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ARTICLE
doi:10.1038/nature14098
A new antibiotic kills pathogens without
detectable resistance
LoseeL.Ling1*,TanjaSchneider2,3*,AaronJ.Peoples1,AmyL.Spoering1,InaEngels2,3,BrianP.Conlon4,AnnaMueller2,3,
TillF.Scha¨berle3,5,DallasE.Hughes1,SlavaEpstein6,MichaelJones7,LinosLazarides7,VictoriaA.Steadman7,DouglasR.Cohen1,
CintiaR.Felix1,K.AshleyFetterman1,WilliamP.Millett1,AnthonyG.Nitti1,AshleyM.Zullo1,ChaoChen4&KimLewis4
Antibioticresistanceisspreadingfasterthantheintroductionofnewcompoundsintoclinicalpractice,causingapublic
healthcrisis.Mostantibioticswereproducedbyscreeningsoilmicroorganisms,butthislimitedresourceofcultivable
bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this
platform.Unculturedbacteriamakeupapproximately99%ofallspeciesinexternalenvironments,andareanuntapped
sourceofnewantibiotics.Wedevelopedseveralmethodstogrowunculturedorganismsbycultivationinsituorbyusing
specificgrowthfactors.Herewereportanewantibioticthatwetermteixobactin,discoveredinascreenofuncultured
bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of
peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus
aureusorMycobacteriumtuberculosisresistanttoteixobactin.Thepropertiesofthiscompoundsuggestapathtowards
developingantibioticsthatarelikelytoavoiddevelopmentofresistance.
Widespreadintroductionofantibioticsinthe1940s,beginningwith factorsthroughthechambersenablesgrowthofunculturedbacteriain
penicillin1,2andstreptomycin3,transformedmedicine,providingeffec- theirnaturalenvironment.Thegrowthrecoverybythismethodap-
tivecuresforthemostprevalentdiseasesofthetime.Resistancedevelop- proaches50%,ascomparedto1%ofcellsfromsoilthatwillgrowona
mentlimitstheusefullifespanofantibioticsandresultsintherequirement nutrientPetridish10.Onceacolonyisproduced,asubstantialnumber
foraconstantintroductionofnewcompounds4,5.However,antimicro- ofunculturedisolatesareabletogrowinvitro14.Extractsfrom10,000
bialdrugdiscoveryisuniquelydifficult6,primarilyduetopoorpenetra- isolatesobtainedbygrowthiniChipswerescreenedforantimicrobial
tionofcompoundsintobacterialcells.Naturalproductsevolvedtobreach activityonplatesoverlaidwithS.aureus.Anextractfromanewspecies
thepenetrationbarriersoftargetbacteria,andmostantibioticsintro- ofb-proteobacteriaprovisionallynamedEleftheriaterraeshowedgood
ducedintotheclinicwerediscoveredbyscreeningcultivablesoilmicro- activity.ThegenomeofE.terraewassequenced(SupplementaryDis-
organisms.Overminingofthislimitedresourcebythe1960sbrought cussion).Basedon16SrDNAandinsilicoDNA/DNAhybridization,
anendtotheinitialeraofantibioticdiscovery7.Syntheticapproaches thisorganismbelongstoanewgenusrelatedtoAquabacteria(Extended
wereunabletoreplacenaturalproducts6. DataFig.2,SupplementaryDiscussion).ThisgroupofGram-negative
Approximately99%ofallspeciesinexternalenvironmentsareun- organismsisnotknowntoproduceantibiotics.Apartiallypurifiedac-
cultured(donotgrowunderlaboratoryconditions),andareaprom- tivefractioncontainedacompoundwithamolecularmassof1,242Da
isingsourceofnewantibiotics8.Wedevelopedseveralmethodstogrow determinedbymassspectrometry,whichwasnotreportedinavailable
unculturedorganismsbycultivationintheirnaturalenvironment9,10, databases.Thecompoundwasisolatedandacompletestereochemical
orbyusingspecificgrowthfactorssuchasiron-chelatingsiderophores11. assignmenthasbeenmadebasedonNMRandadvancedMarfey’sanal-
Unculturedorganismshaverecentlybeenreportedtoproduceinterest- ysis(Fig.1,ExtendedDataFigs3and4andSupplementaryDiscussion).
ingcompoundswithnewstructures/modesofaction—lassomycin,an Thismolecule,whichwenamedteixobactin,isanunusualdepsipep-
inhibitoroftheessentialmycobacterialproteaseClpP1P2C1(ref.12); tide which contains enduracididine, methylphenylalanine, and four
anddiversesecondarymetabolitespresentinamarinespongeTheonella D-aminoacids.Thebiosyntheticgenecluster(GenBankaccessionnum-
swinhoeiwhichareactuallymadebyanunculturedsymbioticEntothe- berKP006601)wasidentifiedusingahomologysearch(Supplementary
onellasp.13. Discussion).Itconsistsoftwolargenon-ribosomalpeptidesynthetase
Herewereportthediscoveryofanewcellwallinhibitor,teixobactin, (NRPS)-coding genes, which we named txo1 and txo2, respectively
fromascreenofunculturedbacteriagrownindiffusionchambersinsitu. (Fig.1).Inaccordancewiththeco-linearityrule,11modulesareencoded.
Theinsilicopredictedadenylationdomainspecificityperfectlymatches
Identificationofteixobactin theaminoacidorderofteixobactin(Fig.1),andallowedustopredict
Amultichanneldevice,theiChip10,wasusedtosimultaneouslyisolate thebiosyntheticpathway(ExtendedDataFig.5).
andgrowunculturedbacteria.Asampleofsoilisdilutedsothatapprox-
imatelyonebacterialcellisdeliveredtoagivenchannel,afterwhich Resistanceandmechanismofaction
thedeviceiscoveredwithtwosemi-permeablemembranesandplaced TeixobactinhadexcellentactivityagainstGram-positivepathogens,
backinthesoil(ExtendedDataFig.1).Diffusionofnutrientsandgrowth includingdrug-resistantstrains(Table1andExtendedDataTable1).
1NovoBioticPharmaceuticals,Cambridge,Massachusetts02138,USA.2InstituteofMedicalMicrobiology,ImmunologyandParasitology—PharmaceuticalMicrobiologySection,UniversityofBonn,Bonn
53115,Germany.3GermanCentreforInfectionResearch(DZIF),PartnerSiteBonn-Cologne,53115Bonn,Germany.4AntimicrobialDiscoveryCenter,NortheasternUniversity,DepartmentofBiology,
Boston,Massachusetts02115,USA.5InstituteforPharmaceuticalBiology,UniversityofBonn,Bonn53115,Germany.6DepartmentofBiology,NortheasternUniversity,Boston,Massachusetts02115,USA.
7Selcia,Ongar,EssexCM50GS,UK.
*Theseauthorscontributedequallytothiswork.
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©2015Macmillan Publishers Limited. All rights reserved
RESEARCH ARTICLE
a txo1 txo2 Figure1|Thestructureofteixobactinandthe
Genes predictedbiosyntheticgenecluster. a,Thetwo
NRPSgenes,thecatalyticdomainstheyencode,
Domains A MT T C A T C A T C A T C A T C A T C A T C A T C A T C A T C A T C TE TE
andtheaminoacidsincorporatedbythe
Amino acids NmPhe Ile Ser Gln Ile Ile Ser Thr Ala End Ile respectivemodules.Domains:A,adenylation;
b C,condensation;MT,methylation(of
NmPhe Ile Ser Gln Ile Ile Ser Thr Ala End Ile phenylalanine);T,thiolation(carrier);andTE,
c O NH2 thioesterase(Ile-Thrringclosure).NmPhe,
H O H O H OH N-methylatedphenylalanine.b,Schematic
HN N N N N N structureofteixobactin.TheN-methylationof
OO OHH O H OHN O thefirstphenylalanineiscatalysedbythe
HN O methyltransferasedomaininmodule1.Thering
NH O O closurebetweenthelastisoleucineandthreonineis
O NHHN catalysedbythethioesterasedomainsduring
moleculeoff-loading,resultinginteixobactin.
O
c,Teixobactinstructure.
HN NH
HN
Potencyagainstmostspecies,includingdifficult-to-treatenterococci RNAandprotein(Fig.3a).Thissuggestedthatteixobactinisanew
andM.tuberculosiswasbelow1mgml21.Teixobactinwasexception- peptidoglycansynthesisinhibitor.
allyactiveagainstClostridiumdifficileandBacillusanthracis(minimal Resistancehasnotdevelopedtothiscompound,suggestingthatthe
inhibitoryconcentration(MIC)of5and20ngml21,respectively).Tei- targetisnotaprotein.Theessentiallackofresistancedevelopment
xobactinhadexcellentbactericidalactivityagainstS.aureus(Fig.2a), throughmutationshasbeendescribedforvancomycinwhichbinds
wassuperiortovancomycininkillinglateexponentialphasepopula- lipidII,theprecursorofpeptidoglycan.Wereasonedthatteixobactin
tions(Fig.2b),andretainedbactericidalactivityagainstintermediate couldbeactingagainstthesametarget.Treatmentofwholecellsof
resistanceS.aureus(VISA)(ExtendedDataFig.6a).Notethatfrequent S.aureuswithteixobactin(1–53MIC)resultedinsignificantaccumu-
clinicalfailureinpatientswithS.aureusMRSAtreatedwithvancomycin lationofthesolublecellwallprecursorundecaprenyl-N-acetylmuramic
hasbeenlinkedtothepoorbactericidalactivityofthiscompound15,16. acid-pentapeptide (UDP-MurNAc-pentapeptide), similar to the
TeixobactinwasineffectiveagainstmostGram-negativebacteria,but vancomycin-treated control cells (Fig. 3b), showing that one of the
showedgoodactivityagainstastrainofE.coliasmB1withadefective membrane-associatedstepsofpeptidoglycanbiosynthesisisblocked.
outermembranepermeabilitybarrier(Table1). Teixobactininhibitedpeptidoglycanbiosynthesisreactionsinvitroin
WewereunabletoobtainmutantsofS.aureusorM.tuberculosis adose-dependentmannerwitheitherlipidI,lipidIIorundecaprenyl-
resistanttoteixobactinevenwhenplatingonmediawithalowdose pyrophosphate (Fig. 3c) as a substrate. Quantitative analysis of the
(43MIC)ofthecompound.SerialpassageofS.aureusinthepresence
ofsub-MIClevelsofteixobactinoveraperiodof27daysfailedtopro-
a b
duce resistant mutants as well(Fig. 2d, Supplementary Discussion). 10 10
Thisusuallypointstoanon-specificmodeofaction,withaccompany- 9 9
iNcoewnofrafIgdmeHyectsprot/3oooxtTounifc3nSdtithd.aeyenta.serduHhrarmoHtoeewuwieonespefe.dGvlTasen2pbreo,eecixtcleheoiilfnliabisxcceaoaiomcttbryt1aopino0cloyt0fritsaanimtctcrgihtooaimanoncdtnglii2lnvnyoit1otfoiy(ntttteohhahixnxeeiibodhmcibiittdgaeayijhdcdoaterisgsnnybta,onidiwonttoshsbesyteeiennmstxtidehsaasmemDtotefiidNmcnp)peA.aedaTlp.itahtihItinnes-- Log(c.f.u. per ml)1045678 COVTeaoxinanxccotroibollmialncytcinin Log(c.f.u. per ml)1045678 COoxanctriolliln
doglycan,buthadvirtuallynoeffectonlabelincorporationintoDNA, 3 3 Vancomycin
Teixobactin
2 2
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Time (h) Time (h)
c d
Table1|Activityofteixobactinagainstpathogenicmicroorganisms Con Ox Van Teix 256
Organismandgenotype TeixobactinMIC(mgml21) 128 Teixobactin
S.aureus(MSSA) 0.25 C 64 Ofloxacin
S.aureus110%serum 0.25 n MI 32
S.aureus(MRSA) 0.25 e i 16
g
EEnntteerrooccooccccuussffaaeeccaiulims((VVRREE)) 00..55 chan 8
Streptococcuspneumoniae(penicillinR) #0.03 old 4
Streptococcuspyogenes 0.06 F 2
Streptococcusagalactiae 0.12 1
Viridansgroupstreptococci 0.12
0.5
B.anthracis #0.06 0 5 10 15 20 25
Clostridiumdifficile 0.005 Time (days)
Propionibacteriumacnes 0.08 Figure2|Time-dependentkillingofpathogensbyteixobactin.a,b,S.aureus
M.tuberculosisH37Rv 0.125
weregrowntoearly(a),andlate(b)exponentialphaseandchallengedwith
Haemophilusinfluenzae 4
antibiotics.Dataarerepresentativeof3independentexperiments6s.d.
Moraxellacatarrhalis 2
c,Teixobactintreatmentresultedinlysis.Thefigureisrepresentativeof
Escherichiacoli 25
Escherichiacoli(asmB1) 2.5 3independentexperiments.d,Resistanceacquisitionduringserialpassaging
Pseudomonasaeruginosa .32 inthepresenceofsub-MIClevelsofantimicrobials.Theyaxisisthehighest
Klebsiellapneumoniae .32 concentrationthecellsgrewinduringpassaging.Forofloxacin,2563MIC
wasthehighestconcentrationtested.Thefigureisrepresentativeof3
TheMICwasdeterminedbybrothmicrodilution.MSSA,methicillin-sensitiveS.aureus;VRE,
vancomycin-resistantenterococci. independentexperiments.
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ARTICLE RESEARCH
a b 1,149.675 d Figure3|Teixobactinbindstocellwall
6,000
110200 00..68 Intens. (a.u.) 12345,,,,,000000000000000 -PP-PCC5555 upmInrraciedcocirurnopremoso(roRarltseNi.coAunal),ao,Irf3mHb3Hpi-oals-ectyuthncoytimhfneteiesd(iexipsnorieobnta(eDScit.nNian),uA(ar)Tne,uEd3sHI.3XH-)-on
Percentage incorporation 468000 Ciprofloxacin Rifampicin Vancomycin Erythromycin A260 00..24 UDP-M10u,0rN00Acm-p/eznt1a,p2e5p0tide Lipid II(Lipid IILipid I-Lac)D gcb(e(e4werxlauylpr3hlctssehio)trtM.resirmoCaebImmaiCeaptnryei)rnsd,cto)seivf.nw6la(Dopni(xta2secha.tpod3cattmi.einadbMiyrxo,(eco8IgIiCmbnnl3yat)ec(rc2awaMatnicn3eneIs)rClealoMwu)tfu,la1Ias4rsCrei3fdid)aanecmaMdatcneseuprIdpcCmmieconiinu(nndgtlaerreodteniylotsinn
c 200 DNA RNPAeptidoglycan Protein 0U0TEnItXre (a2t.e5d× M1IC0) VANT i(m1T0Ee2×I 0X( mM (5iInC×) )MIC)30TEIXe (1× M40IC) MTEoIXlaLipid III:rl ipraidtio 0:1 T 2:1 1:1 UmttoperfeeiDanxtashoPttsaeeb-pdsMacpece(eput1lciltr0ntiNwdr.3oeaUAmlaMlcnfe-ptttpIrerrCeereyanc)ttaurtceaesredpasilotlneasmrpdnwtUeidicndeaDvrteteaePowndu-fcaMsSbose.ymduiadrtyauhNecsrneiAecntupiocfse(-inVeawtdkrAiotbNhlsy.)-
CM
atm/z1,149.675.Theexperimentisrepresentative
TEIX:substrate 0.25:1 0.5:1 1:1 CW
100 molar ratio 2:1 5:1 10:1 OM ooff3teiixnodbeapcetnindeonntpexrepceurirmsoernctos.ncs,uTmhiengefrfeecatctions.
90 Experimentswereperformedin3biological
80 replicatesanddataarepresentedasmean6s.d.
med (%) 6700 T dpu,Crifoimedpcleelxlwfoarlmlparteiocunrosofrtse.ixBoinbdacintignowfittehixobactin
Product for 345000 WTA ilcishpirindodmiincatatetoregmdraebmdyi)aa.terTeshd(euvicfsitgiiboulnreeooifnstrtheheperatemhseionnu-tlnaattyivoeerfof
20 PG
twoindependentexperiments.e,Amodelof
10
CW teixobactintargetingandresistance.The
MurG FemX PBP2 YbjG teixobactinproducerisaGram-negativebacterium
Substrate Lipid I Lipid II Lipid II C55PP CM protectedfromthiscompoundbyexporting
Product Lipid II Lipid II-Gly C55PP C55P itacrosstheoutermembranepermeability
barrier(upperpanel).IntargetGram-positive
organismslackinganoutermembrane,the
targetsarereadilyaccessibleontheoutside
whereteixobactinbindsprecursorsof
peptidoglycan(PG)andWTA.CM,cytoplasmic
membrane;CW,cellwall;OM,outermembrane;
T,teixobactin.
MurG-,FemX-,andPBP2-catalysedreactionsusingradiolabelledsub- contributingtotheexcellentlyticandkillingactivityofthisantibiotic.
strates,showedanalmostcompleteinhibitionatatwofoldmolarexcess Teixobactinwasalsoabletobindundecaprenyl-pyrophosphate,but
ofteixobactinwithrespecttothelipidsubstrate(Fig.3c).Theaddition not undecaprenyl-phosphate (Fig. 3d and Extended Data Table 2).
of purified lipid II prevented teixobactinfrom inhibitinggrowth of AlthoughteixobactinefficientlybindslipidIinvitro,thisisprobably
S.aureus(ExtendedDataTable2).Theseexperimentsshowedthat lesssignificantforantimicrobialactivity,asthisistheintracellularform
teixobactinspecificallyinteractswiththepeptidoglycanprecursor,rather oftheprecursor,unlikesurface-exposedlipidIIandtheundecaprenyl-
thaninterferingwiththeactivityofoneoftheenzymes.Inorderto PP-GlcNAcWTAprecursors(Fig.3eandExtendedDataFig.7).Bind-
evaluatetheminimalmotifrequiredforhighaffinitybindingofteix- ingtothetargetprimarilyreliesontheinteractionoftheantibioticwith
obactin,thedirectinteractionwithseveralundecaprenyl-coupledcell thepyrophosphatemoiety,andthefirstsugarmoietyattachedtothelipid
envelopeprecursorswasinvestigated.Purifiedprecursorswereincu- carrier,ashigherconcentrationsofteixobactinwererequiredtocomple-
batedwithteixobactinatdifferentmolarratios,followedbyextraction telyinhibittheYbjG-catalysedmonophosphorylationofundecaprenyl-
andsubsequentthin-layerchromatographyanalysis(Fig.3d).Inagree- pyrophosphate,involvedintherecyclingprocessoftheessentiallipid
mentwiththeresultsobtainedfromtheinvitroexperiments,lipidIand carrier(Fig.3candExtendedDataFig.7).Corroboratingthisresult,a
lipidIIwerefullytrappedinastablecomplexthatpreventedextraction tenfoldhigherconcentrationofundecaprenyl-pyrophosphatewasre-
ofthelipidfromthereactionmixtureinthepresenceofatwofoldmolar quiredtoantagonizetheantimicrobialactivityofteixobactincompared
excessoftheantibiotic,leadingtotheformationofa2:1stoichiometric tolipidII(ExtendedDataTable2).Theexactnatureofthisfirstsugar
complex.Teixobactinwasactiveagainstvancomycin-resistantenter- isthereforenotimportant,explainingwhyteixobactinisactiveagainst
ococcithathavemodifiedlipidII(lipidII-D-Ala-D-LacorlipidII-D- M.tuberculosis,whereitprobablybindstodecaprenyl-coupledlipid
Ala-D-SerinsteadoflipidII-D-Ala-D-Ala)17–19.Thissuggestedthat,unlike intermediatesofpeptidoglycanandarabinogalactan.Teixobactinisalso
vancomycin,teixobactinisabletobindtothesemodifiedformsoflipid likelytobindtoprenyl-PP-sugarintermediatesofcapsularpolysacchar-
II.Indeed,teixobactinboundtolipidII-D-Ala-D-LacandlipidII-D-Ala- idebiosynthesiswhichisimportantforvirulenceinstaphylococci22and
D-Ser(ExtendedDataFig.6b).Moreover,teixobactinefficientlybound whoseinhibitionofbiosynthesisislethalinstreptococci23.
tothewallteichoicacid(WTA)precursorundecaprenyl-PP-GlcNAc
(lipid III). Although WTA is not essential per se, inhibition of late Invivoefficacy
membrane-boundWTAbiosynthesisstepsislethalduetoaccumulation Giventheattractivemodeofactionofthiscompound,weinvestigated
oftoxicintermediates20.Furthermore,teichoicacidsanchorautolysins, itspotentialasatherapeutic.Thecompoundretaineditspotencyin
preventinguncontrolledhydrolysisofpeptidoglycan21.Inhibitionof thepresenceofserum,wasstable,andhadgoodmicrosomalstability
teichoicacidsynthesisbyteixobactinwouldhelpliberateautolysins, andlowtoxicity(SupplementaryDiscussion).Thepharmacokinetic
00 MONTH 2015 | VOL 000 | NATURE | 3
©2015Macmillan Publishers Limited. All rights reserved
RESEARCH ARTICLE
a 100 *** *** *** *** andshowedgoodefficacyaswell(Fig.4b).Teixobactinwasalsohighly
efficaciousinmiceinfectedwithStreptococcuspneumoniae,causinga
al 80 6log10reductionofc.f.u.inlungs(Fig.4c).
e surviv 60 * Discussion
ag Thisstudy,aswellaspreviouswork12,13,24suggeststhatneworganisms
nt 40
ce suchasunculturedbacteriaarelikelytoharbournewantimicrobials25.
er
P Thisisconsistentwithresistancemechanismsinsoilbacteriabeingstrat-
20
ifiedbyphylogeny,withhorizontaltransmissionlimited26(ascompared
0 topathogens)andthepatternofantibioticproductioncorrelatingwith
Infecctioonntrol Va0n.5comy5cin 0.1 0.25Teixo0.5bacti1n 5 rpelsaitsftoarnmceo.Efnxpaltouirtainlgpruondcuuclttudrreudgbdacistecroivaeirsyli7k.eTleyitxoorbeavcivtienthiseaWpraoxmmiasn-
(mg per kg) (mg per kg) ingtherapeuticcandidate;itiseffectiveagainstdrug-resistantpatho-
b
10 gensinanumberofanimalmodelsofinfection.Bindingofteixobactin
9 toWTAprecursorcontributestoefficientlysisandkilling,duetodiges-
h) 8 tionofthecellwallbyliberatedautolysins.Thisisakintotheactionof
hig anothernaturalproductwithexcellentkillingability,acyldepsipeptide,
er t 7 whichconvertstheClpPproteaseintoanon-specifichydrolasethat
p
u. 6 digeststhecell27.Theseexamplesshowthatnaturalproductsevolved
g(c.f.10 5 toexploittheinherentweaknessesofbacteria28,andadditionalcom-
Lo 4 poundsthatsubvertimportantenzymesintokillingdevicesarelikely
tobediscovered.Teixobactinbindstomultipletargets,noneofwhich
3
isaprotein(Fig.3eandExtendedDataFig.7).Polyprenyl-coupledcell
Icnfoentcrtiolo n2 hcIonfnterctoil o2n6 h 0.5 V(ma1n.g0c poemr2. yk5cgin) 5.0 0.5 T(m1.e0igx opbe2.ar5 cktgin)5.0 esointdivceeaoltoftpGacerkap2mr8.e-Tcpuhoressitotairvrsge,esbtuaoccfhteteraiisxaloaipbniaddcrtIeiInp,,raterhseeernpetyaardnoilp‘yAhacochscpilehlseasstihbe-eleseulo’gnfaortrhmaenootiiuebttiy--
c 10 ofthesemolecules,ishighlyconservedamongeubacteria.Theproducer
isaGram-negativebacterium,anditsoutermembranewillprotectit
9
fromre-entryofthecompound(Fig.3eandExtendedDataFig.7).
g) 8
un Thissuggeststhattheproducerdoesnotemployanalternativepath-
er l 7 wayforcellwallsynthesisthatwouldprotectitfromteixobactin,and
p
g(c.f.u. 10 56 wfrohmichhootrhiezrobnatacltetrriaancsomuildssbioonrroofwa.Rreessiissttaanncceecmouecldhaenveisnmtuafrlloymemsoemrgee
Lo 4 soilbacterium,andgiventhehighlyconservedteixobactinbindingmotif,
thiswouldlikelytaketheformofanantibioticmodifyingenzyme.How-
3
ever,althoughdeterminantscodingforenzymesattackingfrequently
Icnfoentcrtiolo n2 h1A0 momxgi ciplleirn kg 0.5 Teixo1bacti2n.5 (mg pe5r kg) 10 fttoheiuexynodbaraaecntutininbkiisonteoivcwesnnsulfecoshrsatchsoembr-malarocetnavmtahnsacnoormvaaymncciionnm.oTgylhcyiecnor.seAicdefetnestralyirtesdciinsoctmorovmderuoecnd-,
Figure4|Teixobactinisefficaciousinthreemousemodelsofinfection. tionintotheclinic,ittook30yearsforvancomycinresistancetoappear29.
a,Singledosetreatment(i.v.,1hpost-infection,6micepergroup)with ThelipidIImodificationpathwayresultinginvancomycinresistance
teixobactinandvancomycininsepticemiaprotectionmodelusingMRSA. probably originated in the producer of vancomycin, Amycolatopsis
Survivalisdepicted48hafterinfection.b,Singledose(i.v.,2hpost-infection, orientalis19. It will probably take even longer for resistance to the
4micepergroup)treatmentwithteixobactinandvancomycininneutropenic better-protectedteixobactintoemerge.Teixobactinisthefirstmem-
mousethighinfectionmodelusingMRSA.Fordrug-treatedanimals,thigh
berofanewclassoflipidIIbindingantibiotics,structurallydistinct
colony-formingunits(c.f.u.)weredeterminedat26hpost-infection.For
fromglycopeptides,lantibiotics30,31,anddefensins32.Thepropertiesof
controls,c.f.u.inthighsweredeterminedat2hand26hpost-infection.c,Two
teixobactinsuggestthatitevolvedtominimizeresistancedevelopment
dosetreatment,5micepergroup,withteixobactin(i.v.,24hand36hpost-
bytargetmicroorganisms.Itislikelythatadditionalnaturalcompounds
infection)andsingledosetreatmentwithamoxicillin(subcutaneous,24hpost-
infection)inimmunocompetentlunginfectionmodelusingS.pneumoniae. withsimilarlylowsusceptibilitytoresistancearepresentinnatureand
Lungc.f.u.weredeterminedat48hpost-infection.Thec.f.u.fromeach arewaitingtobediscovered.
mouseareplottedasindividualpointsanderrorbarsrepresentthedeviation
withinanexperimentalgroup.*P,0.05,***P,0.001(determinedby OnlineContentMethods,alongwithanyadditionalExtendedDatadisplayitems
andSourceData,areavailableintheonlineversionofthepaper;referencesunique
non-parametriclog-ranktest).
tothesesectionsappearonlyintheonlinepaper.
parametersdeterminedafteri.v.injectionofasingle20mgperkgdose Received29July;accepted19November2014.
inmicewerefavourable,asthelevelofcompoundinserumwasmain- Publishedonline7January2015.
tainedabovetheMICfor4h(ExtendedDataFig.8).Ananimalefficacy
1. Fleming,A.Ontheantibacterialactionofculturesofapenicillium,withspecial
studywasthenperformedinamousesepticemiamodel.Micewerein-
referencetotheiruseintheisolationofB.influenzae.Br.J.Exp.Pathol.10,226–236
fectedintraperitoneallywithmethicillin-resistantS.aureus(MRSA)at (1929).
adosethatleadsto90%ofdeath.Onehourpost-infection,teixobactin 2. Kardos,N.&Demain,A.L.Penicillin:themedicinewiththegreatestimpacton
therapeuticoutcomes.Appl.Microbiol.Biotechnol.92,677–687(2011).
wasintroducedi.v.atsingledosesrangingfrom1to20mgperkg.All
3. Schatz,A.,Bugie,E.&Waksman,S.A.Streptomycin,asubstanceexhibiting
treatedanimalssurvived(Fig.4a),andinasubsequentexperimentthe antibioticactivityagainstgram-positiveandgram-negativebacteria.Proc.Soc.Exp.
PD (protectivedoseatwhichhalfoftheanimalssurvive)wasdeter- Biol.Med.55,66–69(1944).
50
minedtobe0.2mgperkg,whichcomparesfavourablytothe2.75mg 4. Spellberg,B.&Shlaes,D.Prioritizedcurrentunmetneedsforantibacterial
therapies.Clin.Pharmacol.Ther.96,151–153(2014).
perkgPD ofvancomycin,themainantibioticusedtotreatMRSA.
50 5. Bush,K.etal.Tacklingantibioticresistance.NatureRev.Microbiol.9,894–896
TeixobactinwasthentestedinathighmodelofinfectionwithS.aureus, (2011).
4 | NATURE | VOL 000 | 00 MONTH 2015
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ARTICLE RESEARCH
6. Payne,D.J.,Gwynn,M.N.,Holmes,D.J.&Pompliano,D.L.Drugsforbadbugs: 24. Degen,D.etal.Transcriptioninhibitionbythedepsipeptideantibioticsalinamide
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15. Sakoulas,G.etal.RelationshipofMICandbactericidalactivitytoefficacyof
vancomycinfortreatmentofmethicillin-resistantStaphylococcusaureus AcknowledgementsThisworkwassupportedbyNIHgrantT-RO1AI085585toK.L.,by
bacteremia.J.Clin.Microbiol.42,2398–2402(2004). NIHgrantAI085612toA.L.S.,bytheCharlesA.KingTrusttoB.P.C.,andbytheGerman
16. Kollef,M.H.Limitationsofvancomycininthemanagementofresistant ResearchFoundation(DFG;SCHN1284/1-2)andtheGermanCenterforInfection
staphylococcalinfections.Clin.Infect.Dis.45(Suppl3),S191–S195(2007). Research(DZIF)toT.S.andI.E.TheNRSstrainswereprovidedbytheNetworkon
17. Arthur,M.,Depardieu,F.,Reynolds,P.&Courvalin,P.Quantitativeanalysisofthe AntimicrobialResistanceinStaphylococcusaureusfordistributionbyBEIResources,
metabolismofsolublecytoplasmicpeptidoglycanprecursorsofglycopeptide- NIAID,NIH.PreclinicalServicesofferedbyNIAIDaregratefullyacknowledged.Wethank
resistantenterococci.Mol.Microbiol.21,33–44(1996). H.G.Sahlforreadingthemanuscriptandmakingcomments,A.Makriyannisfor
18. Bugg,T.D.etal.MolecularbasisforvancomycinresistanceinEnterococcus suggestions,P.Muller,B.BerdyandS.Kaluziakfortaxonomyanalysis,andM.Jostenfor
faeciumBM4147:biosynthesisofadepsipeptidepeptidoglycanprecursorby performingmassspectrometryanalysis.
vancomycinresistanceproteinsVanHandVanA.Biochemistry30,10408–10415
AuthorContributionsK.L.andT.S.designedthestudy,analysedresults,andwrotethe
(1991).
paper.L.L.L.designedthestudyandanalysedresults.A.J.P.designedthestudy,
19. Marshall,C.G.,Broadhead,G.,Leskiw,B.K.&Wright,G.D.D-Ala-D-Alaligasesfrom performedcompoundisolationandstructuredeterminationandanalyseddata.B.P.C.
glycopeptideantibiotic-producingorganismsarehighlyhomologoustothe
designedthestudy,performedsusceptibilityexperimentsandwrotethepaper.D.E.H.
enterococcalvancomycin-resistanceligasesVanAandVanB.Proc.NatlAcad.Sci.
oversawpreclinicalworkincludingdesigningstudiesandanalysingdata.S.E.designed
USA94,6480–6483(1997).
cultivationexperimentsandanalyseddata.M.J.,L.L.andV.A.S.designedand
20. D’Elia,M.A.etal.LesionsinteichoicacidbiosynthesisinStaphylococcusaureus
performedexperimentsonstructuredeterminationandanalyseddata.I.E.andA.M.
leadtoalethalgainoffunctionintheotherwisedispensablepathway.J.Bacteriol.
designedandperformedexperimentsonmechanismofaction.A.L.S.,D.R.C.,C.R.F.,
188,4183–4189(2006).
K.A.F.,W.P.M.,A.G.N.,A.M.Z.andC.C.performedexperimentsoncompoundproduction,
21. Bierbaum,G.&Sahl,H.G.Inductionofautolysisofstaphylococcibythebasic
isolation,susceptibilitytestinganddataanalysis.T.F.S.identifiedthebiosynthetic
peptideantibioticsPep5andnisinandtheirinfluenceontheactivityofautolytic
cluster.
enzymes.Arch.Microbiol.141,249–254(1985).
22. O’Riordan,K.&Lee,J.C.Staphylococcusaureuscapsularpolysaccharides.Clin. AuthorInformationThebiosyntheticgeneclusterforteixobactinhasbeendeposited
Microbiol.Rev.17,218–234(2004). withGenBankunderaccessionnumberKP006601.Reprintsandpermissions
23. Xayarath,B.&Yother,J.Mutationsblockingsidechainassembly,polymerization, informationisavailableatwww.nature.com/reprints.Theauthorsdeclarecompeting
ortransportofaWzy-dependentStreptococcuspneumoniaecapsulearelethalin financialinterests:detailsareavailableintheonlineversionofthepaper.Readersare
theabsenceofsuppressormutationsandcanaffectpolymertransfertothecell welcometocommentontheonlineversionofthepaper.Correspondenceand
wall.J.Bacteriol.189,3369–3381(2007). requestsformaterialsshouldbeaddressedtoK.L.([email protected]).
00 MONTH 2015 | VOL 000 | NATURE | 5
©2015Macmillan Publishers Limited. All rights reserved
RESEARCH ARTICLE
METHODS andthesolutionwasallowedtoeluteslowly.Thissolutionwasthenpouredovera
fresh 10 g Dowex (1x4 Cl- form) column and the resulting solution filtered
Isolationandcultivationofproducingstrains.Asampleof1gofsoilsample
throughaPall3KMolecularWeightCentrifugalfilter.Theclearsolutionwasthen
collectedfromagrassyfieldinMainewasagitatedvigorouslyin10mlofdeionized
lyophilizedtoleaveawhitepowder.
HOfor10min.Afterlettingthesoilparticulatessettlefor10min,thesupernatant
2
wasdilutedinmoltenSMSmedia(0.125gcasein,0.1gpotatostarch,1gcasamino Minimuminhibitoryconcentration(MIC).MICwasdeterminedbybrothmicro-
acids,20gbacto-agarin1litreofwater)toachieveanaverageconcentrationofone dilutionaccordingtoCLSIguidelines.Thetestmediumformostspecieswascation-
cellper20mlofmedium.Then20mlaliquotswerethendispensedintothewellsof adjustedMueller-Hintonbroth(MHB).Thesametestmediumwassupplemented
aniChip.TheiChipwasplacedindirectcontactwiththesoil.Afteronemonthof with3%lysedhorseblood(ClevelandScientific,Bath,OH)forgrowingStrepto-
incubation,theiChipsweredisassembledandindividualcolonieswerestreaked cocci.HaemophilusTestMediumwasusedforH.influenzae(Teknova,Hollister,
ontoSMSagartotestfortheabilitytopropagateoutsidetheiChipandforcolony CA), Middlebrook 7H9 broth (Difco) was used for mycobacteria, Schaedler-
purification. anaerobebroth(Oxoid)wasusedforC.difficile,andfetalbovineserum(ATCC)
Extractpreparationandscreeningforactivity.Isolatesthatgrewwelloutside wasaddedtoMHB(1:10)totesttheeffectofserum.Alltestmediaweresupple-
theiChipwereculturedinseedbroth(15gglucose,10gmaltextract,10gsoluble mentedwith0.002%polysorbate80topreventdrugbindingtoplasticsurfaces39,
starch,2.5gyeastextract,5gcasaminoacids,and0.2gCaCl N2HOper1litreof andcellconcentrationwasadjustedtoapproximately53105cellsperml.After
2 2
deionizedHO,pH7.0)toincreasebiomass,followedby1:20dilutioninto4dif- 20hofincubationat37uC(2daysforM.smegmatis,and7daysforM.tuberculosis),
2
ferentfermentationbroths.After11daysofagitationat29uC,thefermentations theMICwasdefinedasthelowestconcentrationofantibioticwithnovisiblegrowth.
weredriedandresuspendedinanequalvolumeof100%DMSO.Then5mlofex- ExpandedpanelantibacterialspectrumofteixobactinwastestedatMicromyx,Kal-
tractswerespottedontoalawnofgrowingS.aureusNCTC8325-4cellsinMueller- amazoo,MI,inbrothassays.Experimentswereperformedwithbiologicalreplicates.
Hintonagar(MHA)plates.After20hofincubationat37uC,visibleclearingzones Minimumbactericidalconcentration(MBC).S.aureusNCTC8325-4cellsfrom
indicatedantibacterialactivity.Theextractfromthisisolate,whichwasprovision- thewellsfromanMICmicrobrothplatethathadbeenincubatedfor20hat37uC
allynamedEleftheriaterraesp.,producedalargeclearingzone.AlthoughE.terrae werepelleted.AnaliquotoftheinitialinoculumfortheMICplatewassimilarly
sp.producedantibacterialactivityunderseveralgrowthconditions,thebestactivity processed.Thecellswereresuspendedinfreshmedia,platedontoMHA,andthe
(thatis,largestclearingzone)wasseenwithR4fermentationbroth(10gglucose, coloniesenumeratedafterincubatingfor24hat37uC.TheMBCisdefinedasthe
1gyeastextract,0.1gcasaminoacids,3gproline,10gMgCl-6HO,4gCaCl- firstdrugdilutionwhichresultedina99.9%decreasefromtheinitialbacterialtitre
2 2 2
2HO,0.2gKSO,5.6gTESfreeacid(2-[[1,3-dihydroxy-2-(hydroxymethyl) ofthestartinginoculum,andwasdeterminedtobe23MICforteixobactin.Ex-
2 2 4
propan-2-yl]amino]ethanesulfonicacid)per1litreofdeionizedHO,pH7). perimentswereperformedwithbiologicalreplicates.
2
Sequencingofthestrain.GenomicDNAofE.terraewasisolated.Sequencing Time-dependentkilling.Anovernightcultureofcells(S.aureusHG003;vanco-
wasperformedattheTuftsUniversityCoreFacility(Boston,MA).Apaired-end mycinintermediateS.aureusSA1287)wasdiluted1:10,000inMHBandincubated
librarywithaninsertsizeofapproximately800baseswasgeneratedandsequenced at37uCwithaerationat225r.p.m.for2h(earlyexponential)or5h(lateexponen-
usingIlluminatechnology.Thereadlengthwas251basesperread. tial).Bacteriawerethenchallengedwithantibioticsat103MIC(adesirablecon-
Strainidentification.Asuspensionofcellswasdisruptedbyvigorousagitation centrationatthesiteofinfection),oxacillin(1.5mgml21),vancomycin(10mgml21)
withglassbeads(106nmorsmaller)andthesupernatantusedastemplatetoam- orteixobactin(3mgml21)inculturetubesat37uCand225r.p.m.Atintervals,
plifythe16SrRNAgene,usingGoTaqGreenMasterMix(PromegaM7122),and 100mlaliquotswereremoved,centrifugedat10,000gfor1minandresuspendedin
theuniversalprimersE8FandU1510R33.Thethermocyclerparametersincluded 100mlofsterilephosphatebufferedsaline(PBS).Tenfoldseriallydilutedsuspen-
30cyclesof95uCfor30s,45uCfor30sand72uCfor105s.TheamplifiedDNA sionswereplatedonMHAplatesandincubatedat37uCovernight.Colonieswere
fragmentwassequencedbyMacrogenUSA(Cambridge,MA),andthesequence countedandc.f.u.permlwascalculated.Foranalysisoflysis,12.5mlofcultureat
comparedbyBLASTtoculturedisolatesintheRibosomalDatabaseProject. A600nm(OD600)of1.0wastreatedwith103MICofantibioticsfor24h,afterwhich,
TheassembledgenomeforE.terraewassubmittedtotheRASTgenomeanno- 2mlofeachculturewasaddedtoglasstesttubesandphotographed.Experiments
tationserverat(http://rast.nmpdr.org/)34whichproducedalistofclosestrelatives wereperformedwithbiologicalreplicates.
withpublishedgenomes.TheseareAlicycliphilusdenitrificans,Leptothrixcholodnii, Resistancestudies.Forsinglestepresistance,S.aureusNCTC8325-4at1010c.f.u.
Methylibiumpetroleiphilum,andRubrivivaxgelatinosus,andtheirgenomeswere wereplatedontoMHAcontaining23,43,and103MICofteixobactin40.After
downloadedfromtheNCBIftpsite(ftp://ftp.ncbi.nih.gov/genomes/ASSEMBLY_ 48hofincubationat37uC,noresistantcoloniesweredetected,givingthecalcu-
BACTERIA/).DNA–DNAhybridization(DDH)valuesofthesegenomestoE.terrae latedfrequencyofresistancetoteixobactinof,10210.ForM.tuberculosis,cells
werethenpredictedbytheGenome-to-GenomeDistancecalculator2.0,formula2, wereculturedin7H9mediumandplatedat109cellspermlon10platesandin-
(http://ggdc.dsmz.de/)35–37.NotethatM.petroleiphilumandR.gelatinosusarepres- cubatedfor3weeksat37uCforcolonycounts.Nocoloniesweredetected.
entonthephylogenytreeofE.terrae(ExtendedDataFig.2). Forresistancedevelopmentbysequentialpassaging40,41,S.aureusATCC29213
Biosyntheticgeneclusteridentification.ByscreeningthedraftgenomeofE.terrae, cellsatexponentialphaseweredilutedtoanA600nm(OD600)of0.01in1mlof
obtainedbyIlluminasequencing,manygenefragmentsputativelybelongingto MHBsupplementedwith0.002%polysorbate80containingteixobactinoroflox-
NRPScodinggeneswereidentified.Theassemblywasmanuallyeditedandgap acin.Cellswereincubatedat37uCwithagitation,andpassagedat24hintervalsin
closurePCRswereperformed.Sangersequencingoftheresultingfragmentsallowed thepresenceofteixobactinorofloxacinatsubinhibitoryconcentration(seeSup-
theclosureofthegenelocuscorrespondingtotheteixobactinbiosyntheticgene plementaryDiscussionfordetails).TheMICwasdeterminedbybrothmicrodi-
cluster.Thespecificityoftheadenylationdomainswasdeterminedusingtheon- lution.Experimentswereperformedwithbiologicalreplicates.
linetoolNRPSpredictor2(ref.38). Mammaliancytotoxicity.TheCellTiter96AQueousOneSolutionCellProlif-
Strainfermentationandpurificationofteixobactin.Homogenizedcolonies erationAssay(Promega)wasusedtodeterminethecytotoxicityofteixobactin.Ex-
werefirstgrownwithagitationinseedbroth.After4daysat28uC,theculturewas ponentiallygrowingNIH/3T3mouseembryonicfibroblast(ATCCCRL-1658,in
diluted5%(v/v)intotheR4fermentationmedia,andproductionmonitoredwith Dulbecco’sModifiedEagle’smediumsupplementedwith10%bovinecalfserum),
analyticalHPLC.Forscale-upisolationandpurificationofteixobactin,40litresof andHepG2cells(ATCCHB-8065,inDulbecco’sModifiedEagle’smediumsup-
cellsweregrowninaSartoriusBiostatCultibagSTR50/200Bioreactorforabout plementedwith10%fetalcalfserum)wereseededintoa96-wellflatbottomplate,
7days.Theculturewascentrifugedandthepelletextractedwith10litresof50% andincubatedat37uC.After24h,themediumwasreplacedwithfreshmedium
aqueousacetonitrileandthesuspensionagaincentrifugedfor30min.Theacet- containingtestcompounds(0.5mlofatwofoldserialdilutioninDMSOto99.5ml
onitrilewasremovedfromthesupernatantbyrotaryevaporationunderreduced ofmedia).After48hofincubationat37uC,reportersolutionwasaddedtothecells
pressureuntilonlywaterremained.Themixturewasthenextractedtwicewith5 andafter2h,theA490nm(OD490)wasmeasuredusingaSpectramaxPlusSpectro-
litresofn-BuOH.Theorganiclayerwastransferredtoaroundbottomflaskand photometer.Experimentswereperformedwithbiologicalreplicates.
then-BuOHremovedbyrotaryevaporationunderreducedpressure.Theresult- Haemolyticactivity.FreshhumanredbloodcellswerewashedwithPBSuntilthe
ingyellowsolidwasdissolvedinDMSOandsubjectedtopreparatoryHPLC(SP: upperphasewasclearaftercentrifugation.ThepelletwasresuspendedtoanA600nm
C18,MP:H2O/MeCN/0.1%TFA).Thefractionscontainingteixobactinwerethen (OD600)of24inPBS,andaddedtothewellsofa96-wellU-bottomplate.Tei-
pooledandtheacetonitrileremovedbyrotaryevaporationunderreducedpres- xobactinwasseriallydilutedtwofoldinwaterandaddedtothewellsresultingina
sure.Theremainingaqueousmixturewasthenlyophilizedtoleaveawhitepow- finalconcentrationrangingfrom0.003to200mgml21.Afteronehourat37uC,
der(trifluoroacetatesalt).Teixobactinwasthenconvertedtoahydrochloridesalt, cellswerecentrifugedat1,000g.ThesupernatantwasdilutedandA450nm(OD450)
andendotoxinremovedasfollows.100mgofteixobactin(TFAsalt)wasdissolved measuredusingaSpectramaxPlusSpectrophotometer.Experimentswereper-
in100mlofHOand5gofDowex(134Cl2form)wasaddedandthemixture formedwithbiologicalreplicates.
2
incubatedfor20minwithoccasionalshaking.A10gDowex(134Cl2form)col- Macromolecularsynthesis.S.aureusNCTC8325-4cellswereculturedinminimal
umnwaspreparedandthemixturewasthenpouredontothepreparedcolumn medium(0.02MHEPES,0.002MMgSO,0.0001MCaCl,0.4%succinicacid,
4 2
©2015Macmillan Publishers Limited. All rights reserved
ARTICLE RESEARCH
0.043MNaCl,0.5%(NH) SO)supplementedwith5%trypticsoybroth(TSB). Synthesisandpurificationoflipidintermediates.Largescalesynthesisandpu-
2 42 4
Cellswerepelletedandresuspendedintofreshminimalmediumsupplemented rificationofthepeptidoglycanprecursorslipidIandIIwasperformed45.Radio-
with5%TSBcontainingtestcompoundsandradioactiveprecursorstoadensityof labelledlipidIIwassynthesizedusing[14C]-UDP-GlcNAc(9.25GBqmmol21;
108cellsperml.Theradioactiveprecursorswereglucosaminehydrochloride, ARC)assubstrate.ForsynthesisofthelipidIIvariantwithaterminalD-Lacres-
D-[6-3H(N)](1mCiml21),leucine,L-[3,4,5-3H(N)](1mCiml21),uridine,[5-3H] idue,UDP-MurNAc-depsipeptide(Ala-Glu-Lys-Ala-Lac)waspurifiedfromLac-
(1mCiml21),orthymidine,[methyl-3H](0.25mCiml21)tomeasurecellwall, tobacilluscaseiATCC393.Briefly,L.caseiwasgrowninMRSbrothtoanA
600nm
protein,RNA,andDNAsynthesis,respectively.After20minofincubationat37uC, (OD600)of0.6andincubatedwith65mgml21ofchloramphenicolfor15min.In-
aliquotswereremoved,addedtoicecold25%trichloroaceticacid(TCA),andfil- tracellularaccumulationwasachievedbyincubationwithBacitracin(103MIC,
teredusingMultiscreenFilterplates(MilliporeCat.MSDVN6B50).Thefilterswere 40mgml21)inthepresenceof1.25mMzincforanother60min.Forsynthesisof
washedtwicewithicecold25%TCA,twicewithice-coldwater,driedandcounted lipidIIendingD-Ala-D-SertheUDP-MurNAc-pentapeptide(Ala-Glu-Lys-Ala-Ser)
withscintillationfluidusingPerkinElmerMicroBetaTriLuxMicroplateScintil- wasused.ThewallteichoicacidprecursorlipidIII(undecaprenyl-PP-GlcNAc)
lationandLuminescencecounter.Experimentswereperformedwithbiological waspreparedusingpurifiedTarOenzyme44.Inshort,purifiedrecombinantTarO
replicates. proteinwasincubatedinthepresenceof250nmolC -P,2.5mmolofUDP-GlcNAc
55
IntracellularaccumulationofUDP-N-acetyl-muramicacidpentapeptide.Ana- in83mMTris-HCl(pH8.0),6.7mMMgCl2,8.3%(v/v)dimethylsulfoxide,and
lysisofthecytoplasmicpeptidoglycannucleotideprecursorpoolwasexamined 10mMN-lauroylsarcosine.Thereactionwasinitiatedbytheadditionof150mgof
usingS.aureusATCC29213grownin25mlMHB.CellsweregrowntoanA600nm TarO-His6andincubatedfor3hat30uC.Lipidintermediateswereextractedfrom
(OD )of0.6andincubatedwith130mgml21ofchloramphenicolfor15min. thereactionmixtureswithn-butanol/pyridineacetate(pH4.2)(2:1;vol/vol),ana-
600
Teixobactinwasaddedat1,2.5and53MICandincubatedforanother60min. lysedbyTLCandpurified.C55-PandC55-PPwerepurchasedfromLarodanFine
Vancomycin(VAN;103MIC),knowntoformacomplexwithlipidII,wasused Chemicals,Sweden.[14C]-C55-PPwassynthesizedusingpurifiedS.aureusUppS
aspositivecontrol.Cellswerecollectedandextractedwithboilingwater.Thecell enzymebasedonaprotocolelaboratedforE.coliundecaprenylpyrophosphate
extractwasthencentrifugedandthesupernatantlyophilized42.UDP-linkedcellwall synthase47.Synthesiswasperformedusing0.5nmol[14C]-farnesylpyrophosphate
precursorswereanalysedbyRP18-HPLC43andconfirmedbyMALDI-ToF44mass (ARC;2.035GBqmmol21),5nmolisopentenylpyrophosphate(Sigma–Aldrich)
spectrometry.Experimentswereperformedwithbiologicalreplicates. and5mgUppSenzymein100mMHEPES,pH7.6,50mMKCl,5mMMgCl2,and
Cloning,overexpressionandpurificationofS.aureusUppSandYbjGasHis6- 0.1%TritonX-100.After3hofincubationat30uCradiolabelledC55-PPwasex-
tagfusions.S.aureusN315uppS(SA1103)andybjB(SA0415)wereamplified tractedfromthereactionmixturewithBuOHanddriedundervacuum.Product
usingforwardandreverseprimersuppS_FW-59-TCGGAGGAAAGCATATGT identitywasconfirmedbyTLCanalysis.Experimentswereperformedwithbio-
TTAAAAAGC-39,uppS_RV-59-ATACTCTCGAGCTCCTCACTC-39,SA0415_ logicalreplicates.
FW-59-GCGCGGGATCCATGATAGATAAAAAATTAACATCAC-39andSA0415_ Antagonizationassays.Antagonizationoftheantibioticactivityofteixobactin
RV-59-GCGCGCTCGAGAACGCGTTGTCGTCGATGAT-39,respectivelyand bypotentialtargetmoleculeswasperformedbyanMIC-basedsetupinmicrotitre
clonedintoamodifiedpET20vector44usingrestrictionenzymesNdeI(uppS)or plates.Teixobactin(83MIC)wasmixedwithpotentialHPLC-purifiedantagonists
BamHI(ybjG)andXhoI,togenerateC-terminalHis6-fusionproteins.Recombi- (C55-P,farnesyl-PP[C15-PP;SigmaAldrich],C55-PP,UDP-MurNAc-pentapeptide,
nantUppS-His enzymewasoverexpressedandpurifiedasdescribedforMurG32. UDP-GlcNAc[SigmaAldrich],lipidI,lipidII,andlipidIII)atafixedmolarratio
6
ForoverexpressionandpurificationofYbjG-His E.coliBL21(DE3)C43cellstrans- (fivefoldmolarexcess)oratincreasingconcentrationswithrespecttotheantibiotic,
6
formedwiththeappropriaterecombinantplasmidweregrownin2YT-medium andthelowestratioleadingtocompleteantagonizationofteixobactinactivitywas
(50mgml21ampicillin)at25uC.AtanA600nm(OD600)of0.6,IPTGwasaddedata determined.S.aureusATCC29213(53105c.f.u.perml)wereaddedandsamples
concentrationof1mMtoinduceexpressionoftherecombinantproteins.After16h, wereexaminedforvisiblebacterialgrowthafterovernightincubation.Vancomycin
cellswereharvestedandresuspendedinbufferA(25mMTris/HCl,pH7.5,150mM (83MIC)wasusedasacontrol.Experimentswereperformedwithbiological
NaCl,2mMb-mercaptoethanol,30%glycerol,and1mMMgCl).2mgml21lyso- replicates.
2
zyme,75mgml21DNaseand75mgml21RNasewereadded;cellswereincubated Complexformationofteixobactin.BindingofteixobactintoC -P,C -PP,
55 55
for1honice,sonicatedandtheresultingsuspensionwascentrifuged(20,000g, lipidI,lipidII,lipidII-D-Ala-D-Ser,lipidII-D-Ala-D-LacandlipidIIIwasanal-
30min,4uC).Pelletedbacterialmembraneswerewashedthreetimestoremove ysedbyincubating2nmolofeachpurifiedprecursorwith2to4nmolesofteixo-
remainingcytoplasmiccontent.Membraneproteinsweresolubilizedintwosuc- bactinin50mMTris/HCl,pH7.5,for30minatroomtemperature.Complex
cessivestepswithbufferAcontaining17.6mMn-dodecyl-b-D-maltoside(DDM). formationwasanalysedbyextractingunboundprecursorsfromthereactionmix-
Solubilizedproteinswereseparatedfromcelldebrisbycentrifugation(20,000g, turewithn-butanol/pyridineacetate(pH4.2)(2:1;vol/vol)followedbyTLCana-
30min,4uC)andthesupernatantcontainingrecombinantproteinswasmixedwith lysisusingchloroform/methanol/water/ammonia(88:48:10:1,v/v/v/v)asthesolvent
Talon-agarose(Clontech)andpurificationwasperformed42.Puritywascontrolled anddetectionoflipid-containingprecursorsbyphosphomolybdicacidstaining48.
bySDS–PAGEandproteinconcentrationwasdeterminedusingBradfordprotein Experimentswereperformedwithbiologicalreplicates.
assay(Biorad). hERGinhibitiontesting.TeixobactinwastestedforinhibitionofhERGactivity
Invitropeptidoglycansynthesisreactions.Invitropeptidoglycanbiosynthesis usinganIonWorksTMHTinstrument(MolecularDevicesCorporation),which
reactionswereperformedasdescribedusingpurifiedenzymesandsubstrates32,45. performselectrophysiologymeasurementsina384-wellplate(PatchPlate).Chinese
TheMurGactivityassaywasperformedinafinalvolumeof30mlcontaining hamsterovary(CHO)cellsstablytransfectedwithhERG(cell-lineobtainedfrom
2.5nmolpurifedlipidI,25nmolUDP-N-acetylglucosamine(UDP-GlcNAc)in Cytomyx,UK)werepreparedasasingle-cellsuspensioninextracellularsolution
200mMTris-HCl,5.7mMMgCl,pH7.5,and0.8%TritonX-100inthepresence (Dulbecco’sphosphatebufferedsalinewithcalciumandmagnesiumpH7),and
2
of0.45mgofpurified,recombinantMurG-His enzyme.Reactionmixtureswere aliquotsaddedtoeachwelloftheplate.Thecellswerepositionedoverasmallhole
6
incubatedfor60minat30uC.Forquantitativeanalysis0.5nmolof[14C]-UDP- atthebottomofeachwellbyapplyingavacuumbeneaththeplatetoformanelec-
GlcNAc(9.25GBqmmol21;ARC)wasaddedtothereactionmixtures.Theassay tricalseal.Theresistanceofeachsealwasmeasuredviaacommonground-electrode
forsynthesisoflipidII–Gly catalysedbyFemXwasperformedasdescribedpre- intheintracellularcompartmentandindividualelectrodesplacedintoeachofthe
1
viouslywithoutanymodifications32,45.EnzymaticactivityofS.aureusPBP2was upperwells.Experimentswereperformedwiththreebiologicalreplicates.
determinedbyincubating2nmol[14C]-lipidIIin100mMMES,10mMMgCl, CytochromeP450inhibition.Teixobactinandcontrolcompoundswereincu-
2
pH5.5inatotalvolumeof50ml.Thereactionwasinitiatedbytheadditionof5mg batedwithhumanlivermicrosomesat37uCtodeterminetheireffectonfivemajor
PBP2–His6andincubatedfor2.5hat30uC.MonophosphorylationofC55-PPwas humancytochromesP450s(CYP).Theassayincludedprobesubstrates(midazolam
carriedoutusingpurifiedS.aureusYbjG–His enzymeasdescribedpreviouslyfor forCyp3A4,testosteroneforCyp3A4,tolbutamideforCyp2C9,dextro-methorphan
6
E.colipyrophosphatase46,withmodifications.0.5nmol[14C]-C -PP(1.017kBq) forCyp2D6,S-mephenytoinforCyp2C19,andphenacetinforCyp1A2,2mM
55
wasincubatedwith0.6mgYbjG-His6in20mMTris/HCl,pH7.5,60mMNaCl, NADPH,3mMMgCl in50mMpotassiumphosphatebuffer,pH7.4.Thefinal
2
0.8%TritonX-100for10minat30uC. microsomalconcentrationwas0.5mgml21.NADPHwasaddedlasttostartthe
Inallinvitroassaysteixobactinwasaddedinmolarratiosrangingfrom0.25to assay.Aftertenminutesofincubation,theamountofprobemetaboliteinthesuper-
8withrespecttotheamountof[14C]-C -PP,lipidIorlipidIIand[14C]-lipidII, natantwasdeterminedbyLC/MS/MSusinganAgilent6410massspectrometer
55
respectively.Synthesizedlipidintermediateswereextractedfromthereactionmix- coupledwithanAgilent1200HPLCandaCTCPALchilledautosampler,allcon-
tureswithn-butanol/pyridineacetate,pH4.2(2:1;vol/vol)aftersupplementingthe trolledbyMassHuntersoftware(Agilent).Experimentswereperformedwiththree
reaction mixture with 1M NaCl and analysed by thin-layer chromatography biologicalreplicates.
(TLC).Quantificationwascarriedoutusingphosphoimaging(Stormimagingsys- Invitrogenotoxicity.Teixobactinwastestedinaninvitromicronucleustestthat
tem,GEHealthcare)asdescribed32,45.Experimentswereperformedwithbiological employsfluorescentcellimagingtoassesscytotoxicityandquantifymicronuclei.
replicates. TheassaywasperformedwithCHO-K1cellsinthepresenceorabsenceofAroclor
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RESEARCH ARTICLE
(toinduceCYPactivity)-treatedratliverS9fraction(containsphaseIandphaseII neutropenicbycyclophosphamide(twoconsecutivedosesof150and100mgper
metabolizingenzymes)todetermineifanygenotoxicmetabolitesareproduced. kgdeliveredon4and1daysbeforeinfection).Bacteriawereresuspendedinsterile
Noevidenceofgenotoxicitywasobservedwithteixobactinupto125mgml21(the saline,adjustedtoanA (OD )of0.1,anda0.1mlinoculum(2.83105c.f.u.
625nm 625
highestconcentrationtested)undereithercondition.Experimentswereperformed permouse)injectedintotherightthighsofmice.At2hpost-infection,micere-
withthreebiologicalreplicates. ceivedtreatmentwithteixobactinat1,2.5,5,10or20mgperkgadministeredina
DNAbinding.Compoundswereseriallydilutedandmixedwithshearedsalmon singledose,intravenousinjection(fourmicepergroup).Onegroupofinfectedmice
spermDNA(6.6mgml21finalconcentration).Analiquotwasspottedontoa waseuthanizedandthighsprocessedforc.f.u.toserveasthetimeoftreatment
lawnofgrowingS.aureusNCTC8325-4cells,andthezonesofgrowthinhibition controls.At26hpost-infectionmicewereeuthanizedbyCO inhalation.Theright
2
measuredafter20hofgrowthat37uC.Areductionintheinhibitionzonesizein thighswereasepticallyremoved,weighed,homogenized,seriallydiluted,andpla-
thepresenceofDNAwouldindicatelossofantibacterialactivityduetobindingto tedontrypticasesoyagarforc.f.u.titres.
theDNA.Experimentswereperformedwiththreebiologicalreplicates. Mouselunginfectionmodel.TeixobactinwastestedagainstStreptococcuspneu-
Plasmaproteinbinding.Proteinbindingofteixobactininratplasmawasdeter- moniaeATCC6301(UNT012-2)inanimmunocompetentmousepneumonia
minedusingaRapidEquilibriumDialysis(RED)kit(Pierce)withLC–MS/MSana- modeltodeterminethecompound’spotentialtotreatacuterespiratoryinfections.
lysis.Teixobactin(10mgml21)andratplasmain5%dextrosecontaining0.005% CD-1micewereinfectedintranasally(1.53106c.f.u.permouse).Thecompound
polysorbate80wereaddedtoonesideofthesingle-useREDplatedialysischamber wasdeliveredintravenouslyat24and36hpost-infection,whereasamoxicillinwas
havingan8kDMWcutoffmembrane.Followingfourhoursofdialysisthesamples deliveredsubcutaneouslyatasingleconcentrationtoserveaspositivecontrol.Teix-
frombothsideswereprocessedandanalysedbyLC/MS/MS.Theteixobactincon- obactinwasdeliveredatdosesrangingfrom0.5to10mgperkgperdose(5miceper
centrationwasdetermined,andthepercentageofcompoundboundtoproteinwas dose).At48hpost-infection,treatedmicewereeuthanized,lungsasepticallyre-
calculated.Teixobactinexhibited84%plasmaproteinbinding.Experimentswere movedandprocessedforc.f.u.counts.
performedwiththreebiologicalreplicates.
Microsomalstability.Themetabolicstabilityofteixobactinwasmeasuredinrat 33. Baker,G.C.,Smith,J.J.&Cowan,D.A.Reviewandre-analysisofdomain-specific
16Sprimers.J.Microbiol.Methods55,541–555(2003).
livermicrosomes(Invitrogen/LifeTechnologies,CA)usingNADPHRegeneration
34. Aziz,R.K.etal.TheRASTServer:rapidannotationsusingsubsystemstechnology.
System(Promega)bymonitoringthedisappearanceofthecompoundoveran BMCGenomics9,75(2008).
incubationperiodoftwohours.Teixobactin(60mgml21)orverapamil(5mM) 35. Auch,A.F.,Klenk,H.P.&Goker,M.Standardoperatingprocedureforcalculating
servingaspositivecontrolwereaddedto1mgml21microsomesat37uC.Aliquots genome-to-genomedistancesbasedonhigh-scoringsegmentpairs.Stand.
wereremovedat0h,0.5h,1hand2h,andthereactionsstoppedbyadditionof3 GenomicSci.2,142–148(2010).
36. Auch,A.F.,vonJan,M.,Klenk,H.P.&Goker,M.DigitalDNA–DNAhybridizationfor
volumesofice-coldacetonitrile.SampleswereanalysedbyLC/MS/MS.Experi-
microbialspeciesdelineationbymeansofgenome-to-genomesequence
mentswereperformedwiththreebiologicalreplicates.
comparison.Stand.GenomicSci.2,117–134(2010).
Animalstudies.AllanimalstudieswerecarriedoutatVivisourceLaboratories, 37. Meier-Kolthoff,J.P.,Auch,A.F.,Klenk,H.P.&Goker,M.Genomesequence-based
(Waltham,MA),andUniversityofNorthTexasHealthScienceCenter(Houston, speciesdelimitationwithconfidenceintervalsandimproveddistancefunctions.
TX),andconformedtoinstitutionalanimalcareandusepolicies.Neitherrandom- BMCBioinformatics14,60(2013).
izationnorblindingwasdeemednecessaryfortheanimalinfectionmodels,andall 38. Ro¨ttig,M.etal.NRPSpredictor2–awebserverforpredictingNRPSadenylation
domainspecificity.NucleicAcidsRes.39,W362–W367(2011).
animalswereused.AllanimalstudieswereperformedwithfemaleCD-1mice,
39. Arhin,F.F.etal.Effectofpolysorbate80onoritavancinbindingtoplasticsurfaces:
6–8-weeksold. implicationsforsusceptibilitytesting.Antimicrob.AgentsChemother.52,
Pharmacokineticanalysis.CD-1femalemicewereinjectedintravenouslywitha 1597–1603(2008).
singledoseof20mgperkginwaterandshowednoadverseeffects.Plasmasam- 40. Bogdanovich,T.,Ednie,L.M.,Shapiro,S.&Appelbaum,P.C.Antistaphylococcal
plesweretakenfrom3micepertimepoint(5,15,30min;1,2,4,8and24hpost- activityofceftobiprole,anewbroad-spectrumcephalosporin.Antimicrob.Agents
Chemother.49,4210–4219(2005).
dose).Analiquotofplasmasampleorcalibrationsamplewasmixedwiththree
41. Metzler,K.,Drlica,K.&Blondeau,J.M.Minimalinhibitoryandmutantprevention
volumesofmethanolcontaininginternalstandard,incubatedonicefor5min,and
concentrationsofazithromycin,clarithromycinanderythromycinforclinical
centrifuged.Theprotein-freesupernatantwasanalysedbyLC/MS/MSusingan isolatesofStreptococcuspneumoniae.J.Antimicrob.Chemother.68,631–635
Agilent6410massspectrometercoupledwithanAgilent1200HPLCandaCTC (2013).
PALchilledautosampler,allcontrolledbyMassHuntersoftware(Agilent).After 42. Schneider,T.etal.ThelipopeptideantibioticFriulimicinBinhibitscellwall
separationonaC18reversephaseHPLCcolumn(Agilent)usinganacetonitrile- biosynthesisthroughcomplexformationwithbactoprenolphosphate.Antimicrob.
AgentsChemother.53,1610–1618(2009).
watergradientsystem,peakswereanalysedbymassspectrometryusingESIion-
43. Bro¨tz,H.,Bierbaum,G.,Reynolds,P.E.&Sahl,H.G.Thelantibioticmersacidin
izationinMRMmode.Theproductm/zanalysedwas134.1D,whichprovideda inhibitspeptidoglycanbiosynthesisattheleveloftransglycosylation.Eur.J.
lowlimitofquantificationof1ngml21.Themeanplasmaconcentrationandthe Biochem.246,193–199(1997).
standarddeviationfromall3animalswithineachtimepointwerecalculated.PK 44. Mu¨ller,A.,Ulm,H.,Reder-Christ,K.,Sahl,H.G.&Schneider,T.InteractionoftypeA
parametersoftestagentwerecalculatedwithanon-compartmentalanalysismodel lantibioticswithundecaprenol-boundcellenvelopeprecursors.Microb.Drug
Resist.18,261–270(2012).
basedonWinNonlin.Themeanplasmaconcentrationsfromall3miceateachtime
45. Schneider,T.etal.Invitroassemblyofacomplete,pentaglycineinterpeptide
pointwereusedinthecalculation.
bridgecontainingcellwallprecursor(lipidII-Gly5)ofStaphylococcusaureus.Mol.
Mousesepsisprotectionmodel.Teixobactinwastestedagainstclinicalisolate Microbiol.53,675–685(2004).
S.aureusMRSAATCC33591inamousesepticemiaprotectionassaytoassessits 46. ElGhachi,M.,Derbise,A.,Bouhss,A.&Mengin–Lecreulx,D.Identificationof
invivobioavailabilityandPD (protectivedoseresultingin50%survivalofin- multiplegenesencodingmembraneproteinswithundecaprenylpyrophosphate
50
fectedmiceafter48h).CD-1femalemicewereinfectedwith0.5mlofbacterial phosphatase(UppP)activityinEscherichiacoli.J.Biol.Chem.280,18689–18695
suspension(3.283107c.f.u.permouse)viaintraperitonealinjection,aconcentra- (2005).
47. ElGhachi,M.,Bouhss,A.,Blanot,D.&Mengin–Lecreulx,D.ThebacAgeneof
tionthatachievesatleast90%mortalitywithin48hafterinfection.Atonehour Escherichiacoliencodesanundecaprenylpyrophosphatephosphataseactivity.
post-infection,mice(6pergroup)weretreatedwithteixobactinatsingleintraven- J.Biol.Chem.279,30106–30113(2004).
ousdosesof20,10,5,2.5,and1mgperkg.Infectioncontrolmiceweredosedwith 48. Sham,L.T.etal.Bacterialcellwall.MurJistheflippaseoflipid-linkedprecursorsfor
vehicleorvancomycin.Survivalisobserved48hafterinfectionandtheprobability peptidoglycanbiogenesis.Science345,220–222(2014).
49. Mohammadi,T.etal.IdentificationofFtsWasatransporteroflipid-linkedcellwall
determinedbynon-parametriclog-ranktest.ToobtainthePD theexperiment
50, precursorsacrossthemembrane.EMBOJ.30,1425–1432(2011).
wasrepeatedatlowerdoses5,1,0.5,0.25,and0.1mgperkg.
50. Lazarevic,V.&Karamata,D.ThetagGHoperonofBacillussubtilis168encodesa
Mousethighinfectionmodel.TeixobactinwastestedagainstMRSAATCC33591 two-componentABCtransporterinvolvedinthemetabolismoftwowallteichoic
inaneutropenicmousethighinfectionmodel.FemaleCD-1micewererendered acids.Mol.Microbiol.16,345–355(1995).
©2015Macmillan Publishers Limited. All rights reserved
ARTICLE RESEARCH
ExtendedDataFigure1|TheiChip. a–c,TheiChip(a)consistsofacentral suspensionofcellsinmoltenagar,thethrough-holescapturesmallvolumesof
plate(b)whichhousesgrowingmicroorganisms,semi-permeablemembranes thissuspension,whichsolidifyintheformofsmallagarplugs.Alternatively,
oneachsideoftheplate,whichseparatetheplatefromtheenvironment, moltenagarcanbedispensedintothechambers.Themembranesare
andtwosupportingsidepanels(c).Thecentralplateandsidepanelshave attachedandtheiChipisthenplacedinsoilfromwhichthesampleoriginated.
multiplematchingthrough-holes.Whenthecentralplateisdippedinto
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RESEARCH ARTICLE
ExtendedDataFigure2|16SrRNAgenephylogenyofEleftheriaterrae. theNeighbour-Joining(NJ)clusteringalgorithm,aswellasoptimizedgap
a,ThephylogeneticpositionofE.terraewithintheclassb-proteobacteria.The penalties.Resultingalignmentsweremanuallycuratedandphylogenetictrees
16SrRNAgenesequencesweredownloadedfromEntrezatNCBIusing wereconstructedleveragingPhyML3.0withaTN93substitutionmodel
accessionnumbersretrievedfrompeer-reviewedpublications.b,The and500Bootstrapiterationsofbranchsupport.Topologysearchoptimization
phylogeneticpositionofE.terraeamongitsclosestknownrelatives.The wasconductedusingtheSubtree–Pruning–Regrafting(SPR)algorithmwith
sequencesweredownloadedfromNCBIusingaccessionnumbersretrieved anestimatedTransition–Transversionratioandgammadistribution
fromtheRDPClassifierDatabase.Forbothtrees,multiplesequencealignments parametersaswellasfixedproportionsofinvariablesites.
(MSA)wereconstructedusingClustalW2,implementingadefaultCostMatrix,
©2015Macmillan Publishers Limited. All rights reserved