Key words I
Number of xenobiotic transporters 25 20 5 0 5 0 Pseudomonas aeruginosa Escherichia coli Bacillus subtilis R MFS SMR MATE ABC Mycobacterium tuberculosis Fig.. Numberofputativedrugefluxgenesidentifiedby gemonicanalysis. Putativedrugefluxsystemsareclassifiedintofivetypes. Thedatabaseispostedat[htp:/www.membranetransport.org/]. II β macab III
R OM ABC MF SMR MATE Drug H H H Na / H IM ATP Drug ADP Pi Drug Drug Drug Drug ATP dependent Multi-component 2~ TMs TMs 2 TMs Fig.2. Classificationofdrugefluxsystems. Drugefluxsystemscanbeclassifiedintofivecategoriesbasedontheirstructureandcouplingenergies. ABC:ATPbindingcassete,R:resistancenodulationcel-division,MF:majorfacilitator,SMR:smalmultidrugresistance,MATE:multidrugandtoxiccompoundextrusion Fig.3. SubstratesoftheAcrAB-TolCdrugefluxsystem ine.coli. AcrAB-TolCsystem enhancesmultidrugresistanceofe.coli.thestructuresofcompoundsrecognizedbythissystem areshownin Figure.NumbershowedthechangesMIClevelsandtheirincreasedratiowhenAcrAB-TolCisover-producedintheacrBmutant.
Table. DrugresistanceprofilesofefluxsystemsinE.coli Type Eflux System CP TC MINO EM -(Δ acrb) 0.390.39 0.20 ABC MacAB 0.390.39 0.20 MF Fsr 0.390.39 0.20 MdfA 6.25 0.20 MdtG 0.390.39 0.20 MdtH 0.390.39 0.20 Bcr 0.393.3 0.20 EmrKY 0.390.39 0.20 EmrAB 0.390.39 0.20 EmrD 0.390.39 0.20 MdtL 0.39 0.20 MdtM 0.39 0.20 R AcrAB 3.33.3 3.3 MdtABC 0.390.39 0.20 AcrD 0.39 0.20 AcrEF 6.253.3 3.3 MdtEF 0.390.39 0.20 SMR EmrE 0.390.39 0.20 MdtIJ 0.390.39 0.20 SugE 0.390.39 0.20 MATEMdtK 0.39 0.20 NA NFLX ENX KM FOM 25 0.025 0.05 6.25 3.3 0.20 0.05 6.25 3.3 0.025 0.05 6.25 6.25 3.3 0.05 0.0 6.25 3.3 0.025 0.05 25 6.25 3.3 0.05 0.05 6.25 003.3 0.0 0.39 6.25 3.3 0.05 0.05 6.25 3.3 3.3 0.05 0.052.5 3.3 2003.3 0.0 0.39 6.25 25 0.025 0.05 6.25 3.3 0.025 0.05 6.25 3.3 3.3 0.20 0.39 6.25 3.3 DXR MICforacrBmutant-overproducingefluxsystems(μg/mL) NOV TMP ACR BENZ DOC 3.3 3.3 2.5 3.3,250 0.39 3.3 3.3 2.5 3.3,250 0.39 3.3 6.25 2.5 3.3,250 2.5 3.3 00 3.3,250 0.39 3.3 3.3 2.5 3.3 2,500 3.3 3.3 2.5 3.3,250 3.3 3.3 25 3.3,250 3.3 3.3 2.5 3.3 0,000 0.39 3.3 3.3 2.5 3.3 0,000 0.39 3.3 3.3 2.5 6.25,250 3.3 3.3 2.5 3.3,250 3.3 3.3 50 3.3,250 > 200 00 > 00> 00 00 > 0,000 > 00 3.3 25 3.3 2.5 6.25 0,000 3.3 3.3 6.25 3.3 2.5 3.3 > 0,000 2.5 > 200 50 00 00 50 > 0,000 50 25 3.3 2.5 6.25 5,000 2.5 3.3 3.3 200 6.25,250 0.39 3.3 3.3 2.5 3.3 5,000 3.3 3.3 2.5 3.3,250 25 2.5 2.5 3.3 0,000 0.39 MPIPC MCIPC NFPC CBPC SBPC CXM CMD CAM 00 6.25 2.5 00 2.5 200 6.25 50 200 25 6.25 25 2.5 50 OL AZM FO BI CTP 0.20 0.0 50 0.39 25 20 0.20 0.0 2.5 00 2.5 2.5 0.20 0.0 00 0.20 0.0 0.20 0.0 0.39 0.20 0.0 0.39 0.20 0.39 0.20 0.0 0.20 0.0 60 0.20 0.0 Abbreviations:CP,chloramphenicol;TC,tetracycline;MINO,minocycline;EM,erythromycin;NA,nalidixicacid;NFLX,norfloxacin;ENX,enoxacin;KM,kanamycin;FOM,fosfomycin;DXR,doxorubicin;NOV, novobiocin;tmp,trimethoprim;acr,acriflavine;benz,benzalkonium;doc,deoxycholate;mpipc,oxacilin;mcipc,cloxacilin;nfpc,nafcilin;cbpc,carbenicilin;sbpc,sulbenicilin;cxm,cefuroxime; CMD,cefamandole;CAM,clarithromycin;OL,oleandomycin;AZM,azithromycin;FO,fosmidomycin;BI,bicyclomycin;CTP,cetylpyridinium.,notdetermined.
Fig.. Identifieddrugefluxsystemsandregulationbysignaltransductionsystems. Weidentified20drugefluxsystemsofE.colibyusinggenomicinformation.Wealsodiscoveredanovelresistancemechanism,the two-componentsignaltransductionsystem,whichregulatestheseefluxsystems. β IV Salmonella enterica
3,39 3,350 (kb) tolc 89.7% 529 530 53 532 533 (kb) acrb 9.6% acra 9.7% 2,59 2,595 2,596 2,597 (kb) acrd 9.3% 3,56 3,562 3,563 3,56 3,565 (kb) acre 87.5% acrf 88.7% 2,2 2,25 2,26 2,27 2,28 2,29 2,220 2,22 2,222 (kb) mdta 82.7% mdtb 9.6% mdtc 9.7% 393 39 395 396 397 398 (kb) mdsc ( STM0350 ) Nonexistent in E.coli 5.5% to OprM in P. aeruginosa 2,962 2,963 2,96 (kb) mdsb (STM035 ) Nonexistent in E.coli 62.3% to MexF in P. aeruginosa mdsa (STM0352 ) Nonexistent in E.coli 3.8% to MexE in P. aeruginosa emra 89.7% emrb 95.7% 938 939 90 (kb) mdfa 90.6%,502,503 (kb) mdtk 92.3% Gene encoding Outer Membrane Protein Gene encoding Membrane Fusion Protein Gene encoding R-type Efflux Protein Gene encoding MFS-type Efflux Protein Gene encoding MATE-type Efflux Protein Gene encoding ABC-type Efflux Protein,09,020,02 (kb) maca 83.2% macb 83.3% Fig.5. DrugefluxgenesencodedintheSalmonelaentericaserovarTyphimurium genome. Chromosomalpositionsofgenescodingforputativedrugefluxsystems,outermembranepro teins,andmembranefusionproteinsareindicatedbythekb(kilobasepair)inthes.entericase rovartyphimurium strainlt2genome.arowscorespondtothelengthsanddirectionsofthe genes.aminoacididentitybetweenhomologousproteinsins.entericaande.coliareindicated asnumbersunderthegenenames.
EfluxSystem -(ΔacrB) AcrAB AcrD AcrEF MdtABC MdsABC EmrAB MdfA MdtK MacAB EM 28 28 8 NOV 2 256 6 28 6 8 6 2 2 2 Table2. DrugresistanceprofilesofdrugefluxsystemsinSalmonela TC CP 8 MICfortheΔacrBmutant-overproducingefluxsystems(μg/mL) NA 8 NFLX DXR ACR CV EBR MB R6G TPP 0.06 8 32 2 6 6 8 32 0.3 > 28> 52 28 > 52> 52> 52 52 0.06 8 32 2 6 6 8 32 0.3 > 28 52 32 > 52> 52> 52 52 0.06 8 32 2 6 6 8 32 0.06 8 28 6 28 256 6 6 0.06 8 32 2 6 6 6 32 0.3 32 32 2 6 6 8 32 0.3 6 256 2 6 6 8 32 0.06 8 32 2 6 6 8 32 BENZ SDS DOC 28 20,000 6 > 52> 0,000 > 52 0,000 6 > 52 0,000 52 0,000 8 256 20,000 28> 0,000 28 20,000 28 20,000 28 20,000 Abbreviations:EM,erythromycin;NOV,novobiocin;TC,tetracycline;CP,chloramphenicol;NA,nalidixicacid;NFLX,norfloxacin;DXR, doxorubicin;acr,acriflavine;cv,crystalviolet;ebr,ethidium bromide;mb,methyleneblue;r6g,rhodamine6g;tpp,tetraphenylphosphonium bromide;benz,benzalkonium chloride;sds,sodium dodecylsulfate;doc,sodium deoxycholate. Strain Table3. SusceptibilityofSalmoneladrugtransporter-deletedstrainstotoxiccompounds EM NOV Wild-type 28 256 ΔacrAB acref acrd mdtabc mdsabc emrab mdfamdtkmacab TC NA NFLX DXR 0.06 ACR MIC(μg/mL) CV EBR MB R6G TPP BENZ 0.25 > 28> 52 256 > 52> 52> 52> 52 32 2 32 6 8 32 6 SDS DOC > 52> 0,000 Abbreviations:EM,erythromycin;NOV,novobiocin;TC,tetracycline;NA,nalidixicacid;NFLX,norfloxacin;DXR,doxorubicin;ACR,acriflavine;CV,crystalviolet;EBR,ethidium bromide;mb,methyleneblue;r6g,rhodamine6g;tpp,tetraphenylphosphonium bromide; BENZ,benzalkonium chloride;sds,sodium dodecylsulfate;doc,sodium deoxycholate. MICdeterminationswererepeatedatleastthreetimes. 32 33 Fig.6. RoleofdrugefluxsystemsinSalmonelavirulence. ThisfigureshowsthesurvivalrateofmiceinfectedwithSalmo nelastrains.balb/cmicewereinoculatedoralywith0 5 col onyformingunitofdiferentsalmonelastrainsasindicated. mdsabc mds Salmonella macab β
Fig.7. Transportofdrugsandiron-chelatorbydrugefluxsystems. TheexpressionacrDandmdtABCdrugefluxgenesareregulatedbyFur.FurcontrolsironhomeostasisinmostGram-negative bacteria.wefoundthattheseefluxsystemstransportnotonlydrugsbutalsothesiderophoreenterobactin. V
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