TheWhole-CellMethylerythritolPhosphate(MEP)Pathway-SelectiveAntibacterialScreeningAssay,describedin[1],utilizesageneticallyengineeredstrainofSalmonellatyphimurium(CT31-7d)thatcanutilizeeithertheMEPorthemevalonate(MVA)pathwaysinordertoproduceisopentenyldiphosphate(IPP)anddimethylallyldiphosphate(DMAPP),thetwoessentialprecursorsforallisoprenoids.IntheabsenceofarABInoseandMVA,theMEPpathwayisutilizedforgrowth.WhensupplementedwitharabinoseandMVA,theengineeredMVApathwayisactivated.CompoundsinhibitingMEPpathwaygrowthwithnoeffectonMVApathwaygrowthareselectivefortheMEPpathway.ValidatedusingknownMEPpathwayinhibitorsandotherantibacterials,thescreeningplatformismoresensitivetoMEPpathwayinhibitorsthanwild-typeS.typhimurium.Antibacterialstargetingotherpathwaysdemonstratesimilarminimuminhibitoryconcentrations(MICs)forbothMEPandMVApathwaygrowth.
TheWhole-CellMEPPathway-SelectiveAntibacterialScreeningAssayisperformedina96wellplateandconsistsoftwostages:
1.PrimaryAntibacterialScreen:ThisscreenusesourengineeredcelllineundergrowthconditionswithonlytheMEPpathwayactivetoidentifyinitialantibacterial“hits”.Compoundsareaddedina96-wellplateandscreenedinduplicate.Thisscreentestsasingleconcentration(usually100µg/mL)orauserdeterminedamountagainsteachpathway.Weareabletoscreenupto80compoundsperplateinduplicate.
2.SecondaryMEPPathway-SelectivityScreen:Antibacterial“hits”aresubjectedtodoseresponseanalysisevaluatingMEPgrowthinoneplateandMVAinasecondtodetermineMICsforbothgrowthconditions.UsingCT31-7danddifferentgrowthconditionsallowustodetermineiftheantibacterialhitdisplaysspecificitytowardtheMEPpathway.Dose-responseexperimentsareperformedon4compoundsperplateinduplicateforthesecondaryscreen.Fosmidomycin,aknownMEPpathwayinhibitor,isincludedinbothscreensasapositivecontrol.Theconcentrationused(33μM)isinsufficienttokillwild-typebacteria,butiseffectiveagainstCT31-7dduetoheightenedMEPpathwaysensitivity. |
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