrAislikely
site,inordertoovercomethisenergybarrier,anditrequiresthat tostimulatedirectlythefunctionofAcrD(andAcrB)bysimply
thesiteissmallandcarefullydesignedtobindstringentlyonlyone bindingtothetransporter.
substratespecies.However,withthemultidrugpumpsthatcap- Sincereconstitutionassaysarequitecumbersome,methodsfor
turedrugswithsizeablehydrophobicdomains,thedrugsarenot quantitative,real-timedeterminationofpumpingactivityinin-
stronglystabilizedintheaqueousenvironment,astheirpresence tact cells were needed. Fluorescent probes [e.g., N-phenyl-1-
involvesalargeentropiccostaccompanyingtheorderingofthe naphthylamine, ethidium bromide, and 2-(4-dimethylamino)s-
surrounding water molecules. Hence, the drug binding to the tyryl-N-ethylpyridinium iodide] were preloaded into bacterial
transporterdoesnotrequirethebindingsitetobesmall,tight,and cellsdeenergizedbyuncouplers,andeffluxwasmonitoredbyflu-
stringent.Itcanbeverylargeandcanthusaccommodatealarge orescenceafterreenergizationbyaddinganenergysource,suchas
D
rangeofsubstrateswithsmalldecreasesinthebindingfreeenergy. glucose(60,61).Itisdifficulttoperformassaysofthistypeina
o
Forbiochemicalstudiesofanytransporters,transportstudies reproduciblemannerbecausesomeuncouplersremainafterre- w
ofmembranevesiclesareusuallythepreferredapproach.Never- energization.Anoptimized,semiquantitativemethodforE.coli n
lo
theless, with AcrB, this approach was not fruitful, presumably usingthefluorescentdyeNileredwasreportedin2010(62).A a
becausemostoftheligandstransportedarelipophilicandcannot majorstepintheintact-cellassayofAcrBwasthereal-timeassay d
e
beaccumulatedintheintravesicularspaceduetotheirreadiness ofcephalosporineffluxinE.coliachievedbyNaganoandNikaido d

incrossingthephospholipidbilayerdomainofthemembrane.It in 2009 (63). Those authors measured spectrophotometrically fr
isalsopossiblethattheabsoluterateoftransportdoesnotneedto cephalosporinhydrolysisinintactcellsbyaperiplasmic(cid:2)-lacta- om
behighbecauseofthepresenceoftheOMbarrier,andthisim- mase.BycomparingthehydrolysisratewiththeV andK of
max m h
pedesthedetectionoftransportinvesicles.Thus,thefirstmajor theenzyme,thoseauthorscalculatedtheperiplasmicconcentra- t
t
p
advance had to rely on the reconstitution of purified AcrB into tionsofthecephalosporins,overcomingthemostseriousprob- :
/
proteoliposomes, accomplished by Zgurskaya and Nikaido in lems in intact-cell assays of efflux. They then obtained the ex- /c
m
1999(57).Inordertocircumventtheproblemofthespontaneous pected influx rate (V ) of the drug across the OM from the
in r
diffusionofmostligandsacrossthelipidbilayer,thisstudyusedan permeabilitycoefficientobtainedfromuncoupler-poisonedcells .a
innovativeapproachrelyingontheeffluxoffluorescentlylabeled andfromthedifferenceintheexternalandperiplasmicconcen- s
m
phospholipids into empty “acceptor” vesicles and detected the trationsofthedrug.ThedifferencebetweenV andtheobserved
in .o
efflux of conventional ligands through competition with phos- hydrolysisratethencorrespondstotherateofefflux.Whenthe r
g
pholipidefflux.Inthisway,drugssuchascloxacillin,erythromy- effluxrateofnitrocefinwasplottedagainsttheperiplasmiccon- /

cin,novobiocin,andfusidicacid(butcuriouslynotchloramphen- centrations, a Michaelis-Menten-type saturation curve was ob- o
n
icol)aswellasvariousbileacidswereshowntocompeteagainst tained,showingtheV (0.024nmol/mg/s)andK (5(cid:4)M)ofthe
max m J
phospholipid efflux. Furthermore, the half-maximal concentra- AcrB-catalyzedeffluxprocessforthefirsttime.Sincetheexpres- a
n
tion for inhibition was lowest for bile acid taurocholate ((cid:3)15 sionofAcrBwasincreasedseveralfoldinthestrainused,weesti- u
(cid:4)M),suggestingthatthepropertiesofAcrBwereoptimizedfor matethattheV is(cid:3)6pmol/mg/sinwild-typeE.coliK-12.This a
max ry
theexclusionofbilesalts,majortoxiccomponentsinthemam- assaywasusedwithconventionalcephalosporinsandpenicillins
4
malianintestine,thenormalhabitatforE.coli.Interestingly,the (63–65);onesurprisingfindingwasthatasigmoidalkineticswas ,

additionofAcrAtotheaqueousphase(inthepresenceofMg2(cid:5)) often observed in the plots of velocity versus periplasmic drug 2
0
stronglystimulatedphospholipidtransport:becauselipidshadto concentration,suggestingpositivecooperativity.Recently,asig- 1
9
betransportedfromonevesicletoanother,wehypothesizedthat moidal kinetics was observed for the AcrB-catalyzed efflux of a
b
AcrAmayactbybringingthetwovesiclestogether.Thisapproach compoundofaverydifferentnature,L-arginine-(cid:2)-naphthylam- y

also established that AcrB was a proton/drug antiporter, as the ide(A.Kinana,A.V.Vargiu,andH.Nikaido,unpublisheddata), g
u
transmembrane pH gradient was dissipated accompanying the indicating that this is a common feature of the AcrB-catalyzed e
s
fluxofligands. transportprocess. t
AsimilarreconstitutionassaywassuccessfullyusedforE.coli A major advance in the study of AcrB was made when Mu-
AcrD(23),anAcrBhomologthatalsoworkswithAcrAandTolC rakamietal.(66)solvedthecrystalstructureoftrimericAcrBin
asatripartitetransporter.Thisstudyisimportant,asAcrDtrans- 2002. This symmetric structure showed that each protomer of
ports aminoglycosides, which are very hydrophilic and not ex- AcrBcontainedalargeperiplasmicdomain,aspredictedfromthe
pectedtodiffusespontaneouslyacrossthelipidbilayer.Thus,a primarysequence.Furthermore,theperiplasmicdomainwasseen
conventionalaccumulationassayusingradiolabeledaminoglyco- tohavealargecleftfacingthesurroundingperiplasm.Although
sides indeed proved their accumulation in proteoliposomes. binding of ligands to the area close to the cleft was shown by
Whenstreptomycinwasaddedasthesubstratetoeitherthemore cocrystallization (67–69), this observation did not immediately
acidic,intravesicularspacecorrespondingtotheperiplasmorthe suggestthemechanismsofdrugextrusion(seebelow).Thenext
morealkalineexternalspacecorrespondingtothecytosol,pump- bigadvanceinourunderstandingofAcrBstructureandfunction
ingactivity(asdetectedbythefluxofprotons)wasobservedonly came with crystallographic analysis of the asymmetric trimer
in the former case, showing clearly that the pump captures its structure, where each protomer takes a unique conformation
substrateonlyintheperiplasm.Althoughotheraminoglycosides slightly different from that of its neighbor, elucidated in three
appearedtostimulatethepumpactivityevenwhenaddedtothe laboratories (Fig. 2 ) 7(0–72). The work by Murakami and co-
April2015 Volume28 Number2 ClinicalMicrobiologyReviews cmr.asm.org 341
Lietal.
R,
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Othersubstrate(s)orinformation(reference) Pentoses(1010) CCCP,NAL,TSA(139);CTLM(15)Uncouplers(15);pentoses(1010)HO,MIT,NAL,UV22irradiation(1011) Pentoses(1010) Ironcitrate(inSalmonella)(1012) BindsCHL(1013) Pentoses(1010)
dAgent(s)thatinhibitedgrowth ACR,AMP,AZM,AZT,BAC,BLE,BS,CER,CHIR,CHL,CHO,CIP,CLR,CPZ,DEO,DOX,EB,ERY,FUA,MEC,MIN,NAL,NIT,NOR,NOV,OXA,PUR,SDS,SPT,SPR,STG,TCH,TCS,TET,TMP,TRX,VERPARPHL,VERFUA CIP,SDS BAT,CSD,NAL,PHL CHO,ERY,SMZACR,BAC,CHL,EB EB,SXT AZM,GEN,NIT,OXA,SMT,SPR CEC,SMZ,TET NIT,OXA,SPR,TMPEB,AMX,BAC,MEC,NAL,PARBAC,CAZ,MEC,MTX,PUR,SDS,TLMATM,EBACTFUA,SPTAMK,CHL,CHO,CSD,DEO,ERY,FOX,NOV,PER,PMB,TET,VERNonewithagentstested AMXACR,BAC,CAR,CHIR,CSD,DOX,EB,FOX,GFF,MIT,SDS,STRAZT,BLE,CAL,CHIR,CHL,CIP,CPZ,INH,OXA,SMZ,SPR,TETAMP,DEO htDownloaded from
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aE.coli cOverexpressionphenotype(agent[s],foldincreaseinMIC) (cid:6)ACR,16;BAC,32;CHL,8;CV,8;DEO,(cid:6)(cid:6)32;DOR,64;ERY,32;NAL,4;NOR,(cid:6)8;NOV,64;R6G,64;SDS,8;TMP,(cid:6)32;TPP,16 (cid:6)(cid:6)DEO,32;KAN,2;NOV,4;SDS,8ACR,8;DEO,4;DOX,2;SDS,4CV,2;DEO,4;DOR,8;EB,4;ERY,8;R6G,16;SDS,4;TPP,4(cid:6)DEO,32;FOF,2;NAL,2;NOR,2;NOV,16;SDS,4(cid:6)DEO,32;FOF,2;NAL,2;NOR,2;NOV,16;SDS,4 ACR,2;FOF,4;KAN,2;TET4ACR,8;CHL,16;DOX,4;EB,4;NOR,8;TET,2;TMP,4;TPP,4DEO,32;PAR,2;R6G,2;SDS,2 BAC,2;SDS,2 FOF,4 on January 4//cmr.asm.org/
antsof in CLO,32;UR,6 , 201
umpmut ddecrease 8;CIP,4;256;ERY,OV,64;P8;TPP,25 9 by g
sofprovenandputativeeffluxp bDeletionphenotype(agent[s],folMIC) ACR,128;AMP,4;BAC,32;CHL,(cid:6)(cid:6)(cid:6)2;DAU,128;DEO,2;EB,(cid:6)FUA,128;MTX,8;NAL,2;N(cid:6)64;R6G,512;SDS,128;TET, NochangeNochangeNochange Nochange Nochange BC,4;EB,4 Nochange Nochange Nochange uest
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ABLE1Phen milyandefflune NDacrB acrDacrFyhiV(mdtF) mdtB mdtC FSbcrcmr(cmlA,mdfA)emrB emrD emrY yajRyceByceE(mdtG)yceL(mdtH)ydeB(marC)ydhCyebQ yegB(mdtD) yidY(mdtL)yieO(hsrA) yjiO(mdtM) ynfM
T Fage R M
342 cmr.asm.org ClinicalMicrobiologyReviews April2015 Volume28 Number2
DrugEffluxinGram-NegativeBacteria
MATE(MOP)mdtK(ydhE,BAC,2;CHL,2;DEO,32;DOR,8;EB,2;CPZ,FUA,NOR,PUR,STRnorM)FOF,2;NOR,8;PAR,4;TMP,4;TPP,32yeeOACR,BAC,BIC,CAR,DOR,PUR,VAN,VER SMR(DMT)emrEEB,4;PAR,8ACR,16;BAC,2;EB,8;PAR,2ACR,EB,FOF,PAREB(1014);PAR(1015);ERY(155);cationicosmoprotectants(159)ydgE(mdtI)DEO,4;SDS,2ACR,BAC,CHL,DEO,EB,FUA,MTX,Spermidine(1016)PMB,PUR,TRXydgF(mdtJ)DEO,4;SDS,2CHO,CSD,ERY,NAL,VERSpermidine(1016) ABCmacBNochangeERY,8BAT,BIC,BS,CEC,EB,FOX,NIT,SDS,TCH LysEargOAMP,BS,GFF,INH,NAL,PHL,TMPArginine(1017) UnknownycdZNOR,SDS(notTET)TET,1.7-to2.5-folddecreaseinMICcomparedtothewildtype(28) aAbbreviations:ACR,acriflavine;ACT,actinomycinD;AMK,amikacin;AMP,ampicillin;AMX,amoxicillin;ATM,aztreonam;AZM,azithromycin;AZT,azidothymidine;BAC,benzalkoniumchloride;BAT,bacitracin;BIC,bicyclomycin;BLE,bleomycin;BS,bilesalts;CAL,calcofluor;CAR,carbenicillin;CAZ,ceftazidime;CCCP,carbonylcyanidem-chlorophenylhydrazone;CEC,cefaclor;CER,cerulenin;CHIR,CHIR-900(anLpxCinhibitor);CHL,chloramphenicol;CHO,cholate;CIP,ciprofloxacin;CLO,cloxacillin;CLR,clarithromycin;CPZ,chlorpromazine;CSD,cefsulodin;CV,crystalviolet;DAU,daunomycin;DEO,deoxycholate;DOR,doxorubicin;DOX,doxycycline;EB,ethidiumbromide;ERY,erythromycin;FOF,fosfomycin;FOX,cefoxitin;FUA,fusidicacid;GEN,gentamicin;GFF,glufosfomycin(fosfomycinplusglucose-6-P);INH,isoniazid;KAN,kanamycin;MEC,amdinocillin;MIN,minocycline;MIT,mitomycin;MTX,methotrexate;NAL,nalidixicacid;NIT,nitrofurantoin;NOR,norfloxacin;NOV,novobiocin;OXA,oxacillin;PAR,paraquat(methylviologen);PER,peroxide;PHL,phleomycin;PMB,polymyxinB;PUR,puromycin;R6G,rhodamine6G;SDS,sodiumdodecylsulfate;SMT,sulfamonomethoxine;SMZ,sulfamethizole;SPR,spiramycin;SPT,spectinomycin;STG,streptonigrin;STR,streptomycin;SXT,trimethoprim-sulfamethoxazole;TCH,taurocholate;TCS,triclosan;TDC,taurodeoxycholate;TET,tetracycline;TLM,thiolactomycin;TMP,trimethoprim;TPP,tetraphenylphosphonium;TRX,TritonX-100;TSA,tetrachlorosalicylanilide;VAN,vancomycin;VER,verapamil.bSeereference27.NumbersaftertheabbreviationfortheagentnameindicatethefolddecreaseinMICincomparisonwiththewild-typestrain.(cid:7)cSeereference31.NumbersaftertheabbreviationfortheagentnameindicatesthefoldincreaseinMICincomparisonwiththeacrABparentstrain.(cid:8)(cid:9)dOnlytheantimicrobialagents,dyes,anddetergentsthatsignificantlyinhibitedthegrowthofmutantsmorethanthatofthewildtype,usuallyleadingtogrowthscoresof2(30),arelisted.Furthermore,weconfirmedthattheinhibitionwasreproduciblebyexaminingthedataobtainedwithdifferentconcentrationsofthesameagent. on January 4, 2019 by guesthttp://cmr.asm.org/Downloaded from
April2015 Volume28 Number2 ClinicalMicrobiologyReviews cmr.asm.org 343
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FIG2DrugtransportmechanismofAcrB.ShownistheasymmetriccrystalstructureofAcrB(ProteinDataBankaccessionnumber2DRD),viewedfromoutside m
thecell,withthetopportioncutoffforclarity.Conformationalcyclingof3AcrBprotomers,inaccess(blue),binding(red),andextrusion(green),isseenby .o
cocrystallizationofAcrBwithitssubstrateminocycline,showninayellowstickmodel. rg
/

o
n
workers(70)wasespeciallyimportant,becausetheysucceededin stratessuchasmacrolides,rifampin,andadimerofdoxorubicin
J
cocrystallizingAcrBwiththesubstrateminocyclineordoxorubi- bindtoamoreproximalbindingsiteintheaccessprotomer,pre- a
n
cin. In both cases, the substrates were seen in a predominantly sumablybeforetheireventualmovementtothedistalpocketcon- u
a
hydrophobicpocketwithintheperiplasmicdomain,nowcalled comitantwiththeconformationalchangeoftheproteinintothe
r
y
the distal binding pocket, close to the center of the trimer and bindingprotomer.Interestingly,thisproximalbindingsitesur-
4
locatedinoneparticularprotomer,calledthebindingprotomer. rounded by residues Asp566, Phe664, Phe666, Glu673, Arg717, ,

2
Thepresenceofthreeconformationallydifferentprotomers,the andAsn719overlapsmostlytheperiplasmicbindingsiteidenti-
0
access,binding,andextrusionprotomers(Fig.2)(44),suggesteda fiedbyYuandcollaboratorsseveralyearsearlier(67)inasymmet- 1
9
functionally rotating mechanism, in which each protomer goes ricAcrBcrystalstructure.Yuandassociatesfurthershowedthe
b
through a succession of conformational alterations. The distal importanceofthisbindingsitebysite-directedmutagenesis,find- y

binding pocket becomes collapsed in the extrusion protomer, ingthataPhe666Alamutation,forexample,resultsinadrastic g
u
consistentwiththemovementofthedrugtotheexitgatecloseto decreaseofresistancetoawiderangeofsubstrates(67).Thein- e
s
theendoftheTolCchannel.Thisconceptofconformationalcy- volvementofresiduessurroundingthissitewasalsoshownbythe t
clingorfunctionalrotationwasthensubstantiatedbythefinding factthattheirCys-substitutedmutantswerestronglylabeledbyan
that disulfide cross-linking of nearby residues, although appar- AcrB substrate, boron-dipyrromethene (BODIPY)-maleimide
entlyoccurringinonlyoneortwoprotomers,nearlycompletely (79).Anothersymmetricalcrystalstructurecontainingasubstrate
inactivatedthetrimericcomplex(73).(TheAcrBhomologMexB atthispositionhasadeoxycholatemolecule(80).Theimportance
ofP.aeruginosahasbeencrystallizedwithout[74]andwith[75] ofthisproximalbindingpocketwasfurtheremphasizedbyrecent
anaddedinhibitor,apyridopyrimidinederivative.)Inasimilar studies(81,82).ThepreferenceofAcrDfor(cid:2)-lactamscontaining
vein,whentheAcrBtrimerwasproducedasacovalentlylinked multipleanionicgroups,suchascarbenicillin,sulbenicillin,and
singleprotein,andonlyoneprotomericunitwasinactivatedinthe aztreonam,isessentiallyduetotheresidueswithintheproximal
proton translocation pathway, the entire trimeric complex be- pocket (81), while a region in the MexY aminoglycoside pump
cameinactive(76).Furthermore,whentheCysresidueswerein- thatcorrespondstoaproximalbindingpocketofAcrBplaysarole
troducedintoonlyoneoftheprotomericunits,theircross-linking inaminoglycosiderecognitionandexport(82).
immediately inactivated the function of AcrB trimers, showing Yet another binding site for drugs was identified within the
thatinactivationwasnotduetoafailureofthetrimericassembly. centralcavityofthetrimerbycocrystallization(67,83).Possibly,
Morerecently,twolaboratories(77,78)showedthatlargesub- theinitialbindinghereisfollowedbytheeventualtranslocationof
344 cmr.asm.org ClinicalMicrobiologyReviews April2015 Volume28 Number2
DrugEffluxinGram-NegativeBacteria
thesubstratestothedistalbindingpocket,perhapsthroughthe “bind”withasignificantlylowerbindingenergytothelowerpart
“vestibules”betweentheprotomers(84).Althoughthefunctional ofthepocket,whichwecalleda“cave”(Fig.3)(91).
significanceofthisbindingsitecouldnotbeascertainedbysite- Togetfurtherinsightsintothebinding-and-effluxprocess,we
directedmutagenesis,symmetriccocrystalsofAcrBwithdrugsin examinedpotentialcompetitionbetweensubstrates.Ithadbeen
thecentralcavityhavebeenreportedforampicillin(68)andlin- nearlyimpossibletoshowcompetitionamongsubstratesofAcrB
ezolid(85). byusinganMICassay(92).Still,withareal-timeeffluxassaywith
Dastidaretal.carriedoutthefirstefforttousesubstratecom- thedyeNilered,weshowedthatdoxorubicin,minocycline,and
petitionforcovalentlabelingofselectedresiduesfortheelucida- other tetracyclines as well as tetraphenylphosphonium, but not
tionofthepathofthedrugmoleculeswithinthelargeperiplasmic chloramphenicol, macrolides, deoxycholate, nafcillin, or novo-
domain of AcrB (86). Some residues of Haemophilus influenzae biocin, inhibited dye efflux (62). Also, when a real-time efflux
AcrB were converted to Cys and were labeled with fluorescein assayofnitrocefin(63)wasused,weshowedstronginhibitionby
maleimide.ThelabelingofAla288Cys(correspondingtoGly290 minocycline,predictedtobindtotheupperpartofthepocket,like
ofE.coliAcrBandclosetothedistalbindingsite)wasdecreasedby nitrocefin (91). In contrast, a substrate that is not predicted to
thepresenceofallsubstratestested,exceptethidium.Wefollowed bindtothispartofthepocket,i.e.,chloramphenicol,didnotin-
D
uponthisworkbyselecting48residuesthatlieonthepresumed hibitnitrocefinefflux,andactually,therewassomehintofstim-
o
path(s)ofthedrugs,convertingeachresiduetoCys,andlabeling ulationinstead(91).Thelatterphenomenonisdiscussedinmore w
theCysresidueinintactcellswithahydrophobic,covalent-label- detailbelow. n
lo
ing probe, BODIPY-maleimide (79). Residues outside the pre- Dockingprograms,however,havebeenoptimizedbyusingthe a
dictedpathwerenotlabeledatall,evenwhentheywerelocatedin bindingofsmall,hydrophilicsubstratesmostlytothebindingsites d
e
themiddleofhydrophobicpatches.Incontrast,mostofthetested withinenzymes.Bindingofhydrophobicoramphiphilicligands d

residuesinthedistalbindingpocketwerestronglylabeled,aswere tothelargebindingpocketsoftransportersispredictedtooccurin fr
o
theresiduesliningtheproximalpocketaswellastheentranceand significantlydifferentways(59).Thus,weexaminedindetailthe m
the bottom of the large external cleft between two subdomains bindingof9substrates,2inhibitors,and2nonsubstratestothe
h
(PC1andPC2[66]).Finally,byusingbulkycovalent-labelingre- distalbindingpocketofAcrBbyextensiveMDsimulations(93). t
t
p
agents,withsomeresidues,wehavebeenableto“clog”thesub- Thisintroducedtwomajorimprovementsoverthedockingap- :
/
strate path so that the efflux of a substrate, Nile red, could be proach.First,watermoleculesnowbecameapartofthesystemso /c
m
blocked. thattheinteractionofamphiphilicandmorehydrophilicligands
r
Thisstudyreinforcedtheimportanceofthedrugbindingto couldbepredictedinamuchmorerealisticmanner.Second,the .a
thedistalbindingpocketasamajorstepinefflux.Site-directed distalbindingpocketiscomposedofresiduesthatareonseveral s
m
mutagenesis of Phe residues in this pocket (87) indicated that loopsegmentsinarelativelylooselyconstructedareaofthepro-
.
o
theseresiduesareimportantforefflux,withthePhe610Alamuta- tein,somovementandrotationsofthechainswereexpected.In- r
g
tionshowingthemostwidespreadeffectonmanysubstrates.A deed,withmanyligands,therewasanextensivealterationinthe /

moleculardynamics(MD)simulationstudyofthismutantpro- shape of the binding site to better accommodate diverse sub- o
n
tein(88)revealedthatasubstrate,doxorubicin,stillboundtothe strates.Interestingly,someofthose“cavebinders”inthedocking
J
pocketwithastrongaffinity;theinterpretationoftheseresultsis approachleftthelowerareaandwerefoundtofavortheupper a
n
describedbelow.Site-directedmutagenesisbasedonthesequence areaofthepocket,althoughthebindingappearedtobeweak. u
a
differencebetweenAcrBandMexB,whichshowdifferentprofi- Chloramphenicolwasfoundtoslightlyacceleratetheeffluxof
r
y
cienciesinmacrolideefflux,ledtothediscoveryoftheimportance nitrocefin(91).Thiswasconfirmedbyasubsequentcarefulstudy,
4
ofGly616inAcrBforthisfunction(89);interestingly,thisresidue and it was found that solvents such as benzene or cyclohexane ,

2
isapartoftheGly-richloop(alsocalledtheswitchloop),which produced much more pronounced stimulation of nitrocefin ef-
0
separatesthedistalpocketfromtheproximalpocket(77)andis flux(94).MDsimulationssuggestedthatbenzeneinteractspri- 1
9
thoughttobecriticalfortranslocationofthesubstrates,especially marilywiththePhe-richhydrophobicdomainthatcomprisesthe
b
large molecules such as macrolides. More recently, Eicher et al. lowerportionofthebindingpocketandnotwiththeuppersub- y

showed the coupling of remote alternating-access transport pocket that binds minocycline or nitrocefin (94). Interestingly, g
u
mechanismsforprotonsandAcrBsubstratesthroughamecha- thelowerportionofthebindingpocketiswherethehydropho- e
s
nisminvolvingtworemotealternating-accessconformationalcy- bicpartoftheinhibitorD13-9001bindstightlyandwasnamed t
cleswithineachpromoter(90). a “hydrophobic trap” by Nakashima et al. (75) (see Efflux
Althoughthesemutagenesisstudiesarevaluable,theydonot PumpInhibitors,below).Furthermore,recentMDsimulations
tell us how various substrates bind to the AcrB transporter. As showedthatothereffluxpumpinhibitors(EPIs),suchasphenylal-
statedabove,onlyafewcrystalstructuresofdrug-AcrBcomplexes anine-arginine-(cid:2)-naphthylamide (PA(cid:2)N), 1-(1-naphthylmethyl)-pi-
are currently available. Thus, computational analysis of drug- perazine(NMP),andthenew,potentinhibitorMBX2319(95),allbind
AcrB interactions was first initiated with the docking software tightlytothehydrophobictrapandtherebydistorttheshapeof
AutodockVina(91).VariousknownsubstratesofAcrB,including therestofthepocket,closingthecrevicewhereminocyclineor
minocycline,dockedtotheupperpart(closertotheexitgate)of nitrocefin becomes bound (96). These observations suggest the
thedistalbindingsite,whichcontainsacharacteristiccrevice(Fig. following.(i)Thedistalbindingpocketisverylarge,anddifferent
3).Thisiswhereminocyclineanddoxorubicinboundinthecrys- ligands prefer different areas of the pocket for binding. Thus, a
talstructures(70).Cefazolin,anonsubstrate(63),gratifyinglydid “typical” substrate, like minocycline or nitrocefin, which has a
notbindtothebindingpocket.However,othersubstratesfailedto numberofhydrophilicgroups,tendstobindtotheupper“crev-
bindtothisupperportionofthepocket;asanexample,chloram- ice”area,whichisrichinhydrophilicandchargedresiduesand
phenicol and solvents such as cyclohexane were predicted to was indeed shown to be involved in substrate binding (93). In
April2015 Volume28 Number2 ClinicalMicrobiologyReviews cmr.asm.org 345
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FIG3InteractionofdrugsubstratesandtheAcrB-bindingprotomeranalyzedwithAutodockVinadockingsoftware.Substratesareshowntobindtoeitherthe
.
upperpart(groovebinder)(doxorubicin[A]andtetracycline[B])orthelowerpart(cavebinder)(chloramphenicol[C]andcyclohexane[D])ofthedistal o
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bindingsite.(Modifiedfromreference91.) g
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contrast,forhydrophobicligandssuchascyclohexaneorchlor- Afterthebindingofthesubstratetothedistalbindingpocketin
J
amphenicol,bindingtothisareaisdifficult.(ii)Atightinteraction the binding protomer, the proton(s) must come in from the a
n
with the hydrophobic trap distorts the structure of the crevice, periplasm to bind to the Asp residue(s) in the transmembrane u
a
inhibitingtheeffluxoftypicalsubstrates.(iii)Alooseinteraction domain,causingtheconformationoftheproteintochangeinto
r
y
withthehydrophobictrap,ontheotherhand,mayenhancethe theextrusionprotomer,therebysqueezingoutthesubstratetothe
4
effluxoftypicalsubstrates,byeitherfacilitatingtheinteractionof exitgatebythecollapseofthepocket.Asp407andAsp408appear ,

2
suchsubstrateswiththepocket,speedingupthesequenceofcon- tobeessentialfortheenergytransductionofAcrB,togetherwith
0
formationalchangesneededfortheexportofsubstrates,orboth. Lys940andArg971(105)aswellasThr978(106).Inthebinding 1
9
Inanycase,theinteractionbetweentheAcrBtransporterandits protomer,bothAsp407andAsp408appeartobedeprotonated,as
b
substrates/inhibitors/enhancersappearsquitecomplex.Inassess- thepresumablyprotonatedLys940sidechainissituatedbetween y

ingthebindingofdrugstothepocketoftransporterssuchasAcrB, the two Asp side chains. In the extrusion protomer, Lys940 is g
u
wenowrealizethatbindingfollowsthesameprincipleselucidated movedawayfromtheAspresiduesandnowfacestheThr978side e
s
bythepioneeringearlycrystallographicstudiesbyBrennanand chain(107).ThecarboxylgroupofAsp408wasindeedshownto t
coworkers(forexample,seereferences97and98),carriedoutby haveanunusuallyhighpK of7.4,whichwouldhelpinthefacile
a
using soluble regulators of MDR pumps, such as QacR, rather bindingandreleaseoftheprotonunderphysiologicalconditions
thanthepumpsthemselves. (107).ArecentMDsimulationstudy(108)suggeststhatinthe
Computersimulationhasnowbecomeanimportantapproach extrusionprotomer,Asp408becomesprotonated,butAsp407re-
forstudyingthemechanismofAcrBfunction.Themovementofa mainsdeprotonated.Inthisscheme,thetranslocationofonepro-
substrate,doxorubicin,fromthedistalbindingpockettoaposi- tonacrosstheIMwouldbesufficienttocausetheconformational
tionclosetotheexitgate,accompanyingtheclosureofthepocket, changes in AcrB, resulting in the extrusion of the drug mole-
was shown by a targeted MD simulation (99). Large substrates, cule(s).ThefoldingandassemblyoftheAcrBtrimerwerestudied
found in the proximal binding pocket in AcrB cocrystals (77), mainlybyWeiandassociates,whoshowedthatthefoldingofthe
movedsubstantiallyinthedirectionofthedistalbindingpocket monomeric unit precedes trimerization (109) and analyzed the
duringMDsimulation(100).Movementofwatermoleculeswas functionofaprotrudingloopthatinsertsdeeplyintotheneigh-
analyzed by simulation (101, 102), and coarse-grained models boringsubunit(110).
wereusedtoanalyzetheconformationaltransitions(103)aswell In 2007, when AcrB was crystallized without amplification
asthedrugpathways(104)withinAcrB. fromE.coli,thesymmetriccrystalswerefoundtocontainasmall
346 cmr.asm.org ClinicalMicrobiologyReviews April2015 Volume28 Number2