日本化学療法学会雑誌第56巻第4号

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1 Key words I

2 Number of xenobiotic transporters Pseudomonas aeruginosa Escherichia coli Bacillus subtilis R MFS SMR MATE ABC Mycobacterium tuberculosis Fig.. Numberofputativedrugefluxgenesidentifiedby gemonicanalysis. Putativedrugefluxsystemsareclassifiedintofivetypes. Thedatabaseispostedat[htp:/ II β macab III

3 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.

4 Table. DrugresistanceprofilesofefluxsystemsinE.coli Type Eflux System CP TC MINO EM -(Δ acrb) ABC MacAB MF Fsr MdfA MdtG MdtH Bcr EmrKY EmrAB EmrD MdtL MdtM R AcrAB MdtABC AcrD AcrEF MdtEF SMR EmrE MdtIJ SugE MATEMdtK NA NFLX ENX KM FOM DXR MICforacrBmutant-overproducingefluxsystems(μg/mL) NOV TMP ACR BENZ DOC , , , , , , , , , , , ,250 > > 00> > 0,000 > , > 0, > > 0, , , , , , MPIPC MCIPC NFPC CBPC SBPC CXM CMD CAM OL AZM FO BI CTP 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.

5 Fig.. Identifieddrugefluxsystemsandregulationbysignaltransductionsystems. Weidentified20drugefluxsystemsofE.colibyusinggenomicinformation.Wealsodiscoveredanovelresistancemechanism,the two-componentsignaltransductionsystem,whichregulatestheseefluxsystems. β IV Salmonella enterica

6 3,39 3,350 (kb) tolc 89.7% (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% (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% (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.

7 EfluxSystem -(ΔacrB) AcrAB AcrD AcrEF MdtABC MdsABC EmrAB MdfA MdtK MacAB EM NOV Table2. DrugresistanceprofilesofdrugefluxsystemsinSalmonela TC CP 8 MICfortheΔacrBmutant-overproducingefluxsystems(μg/mL) NA 8 NFLX DXR ACR CV EBR MB R6G TPP > 28> > 52> 52> > > 52> 52> BENZ SDS DOC 28 20,000 6 > 52> 0,000 > 52 0,000 6 > 52 0, , ,000 28> 0, , , ,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 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> 52> 52> 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 Fig.6. RoleofdrugefluxsystemsinSalmonelavirulence. ThisfigureshowsthesurvivalrateofmiceinfectedwithSalmo nelastrains.balb/cmicewereinoculatedoralywith0 5 col onyformingunitofdiferentsalmonelastrainsasindicated. mdsabc mds Salmonella macab β

8 Fig.7. Transportofdrugsandiron-chelatorbydrugefluxsystems. TheexpressionacrDandmdtABCdrugefluxgenesareregulatedbyFur.FurcontrolsironhomeostasisinmostGram-negative bacteria.wefoundthattheseefluxsystemstransportnotonlydrugsbutalsothesiderophoreenterobactin. V

9 Salmonella enterica Escherichia coli Escherichia coli Escherichia coli β Escherichia coli Salmonella Escherichia coli Salmonella enterica Escherichia coli evga Escherichia coli Escherichia coli

10 Escherichia coli Escherichia coli Escherichia coli

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