Systematic and Molecular Basis of the Antibacterial Action of Quinoxaline 1,4-Di-N-Oxides against

18 Pages·2015·1.89 MB·English

RESEARCHARTICLE
Systematic and Molecular Basis of the
N
Antibacterial Action of Quinoxaline 1,4-Di- -
Escherichia coli
Oxides against
GuyueCheng1,3,BeiLi1,ChenxiWang1,HongfeiZhang1,GuixiaLiang1,ZhifeiWeng1,
HaihongHao1,3,XuWang1,3,ZhenliLiu2,3,MenghongDai1,3*,YulianWang2,3*,
ZonghuiYuan1,2,3*
1 MOALaboratoryforRiskAssessmentofQualityandSafetyofLivestockandPoultryProducts,Huazhong
AgriculturalUniversity,Wuhan,Hubei,430070,China,2 NationalReferenceLaboratoryofVeterinaryDrug
Residues(HZAU)andMAOKeyLaboratoryforDetectionofVeterinaryDrugResidues,Huazhong
AgriculturalUniversity,Wuhan,Hubei,430070,China,3 HubeiCollaborativeInnovationCenterforAnimal
NutritionandFeedSafety,HuazhongAgriculturalUniversity,Wuhan,Hubei,430070,China
* [email protected](MD); [email protected](YW);
[email protected](ZY)
OPENACCESS
Abstract
Citation:ChengG,LiB,WangC,ZhangH,LiangG,
WengZ,etal.(2015)SystematicandMolecular
Quinoxaline1,4-di-N-oxides(QdNOs)arewidelyknownaspotentantibacterialagents,but
BasisoftheAntibacterialActionofQuinoxaline1,4-
Di-N-OxidesagainstEscherichiacoli.PLoSONE theirantibacterialmechanismsareincompletelyunderstood.Inthisstudy,thetranscrip-
10(8):e0136450.doi:10.1371/journal.pone.0136450 tomicandproteomicprofilesofEscherichiacoliexposedtoQdNOswereintegrativelyinves-
tigated,andtheresultsdemonstratedthatQdNOsmainlyinducedanSOSresponseand
Editor:DanielRittschof,DukeUniversityMarine
Laboratory,UNITEDSTATES oxidativestress.Moreover,genesandproteinsinvolvedinthebacterialmetabolism,cellular
structuremaintenance,resistanceandvirulencewerealsofoundtobechanged,conferring
Received:April17,2015
bacterialsurvivalstrategies.Biochemicalassaysshowedthatreactiveoxygenspecies
Accepted:August3,2015
wereinducedintheQdNO-treatedbacteriaandthatfreeradicalscavengersattenuatedthe
Published:August21,2015
antibacterialactionofQdNOsandDNAdamage,suggestinganoxidative-DNA-damage
Copyright:©2015Chengetal.Thisisanopen actionofQdNOs.TheQdNOradicalintermediates,likelycarbon-centeredandaryl-type
accessarticledistributedunderthetermsofthe
radicals,asidentifiedbyelectronparamagneticresonance,werethemajorradicalsinduced
CreativeCommonsAttributionLicense,whichpermits
byQdNOs,andxanthineoxidasewasoneoftheQdNO-activatingenzymes.Thisstudypro-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare videsnewinsightsintotheactionofQdNOsinasystematicmannerandincreasesthecur-
credited. rentknowledgeofbacterialphysiologyunderantibioticstresses,whichmaybeofgreat
DataAvailabilityStatement:Themicroarraydata valueinthedevelopmentofnewantibiotic-potentiatingstrategies.
havebeendepositedintheNCBIGeneExpression
Ommibus(GEO)databaseundertheaccession
numberofGSE39607.
Funding:Theresearchwassupportedbythe
NationalHighTechnologyResearchand
Introduction
DevelopmentProgramofChina(2011AA10A214)
(www.most.gov.cn)toMD,theNationalProgramfor Quinoxaline1,4-di-N-oxidederivatives(QdNOs)haveexhibitedmanydiversebiologicalprop-
RiskAssessmentofQualityandSafetyofLivestock
erties,includingantibacterial,antifungal,antiviral,anticancerandinsecticidalactivities[1].
andPoultryProducts(GJFP2015008)(www.moa.gov.
TheQdNOsusedinveterinarypractice,includingquindoxin,carbadox,olaquindox(OLA),
cn)toZY,theNationalNaturalScienceFoundationof
mequindox(MEQ)andquinocetone,areknownaspotentantibacterialagentsforthepreven-
China(31272614)(www.nsfc.gov.cn)toXWandthe
FundamentalResearchFundsfortheCentral tionofcolibacillosis,diarrhea,andentericsalmonellosisinlivestockandpoultry[2].Cyadox
PLOSONE|DOI:10.1371/journal.pone.0136450 August21,2015 1/18
AntibacterialActionofQdNOs
UniversitiesofChina(2013QC002)(www.moe.gov. (CYA)isanovelQdNOderivative,andthemicrobiologicalsafetyevaluationofCYAsuggests
cn)toGC.Thefundershadnoroleinstudydesign, thatitisasafememberoftheQdNOfamily[3].
datacollectionandanalysis,decisiontopublish,or
AlthoughQdNOantibacterialshavebeenwidelyusedformorethanhalfacentury,the
preparationofthemanuscript.
modeofactionunderlyingtheirantibacterialpropertyhasnotyetbeenclearlyelucidated.Pre-
CompetingInterests:Theauthorshavedeclared viousinvestigationshaveshownthatQdNOscaninhibitDNAsynthesisandcauseDNAdam-
thatnocompetinginterestsexist.
age[4,5].Suteretal.[4]hassuggestedthatsomeundefinedbacterialreductase(s)areinvolved
inthemetabolicactivationofQdNOsandthatsomeunstableintermediateproduct,likelyfree
radicalsthatmaymightattackDNA,isproduced.However,themolecularbasisunderlying
QdNOactivationandQdNO-inducedDNAdamagehasnotyetbeenclarified.
Inthisstudy,transcriptomicandproteomicanalyseswereemployedtoexaminetheeffects
ofQdNOsonsusceptiveE.coli.TheresultsdemonstratedthattheSOSresponseandoxidative
stressweregenerallyinducedinthebacteriaexposedtoQdNOs,suggestingthatQdNOskilled
thebacteriamainlybytheirinductionofoxidativeDNAdamage.TheQdNO-activating
enzyme(s)andtheQdNO-inducedradicalswereidentifiedandthemechanismofQdNO-
inducedDNAdamagewasinvestigated.Thisstudynotonlysupportsthepreviousfindingsbut
alsoprovidesnewinsightsintotheantibacterialmechanismofQdNOsfromasystematicper-
spective,whichwillguidefutureresearchanddevelopmentofthistypeofcompounds.
MaterialsandMethods
DrugsandChemicals
CYA,bisdesoxycyadox(Cy1),cyadox1-monoxide(Cy2)andcyadox4-monoxide(Cy10)(Fig
1)weresynthesizedbytheInstituteofVeterinaryPharmaceuticals(HZAU)(Wuhan,Hubei,
China).OLAwasobtainedfromtheChinaInstituteofVeterinaryDrugControl(Beijing,
China).MEQwaspurchasedfromBeijingZhongnongfaPharmaceuticalCo.Ltd.(Huanggang,
Huibei,China).AnROSassaykitanddihydroethidiumwerepurchasedfromBeyotime(Shen-
zhen,China).3'-(p-hydroxyphenyl)fluorescein(HPF)andtheTrizolReagentwereboughtfrom
Invitrogen(Carlsbad,CA,USA).Ciprofloxacin,carbenicillin,enrofloxacin,gentamycin,
Fig1.ChemicalstructuresofQdNOsandtheirmetabolites.
doi:10.1371/journal.pone.0136450.g001
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AntibacterialActionofQdNOs
tirapazamine(TPZ),sodium4,5-dihydroxybenzene-1,3-disulfonate(tiron),β-mercaptoethanol
(β-ME),xanthineoxidaseofE.coliK12,xanthine,oxypurinol,4-methylpyrazole,andraloxifene
werepurchasedfromSigma(StLouis,MO,USA).5,5-Dimethyl-1-pyrroline-N-oxide(DMPO),
N-tert-butyl-alpha-phenylnitrone(PBN)and2,2-dipyridylwereobtainedfromFluka(Buchs,
Switzerland).AuranofinanddicoumarolwerepurchasedfromJ&KChemicalCompany
(Shanghai,China).OxonicacidwasobtainedfromTokyoChemicalIndustry(Shanghai,
China).RotenoneanddimercaprolwerepurchasedfromAladdin(Shanghai,China).Allother
chemicalsandreagentscommerciallyavailablewereofthehighestanalyticalgrade.
Bacteriaandplasmids
EscherichiacoliCVCC196,E.coliCVCC216,E.coliCVCC220,E.coliCVCC223,E.coli
CVCC224,E.coliCVCC1500,E.coliCVCC1502,E.coliCVCC1513,E.coliCVCC1514,E.coli
CVCC1519,E.coliCVCC1496,E.coliCVCC2943,andE.coliAe1wereobtainedfromtheChina
InstituteofVeterinaryDrugControl(IVDC).E.coliATCC25922waspurchasedfromtheAmer-
icanTypeCultureCollection(ATCC).E.coliDH5αwaspurchasedfromBestBio(Shanghai,
China).SupercoiledplasmidpBR322andpUC-19werepurchasedfromTakara(Dalian,China).
Antibacterialsusceptibilitytest
Theminimuminhibitoryconcentration(MIC)ofQdNOsagainstE.coliwasdeterminedby
themicrodilutionmethodaccordingtotheM7-A8CLSIstandard[6].Thetestwasconducted
underaerobicoranaerobicconditions(85%N ,10%H ,and5%CO )at37°Cfor16to18h.
2 2 2
TheMICwasdefinedastheminimumdrugconcentrationintheclearwells.Todeterminate
theminimumbactericidalconcentration(MBC),100μloftheculturesfromtheclearwellsin
theMICtestwereplatedonagarbroth,andtheviablecellswerecounted.TheMBCwas
definedastheminimumdrugconcentrationatwhich99.95%ofthebacterialcellswerekilled.
Scanningelectronmicroscopy(SEM)
Bacteriawereexposedto0.5,1and4μg/mlCYAforindicatedtimesunderanaerobiccondi-
tions.Thebacteriawerethenfixedin2.5%glutaraldehydeat4°Cfor12h,andthensequen-
tiallydehydratedin20%~100%ethanol.Thecellswerethencollectedfreeze-driedfor3h,
gold-platedandsubjectedtoSEManalysis(HitachiS-570,Tokyo,Japan).
Microarrayanalysis
AnovernightcultureofE.coliCVCC2943wasdiluted1:100intoLuria-Bertani(LB)brothand
maintainedunderanaerobicconditionsfor1h.Aftertheadditionof0.5×MICandMBCof
CYA,MICandMBCofOLA,or1%(vol/vol)DMSO,andthecellswereincubatedat37°Cfor
30min.ThetotalbacterialRNAwasextractedusingtheTrizolreagent.
E.coliGenome2.0arrays(Affymetrix,SantaClara,CA,USA)wereusedforhybridization.
ThedatawereanalyzedusingthePartekGenomicsSuite(Partek,StLouis,MO,USA)andnor-
malizedwiththeRobustMultichipAveragealgorithm.Analysisofvariance(ANOVA)was
usedtoanalyzethelog-transformedlevels.Thedataqualitywasconfirmedbyprincipalcom-
ponentanalysis,histogramanalysisandbox-whiskerplots.Genesassociatedwitheither
>2-foldor<-2-foldchangeswithPvaluesoflessthan0.05wereconsidereddifferentially
expressedgenes.
Therelativetranscriptamountwasmeasuredbyquantitativereversetranscription-PCR
(qRT-PCR).TheprimersdesignedforsixselectedgenesarepresentedinTable1.Thefold
changesoftheselectedgeneswerenormalizedtothatofthereference16SrRNAgene.
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AntibacterialActionofQdNOs
Table1. Primersequencesforreal-timePCR.
Gene Sequence(5’-3’) Tm(°C)
Forwardprimer Reverseprimer
16S* GCTACAATGGCGCATACAAA TTCATGGAGTCGAGTTGCAG 56
recA CGGGTAACCTGAAGCAGTCCAA GGATGTCGAGACGAACAGAGGC 65
lexA ATTATGGATGGCGACTTGCTGG CAGTTCGACTTTATTGCCCTGTTTT 65
recN GTGGTGAATTGTCCCGCATCG CCGCTAATCCCTACATCCACTTC 65
sbmC GAAGGAGTGGGTTGCTGTCTATT GAATGACGCCCTCACTGTTTT 65
rbsC ACGAAAGCGTGGCTGATGGAG AATGGCGTTCACTGAGGTTTGC 63
treB GCGTTGATTAGCGGCGGTTTG TCGTAGATCGTTTGCAGGGAAGG 56
fliM CAGCGAAGTCAAAGACGAACCG GGCAAATTCATGGTACGGCTGA 65
Note:
*Primersof16SrRNAgeneweredesignedaccordingtothereference[30].
doi:10.1371/journal.pone.0136450.t001
Proteomicanalysis
E.coliCVCC2943cellsweretreatedwith0.5×MIC/MBCofCYAorMIC/MBCofOLAat
37°Cfor30minasdescribedabovein“Microarrayanalysis”.TheproteinsofQdNO-treatedE.
coliwereextractedmainlyaccordingtoPasqualietal.[7].Two-dimensionalgelelectrophoresis
(2-DE)wasperformedaccordingtoGörgetal.[8].ThegelswereanalyzedusingthePDQuest
software7.0(Bio-Rad).Theproteinspotsthatpresentinqualitativeorquantitativedifferences
wereexcisedfromthegelsanddigestedin-gelwithtrypsin[9].Matrix-assistedlaserdesorption
ionizationtime-of-flighttandemmassspectrometry(MALDI-TOFMS/MS)wasperformed
usinga4800ProteomicsAnalyzer(AppliedBiosystems,FosterCity,CA,USA).TheMSspec-
traweresearchedagainstUniProtKB/Swiss-ProtdatabaseusingMascot(MatrixScience,Lon-
don,U.K.).
DetectionoffreeradicalsfromE.coliexposedtoQdNOs
AnovernightcultureofE.coliCVCC2943wasinoculatedintofreshLBmedium(1:100)and
incubatedat37°Cfor1hwithshakingat250rpm.Thisculturewasusedforsubsequentdrug
treatments.Thebacterialculturewastreatedwith0.5×MIC,MICandMBCofQdNOsand
0.3%(anaerobiccondition)or10%(aerobiccondition)DMSOasacontrol.Afterincubation
withdrugsfortheindicatedtimesat37°C,thereactionswerestoppedat4°C.TodetectROS,
thebacteriacellswereresuspendedin500μlofPBScontaining10μM2’,7’-dichlorfluorescein-
diacetate(DCFH-DA)andincubatedinthedarkat37°Cfor40min.TheQdNO-treatedbacte-
riawerethenresuspendedin350μlofPBSandanalyzedusingafluorescencemicroplatereader
(Bio-Tek)withexcitationandemissionwavelengthsof490and530nm,respectively.Todetect
O -(cid:1)radicals,theQdNO-treatedbacteria(0.5μg/mlciprofloxacinasapositivecontrol[10])
2
wereresuspendedin500μlofPBScontaining10μMdihydroethidiumandincubatedinthe
darkat37°Cfor40min.Thecellpelletwasresuspendedin1mlofPBSforfluorescenceanaly-
siswithexcitationandemissionwavelengthsof310and600nm,respectively.
EffectsofradicalscavengersandenzymeinhibitorsonE.coliexposed
toQdNOs
AnovernightcultureofE.coliCVCC2943wasinoculatedintofreshLBmedium(1:100)and
incubatedat37°Cfor1hwithshakingat250rpm.Thisculturewasthenusedforsubsequent
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AntibacterialActionofQdNOs
drugtreatments.Theindicatedfreeradicalscavengersataconcentrationof100mMwerethen
addedtobacterialculture,andthisstepwasfollowedbytheadditionof4μg/mlCYAunder
anaerobicconditions.Afterincubationat37°Cfor3h,theviablecellcountswerecalculated.
BacteriawerepretreatedwiththeO -(cid:1)scavengertironataconcentrationof100mMandincu-
2
batedat37°Cfor15min.AfteradditionofCYAandincubationat37°Cfortheindicated
times,theviablecellswerecounted.
Thebacteriawereincubatedwith20μMrotenone(topreventelectrontransferfromtheFe-
Scentertoubiquinone)or40μMdimercaprol(toblockelectrontransferfromcytochromeb
tocytochromec )at37°Cfor0.5handthentreatedwith2×MICofenrofloxacin,CYAor
1
MEQat37°Cfor45minunderanaerobicconditionstodetectthelevelofROS.Underaerobic
conditions,thebacteriawereincubatedwith20μMrotenoneor40μMdimercaprolat37°Cfor
0.5handthentreatedwithMICofenrofloxacin,CYAorMEQat37°Cfor45mintodetectthe
levelofO -(cid:1)radicals.
2
Underanaerobicconditions,thebacteriawereincubatedwiththeindicatedenzymeinhibi-
tors,including2,2’-dipyridyl(ironchelator),oxypurinol(xanthineoxidaseinhibitor),4-methy-
pyrazole(alcoholdehydrogenaseinhibitor),dicoumarol(lipoamidedehydrogenaseinhibitor),
raloxifene(aldehydedehydrogenaseinhibitor),auranofin(thioredoxininhibitor)andoxonic
acid(uricaseinhibitor),ataconcentrationof20μMat37°Cfor0.5handthentreatedwiththe
MBCofCYAorMEQat37°Cfor3htocalculatetheviablecellcountsorfor0.5htodetect
thelevelofROS.Underaerobicconditions,thebacteriawereincubatedwith20μMofeach
inhibitorat37°Cfor0.5handthentreatedwith4μg/mlCYAat37°Cfor1htodetectthelevel
ofO -(cid:1)radicals.
2
DetectionofQdNO-inducedDNAdamage
E.coliDH5αcellsweretransformedwiththepUC-19orPBR322plasmid,andthebacteria
containingtheplasmidswerethengrowninLBmediaat37°Cfor8h.Theculturewascentri-
fugedat3,000gfor10min,andthepelletwassuspendedinPBSandincubatedwiththeindi-
catedconcentrationofCYAorOLAorwith128μg/mlTPZasapositivecontrol[11]at37°C
for2hunderanaerobicconditions.Toexaminetheeffectsoffreeradicalscavengersonthe
QdNO-inducedDNAdamage,E.colicellscontainingpUC-19werepretreatedwiththeindi-
catedradicalscavengersataconcentrationof100mM.Aftertheadditionof128μg/mlCYA,
thecellswereincubatedat37°Cfor2h.Theplasmidswereextractedandelectrophoretically
separatedon1%agarosegels.
Inthepresenceorabsenceofxanthineoxidase/xanthine(XO/X),pBR322(10μg/ml)was
incubatedwiththeindicatedconcentrationsofQdNOsat37°Cfor0.5hpriortoelectrophore-
sisanalysis.
Electronparamagneticresonance(EPR)analysis
EPRwasperformedusingaBrukerEMX-plusEPRspectrometer(Bruker,Germany)operated
at9.4GHz.CYAoritsmetabolitesand50μlofPBNorDMPOwerepre-degassedseparately
withN gas.TwohundredmicrolitersofCYA(1mg/ml)wasaddedwith2mlofbacterialcells,
2
extractedbacterialproteins,orXO/XrespectivelyintubesflushedwithN andthemixtures
2
weremaintainedinanoilbathat37°Cfor15min.TheEPRspectraweremonitoredatthefol-
lowingconditions:microwavefrequency,9.4GHz;microwavepower,18mW;modulation
amplitude,3.0G;fieldsweepfrom3000to3800G;timeconstant,80ms;conversiontime,30
ms.ThespectraweresimulatedusingtheWinEPRprogram(Bruker).
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AntibacterialActionofQdNOs
Results
TheantibacterialactivityofQdNOsagainstE.coli
E.colistrainsisolatedfrompigsorchickensweremoresusceptibletoQdNOsintheabsence
thaninthepresenceofoxygen(Table2),andE.coliCVCC2943,whichwaschosenforthesub-
sequentomicexperiments,wasthemostsusceptiblestrain.CYAwasmetabolizedintoCy1,
Cy2andCy10inE.coliCVCC2943underbothaerobicandanaerobicconditions(S1Fig,see
S1Textfordetailsoftheprocedure),indicatingthatQdNOwasmainlyreducedatthetwoN-
oxidegroupsonthequinoxalinering.TheN-deoxymetabolitesofCYAshowednoantibacte-
rialactivity(MIC>128μg/ml),indicatingthatthetwoN-oxidegroupswerenecessaryforthe
antibacterialactivityofQdNOs.
BacteriaexposedtoCYAbecameelongated(Fig2A–2C),indicatingthecelldivisionwas
halted.ThekillingofbacteriabecamemorerapidwiththeincreasingconcentrationofQdNOs
(Fig2D–2E),indicatingthatQdNOsareconcentration-dependentantibacterials.
TranscriptomicprofilesofE.coliCVCC2943exposedtoQdNOs
ThetranscriptomicprofilesofE.coliexposedtoCYAandOLAwerecompared.Twenty-five
geneswereactivatedby0.5×MICCYAbeyondthethresholdlevels(>2-foldchange),andthe
lexA-dependentgenesassociatedwiththeSOSresponserepresented60%ofalloftheup-regu-
latedgenes(Table3).SOSgenesweretriggeredbyCYAinadose-dependentmanner,and
threeotherSOSgenes(umuD,umuC,andruvB)wereup-regulatedbytheMBCofCYA(S1
Table).Morethan100genesclassifiedinsevenfunctionalcategorieswerealteredsignificantly
intheOLAprofiles,andapproximately20geneswereSOSgenes(S1Table).
Table2. AntibacterialactivitiesofCYAorOLAagainstE.colistrainsunderaerobicandanaerobicconditions.
(μg/ml)
E.colistrains Serotype CYA OLA
Aerobic Anaerobic Aerobic Anaerobic
MIC MBC MIC MBC MIC MBC MIC MBC
Control
ATCC25922 8 128 1 2 16 128 2 8
Swine-origin
CVCC196 O8:K87,K88ac 32 64 2 8 16 128 4 8
CVCC216 O8:K87,K88ad 32 64 4 16 32 128 8 16
CVCC220 O101:K32 32 128 2 8 16 128 4 8
CVCC223 O141:K99 32 64 1 8 16 >128 2 16
CVCC224 O149:K91,K88ac 32 64 2 16 32 128 4 8
CVCC1500 O149:K88ac 32 128 4 8 32 128 8 16
CVCC1502 O9:K88 32 128 4 16 32 64 4 16
CVCC1513 O101:K99 32 >128 1 8 16 128 2 8
CVCC1514 O45:K99 32 128 0.5 16 8 16 2 8
CVCC1519 O139 32 >128 2 32 32 128 8 16
Chicken-origin
CVCC1496 O139:K+ 32 128 2 32 NA NA NA NA
CVCC2943 O1 16 128 1 4 16 128 4 16
Ae1 32–64 128 4 32 NA NA NA NA
doi:10.1371/journal.pone.0136450.t002
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AntibacterialActionofQdNOs
Fig2.GrowthofE.coliCVCC2943exposedtoQdNOs.(A-C)MorphologyofE.coliCVCC2943exposed
toCYA.Bacteriaweretreatedwith1%DMSOasacontrolfor24h(A)orwith1μg/mlCYAfor4h(B)and24
h(C)underanaerobicconditions.ThebacterialmorphologywasobservedbySEMasdescribedinmaterials
andmethods.(D,E)KillingcurvesofE.coliCVCC2943exposedtoQdNOs.Thebacteriawereexposedto
0.5(0.5×MIC),1(MIC),and4μg/ml(MBC)CYA(D),and2(0.5×MIC),4(MIC),16μg/ml(MBC)OLA(E),or
with1%DMSOasacontrol.
doi:10.1371/journal.pone.0136450.g002
ThetranscriptionsofstressresponsegeneswasalsochangedintheQdNOprofiles
(Table3).marAencodingaDNA-bindingtranscriptionalregulatorwasup-regulated.yfaE
involvedinthesynthesisofribonucleosidediphosphatereductasewasup-regulated,whereas
treBandtreC,whichareinvolvedinthemetabolismofcarbohydrateoraminoacids,were
down-regulated.napF,whichparticipatesintheactivationofperiplasmicnitratereductase,
andhyaE,whichencodesthehydrogenaseisoenzyme1chaperone,weredown-regulated.
Amongthecellenvelopegenes,proVandrbsCencodingmembranetransportersweredown-
regulated,whereasbhsAandsmpA,whichencodeoutermembraneproteins,wereup-regu-
lated.Amongthevirulencegenes,fliMencodingaflagellaproteinwasdown-regulated.In
addition,yjbJ,whichisinvolvedinosmoticstress,andintTencodingaprophageintegrase
wereup-regulated.IntheOLAprofile,mdaB,dps,hdeAandsodB,whichareinvolvedinthe
oxidativestressresistance,bsmAandbhsA,whichareinvolvedinperoxideresistanceinbio-
film,andsdhC,sdhD,andcyoABC,whichareinvolvedinoxidativephosphorylationwereup-
regulated(S1Table).
FourinducedSOSgenes(recA,recN,sbmCandlexA)andthreedown-regulatedgenes(fliM,
rbsCandtreB)wereverifiedbyqRT-PCR(Table4).
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Table3. SelecteddifferentiallytranscribedgenesofE.coliexposedtoCYAorOLA.
Gene Relativechangeingenetranscription
CYA OLA
0.5×MIC MBC MIC MBC
SOSresponsea
c0943 12.13 44.19 31.84 62.06
recN 9.95 21.52 15.91 22.24
yebG 9.26 18.56 12.35 22.28
sulA 8.59 17.59 13.50 15.36
recA 7.46 11.42 9.70 13.88
yebF 7.22 20.03 9.17 15.67
lexA 4.07 4.54 4.30 4.68
dinI 3.86 5.01 4.57 5.47
recX 3.33 6.11 5.10 6.05
uvrB 3.18 5.15 3.05 6.75
uvrA 2.78 3.75 4.20 4.41
dinD 2.72 4.43 5.48 5.24
c3144 2.52 8.71 6.82 11.16
dinF 2.48 2.44 2.58 2.49
ydjQ 2.41 2.64 2.98 2.22
ruvA 2.39 2.43 2.30 2.43
ydjM 2.33 2.46 2.56 2.31
sbmC 2.24 2.74 4.27 2.91
polB 2.07 2.45 2.79 2.96
Otherstressresponsesb
yjbJ 2.47 3.11 1.69 2.88
intT 2.32 5.31 4.66 6.27
yafO 1.96 2.26 2.10 2.51
smpA 1.77 2.52 2.21 2.94
ydhC 1.61 2.89 1.52 2.10
marA 1.30 2.02 1.01 3.01
yfaE 1.25 2.41 2.54 2.84
bhsA 1.10 1.97 1.67 2.43
gyrA 1.30 1.79 1.57 1.73
gyrB 1.10 1.53 1.12 1.76
proV 1.05 -2.04 1.06 -2.15
treB -1.06 -2.80 -1.54 -2.82
fliM -1.12 -2.21 -1.89 -2.07
treC -1.13 -2.56 -1.59 -2.55
rbsC -1.14 -2.41 -1.40 -2.86
hyaE -1.76 -2.15 -1.77 -2.43
napF -1.78 -2.12 -3.22 -3.43
Numbersinboldindicatea2>-foldchangeor<-2-foldchange.0.5×MIC,MIC,andMBCindicatedrug
concentrations.
aDNAdamageresponsegeneswereselectedwitha>2-foldchangeinalldrugtreatmentgroups.
bStressresponsegeneswereselectedastheconsistentlyup-ordown-regulatedinadose-dependent
mannerforbothdrugs.
doi:10.1371/journal.pone.0136450.t003
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Table4. Validationofgenetranscriptionprofilesbyreal-timePCR.
Expressionpattern Gene Ratiobymicroarrayanalysis RatiobyRT-PCR
0.5×MICCYA MBCCYA MICOLA MBCOLA 0.5×MICCYA MBCCYA MICOLA MBCOLA
Conserved rrn 0.96 0.96 0.96 0.92 1 1 1 1
Up-regulated recA 7.46 11.42 9.70 13.88 5.97 12.41 8.60 19.05
lexA 4.07 4.54 4.30 4.68 2.62 2.53 3.18 2.29
recN 9.95 21.52 15.91 22.24 5.85 9.02 6.03 8.45
sbmC 2.24 2.74 4.27 2.91 2.54 2.70 2.38 2.17
Down-regulated rbsC 0.88 0.41 0.72 0.35 1.22 0.49 0.85 0.49
treB 0.94 0.36 0.65 0.35 1.16 0.40 0.83 0.32
fliM 0.89 0.45 0.53 0.48 1.29 0.46 0.79 0.49
0.5×MIC,MIC,andMBCindicatedrugconcentrations.
doi:10.1371/journal.pone.0136450.t004
ProteomicprofilesofE.coliCVCC2943exposedtoQdNOs
InpH3–10gels,differentialproteinswereobservedonlyintheCYAgroups(S2Fig,S2Table).
TheDNAprotection-relatedproteinDpswassignificantlyup-regulated.Abifunctionalpro-
tein(FolD),aNADH-quinoneoxidoreductasesubunit(NuoC/D),thesingle-strandedDNA-
bindingprotein(Ssb)andtherespirationnitratereductasebetasubunit(NarY)appeared.In
thepH4–7gels,overtwentyproteinspotsshowedalteredexpression(S2Fig,S3Table).Elon-
gationfactorTu1(EftU1)wasabsentordown-regulatedsignificantlyinQdNOprofiles.
Thesynthesisratesofsomeproteins(e.g.,EftU1,NuoC/D,YgfZ,PflB,NanE,IadA,NarY,
andFolD)weremarkedlymorepronouncedthanthoseoftheircorrespondingmRNAs.LexA
repressorwasdown-regulatedattheproteinlevelbutsignificantlyup-regulatedatthemRNA
levelbecauseLexAwasauto-digestedduringSOSresponse.
QdNOinducedfreeradicalsinE.coli
Underanaerobicconditions,ROSweredetectedinbacteriaexposedtoQdNOs(Fig3A).Dif-
ferentfromenrofloxacinandgentamycin,whichpushcellularrespirationintooverdriveto
producedangerousamountsofROS[10],theamountofROSinducedbyQdNOsreachedthe
highestlevelat0.5hbutdeclinedafterwards,indicatingthatROSmaybeobtainedfromtheN-
deoxymetabolismofQdNOs.Underaerobicconditions,QdNOsinducedareleaseofO -(cid:1)radi-
2
calsinbacteria(Fig3B).TheproductionofROSandO -(cid:1)radicalsincreasedwithanincreasein
2
theconcentrationofQdNOs(S3AFig).NoO -(cid:1)radicalswereobservedfortheN-deoxymetab-
2
olites(S3BFig),indicatingthatthereductiveactivationofQdNOisinvolvedinO -(cid:1)formation.
2
(cid:1)OHradicalswerenotdetectedinbacteriaexposedtoCYAunderanaerobicconditions,
whereascarbenicillininducedtheproductionof(cid:1)OHradicals,aspreviouslyreported[10](S4
Fig,seeS1Textfordetailsoftheprocedure).
Underanaerobicconditions,eighttypesoffreeradicalscavengerscouldinhibittheantibac-
terialactivityofCYAtocertainextents(Fig3C),indicatingthatthefreeradicalsplayanimpor-
tantroleintheantibacterialactionofCYA.Inparticular,β-MEandNaN presentedthetwo
3
highestinhibitioneffects.TheO -(cid:1)scavengertironpartiallyinhibitedtheantibacterialactivity
2
ofCYA(Fig3D),suggestingthatO -(cid:1)radicalsareinvolvedintheantibacterialactionofCYA
2
underaerobiccondition.
PLOSONE|DOI:10.1371/journal.pone.0136450 August21,2015 9/18
AntibacterialActionofQdNOs
Fig3.Intracellularfreeradicallevels(A,B)andeffectsoffreeradicalscavengers(C,D)onE.coliCVCC2943exposedtoQdNOs.(A)Under
anaerobicconditions,thebacteriaweretreatedwiththeMBCofCYA(4μg/ml),OLA(16μg/ml),MEQ(2μg/ml),enrofloxacin(0.03μg/ml),andgentamycin
(2μg/ml).Afterincubationfortheindicatedtimes,thelevelofROSwasdetected.(B)Underaerobicconditions,E.coliCVCC2943cellsweretreatedwith
2×MICofCYA(32μg/ml),OLA(32μg/ml),MEQ(4μg/ml),enrofloxacin(0.03μg/ml),andgentamycin(2μg/ml).Afterincubationofbacteriawiththedrugsfor
theindicatedtimes,thelevelofO -(cid:1)radicalswasdetected.(C)Underanaerobicconditions,thebacteriawerepretreatedwiththeindicatedfreeradical
2
scavengersataconcentrationof100mM,andCYAattheMBC(4μg/ml)wasthenadded.Sampleswerecollected3hafteradditionofthedrug,andthe
survivingbacteriawerecounted.(D)Underaerobicconditions,bacteriawerepretreatedornotpretreatedwithtironbeforetheadditionofindicated
concentrationofCYA.Attheindicatedtimes,thesurvivingbacteriawerecounted.Thedataarepresentedasthemeans±SDs(errorbars),n=3.ENR,
enrofloxacin;GET,gentamycin.
doi:10.1371/journal.pone.0136450.g003
OriginofQdNO-inducedradicalsinE.coli
TheexpressionofsomegenesandproteinsinvolvedintheTCAcycleandrespirationwere
alteredintheQdNOprofiles(Table5),thuswhethertheelectrontransferchainwasinterfered
byQdNOswasinvestigatedusingspecificinhibitors.Underanaerobiccondition,rotenoneand
dimercaprolinhibitedtheproductionofROSinalldrug-treatedgroups,buttheinhibition
ratiosofQdNOgroupswerelessthanthatoftheenrofloxacingroup(Fig4A),indicatingthat
theQdNO-inducedROScamefromotherorigin(s)exceptforrespiration-origin.Underaero-
bicconditions,inconsistentwiththeenrofloxacingroup,thetwoinhibitorscouldnotalleviate
thegenerationofO -(cid:1)radicalsintheQdNOgroups(Fig4B),indicatingthatmostoftheO -(cid:1)
2 2
radicalswereyieldedfromthemetabolismofQdNOs.
Theenzymeinhibitors2,2’-dipyridyl,oxypurinoland4-methypyrazoleimpairedtheanti-
bacterialpotencyofQdNOs(Fig4C).Oxypurinol,4-methylpyrazoleandauranofindecreased
PLOSONE|DOI:10.1371/journal.pone.0136450 August21,2015 10/18