Characterization of Antibacterial Activities of Eastern Subterranean Termite, Reticulitermes flavipes

17 Pages·2016·3.93 MB·English

RESEARCHARTICLE
Characterization of Antibacterial Activities of
Eastern Subterranean Termite, Reticulitermes
flavipes, against Human Pathogens
YuanZeng1,XingPingHu1*,Sang-JinSuh2*
1 DepartmentofEntomologyandPlantPathology,AuburnUniversity,Auburn,AL,UnitedStatesofAmerica,
2 DepartmentofBiologicalSciences,AuburnUniversity,AuburnUniversity,Auburn,AL,UnitedStatesof
America
*[email protected](SS);[email protected](XPH)
a11111
Abstract
Theemergenceanddisseminationofmultidrugresistantbacterialpathogensnecessitate
researchtofindnewantimicrobialsagainsttheseorganisms.Weinvestigatedantimicrobial
productionbyeasternsubterraneantermites,Reticulitermesflavipes,againstapanelof
OPENACCESS bacteriaincludingthreemultidrugresistant(MDR)andfournon-MDRhumanpathogens.
Citation:ZengY,HuXP,SuhS-J(2016) Wedeterminedthatthecrudeextractofnaïvetermiteshadabroad-spectrumactivity
CharacterizationofAntibacterialActivitiesofEastern againstthenon-MDRbacteriabutitwasineffectiveagainstthethreeMDRpathogensPseu-
SubterraneanTermite,Reticulitermesflavipes,
domonasaeruginosa,methicillin-resistantStaphylococcusaureus(MRSA),andAcineto-
againstHumanPathogens.PLoSONE11(9):
bacterbaumannii.Heatortrypsintreatmentresultedinacompletelossofactivity
e0162249.doi:10.1371/journal.pone.0162249
suggestingthatantibacterialactivitywasproteinaceousinnature.Theantimicrobialactivity
Editor:ErjunLing,InstituteofPlantPhysiologyand
changeddramaticallywhenthetermiteswerefedwitheitherheat-killedP.aeruginosaor
EcologyShanghaiInstitutesforBiologicalSciences,
CHINA MRSA.Heat-killedP.aeruginosainducedactivityagainstP.aeruginosaandMRSAwhile
maintainingorslightlyincreasingactivityagainstnon-MDRbacteria.Heat-killedMRSA
Received:May24,2016
inducedactivityspecificallyagainstMRSA,alteredtheactivityagainsttwootherGram-posi-
Accepted:August21,2016
tivebacteria,andinhibitedactivityagainstthreeGram-negativebacteria.Neitherthenaïve
Published:September9,2016
termitesnorthetermiteschallengedwithheat-killedpathogensproducedantibacterialactiv-
Copyright:©2016Zengetal.Thisisanopen ityagainstA.baumannii.Furtherinvestigationdemonstratedthathemolymph,notthehind-
accessarticledistributedunderthetermsofthe
gut,wastheprimarysourceofantibioticactivity.Thissuggeststhatthetermiteproduces
CreativeCommonsAttributionLicense,whichpermits
theseantibacterialactivitiesandnotthehindgutmicrobiota.Two-dimensionalgelelectro-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare phoreticanalysesof493hemolymphproteinspotsindicatedthatatotalof38and65pro-
credited. teinsweredifferentiallyexpressedatleast2.5-folduponbeingfedwithP.aeruginosaand
DataAvailabilityStatement:Allrelevantdataare MRSA,respectively.Ourresultsprovidethefirstevidenceofconstitutiveandinducible
withinthepaperanditsSupportingInformationfiles. activitiesproducedbyR.flavipesagainsthumanbacterialpathogens.
Funding:ThisstudywassupportedbytheNational
InstituteofFoodandAgriculture/UnitedStates
DepartmentofAgricultureHatchprogram(ALA015-1-
15005),unrestrictedindustryfunds,theAuburn
UniversityIntramuralGrantProgram,theAuburn
UniversityDetectionandFoodSafetyPeaksof
Excellenceprogram(USDA2005-3439415674A)and
NSFEPSCoR(NSF-EPS11-58862).
PLOSONE|DOI:10.1371/journal.pone.0162249 September9,2016 1/17
ProductionofAntibioticsbyReticulitermesflavipes
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Inrecentyears,insectshavebeenrecognizedforhavingpotentimmunedefensesthatproduce
constitutiveandinducibleantimicrobialcompoundstocombatvariouspathogens[1].Thus,
theyhavebeentargetedasapotentialsourceofantimicrobialcompounds[2,3].Insectspossess
compleximmuneresponsesthatactsynergisticallytoprovideprotectionagainstmicrobial
infections[4].Whenpathogensbreakthroughmorphologicalbarriers,insectsevokeinnate
immuneresponsescomprisedofcellularandhumoralreactions.Cellularreactionsarehemo-
cyte-mediatedandincludephagocytosisandencapsulation,whilehumoralreactionsinvolve
theproductionofantimicrobialproteinsandactivationofenzymaticcascades[5].Overthe
lastfewdecades,morethan150insectantimicrobialpeptides/proteins(AMPs)havebeeniden-
tifiedfromnaïve,microbe-challenged,orinjuredinsects[6].ReportedinsectAMPsinclude
lysozymes,cecropins,attacins,defensins,andprolinerichpeptides[6,7].
Recently,severalconstitutiveantimicrobialproteinsandpeptideshavebeenidentifiedfrom
threetermitefamilies:Termopsidae[8],Rhinotermitidae[9–12],andTermitidae[13–16].The
majorityofthesemoleculeshaveantifungalactivitiesandonlyafew,includingtermicin,defen-
sin-likepeptides,spinigerin,andlysozymes,haveweakantibacterialactivities[13,16].Hussain
etal.[17]reportedinductionofantibacterialactivityfromthewholebodyhomogenatesof
CoptotermesformosanusShirakiuponexposurewithvariousbacteria,includingahumanpath-
ogenStaphylococcusaureus.However,exposuretodifferentbacteriadidnotstimulateactivity
againsttheinducingorganismsexceptforBacillusthuringiensis.
Subterraneantermites(Blattodea:Isoptera:Rhinotermitidae),especiallytheReticulitermes
species,arewidelydistributedintheUnitedStates.Thesetermiteshavedevelopeddiseaseresis-
tancemechanismsthatfacilitatedtheirsurvivalandpropagationastheynestandforageinsoil
[18].Termite-producedAMPs,termicin(initiallyisolatedfromafungus-growingtermite)and
tGNBPs(termitegram-negativebindingproteins),havebeendescribedintheeasternsubterra-
neantermiteR.flavipesandthedarksouthernsubterraneantermiteR.virginicus[9,10,13].
GNBP2hasβ-1,3-glucanaseactivityintermitesandcontributestoexternalantifungaldefense
[13].We previouslyreportedthediscoveryofconstitutiveantibacterialactivityfromthecell-
freeextract(CFE)ofR.flavipesagainstacommonGram-positivesoil-borneentomopathogenic
bacterium,B.subtilis[12].Inthisstudy,wedeterminedthepresence,characteristics,andlevels
ofconstitutiveandinducibleantibacterialactivitiesinR.flavipesagainstapanelofhumanbac-
terialpathogensincludingthreecommonmultidrugresistantnosocomialpathogensandfive
non-MDRpathogens.
MaterialsandMethods
Termite maintenanceandinductionofantimicrobialactivity
R.flavipeswerecollectedontheAuburnUniversitycampusaspreviouslydescribed[12,19]
andworkersweremaintainedinUrbanEntomologyLaboratoryat25±2°Cforatleast20days
beforebeingsubjectedtoexperiments.To examinethepotentiallyinducibleantibacterialactiv-
ity,twoheat-killedMDRpathogens,Gram-negativeP.aeruginosaandGram-positiveMRSA,
wereselectedtostimulatetheimmuneresponse.BacteriaweregrowninLysogenyBroth(LB)
[20]at37°Cwithaerationovernight,subcultured,andgrowninfreshLBtoearly-midlog-
phase(OD =0.3±0.05).Twenty-fourmlofheat-killedbacterialsuspensionwasobtainedas
600
follows:cellsfrom48mlofculturewasharvestedviacentrifugationat13,200rpmfor5min,
washedtwicewith48mlofmilli-Q(MQ)water,resuspendedin24mlofMQwater(~6x108
cells/ml),andheat-killedat100°Cfor10min.R.flavipesworkersweresurfacesterilizedwith
70%ethanoltoeliminatesurfacemicrobesimmediatelybeforebeingsubjectedtotesting.To
PLOSONE|DOI:10.1371/journal.pone.0162249 September9,2016 2/17
ProductionofAntibioticsbyReticulitermesflavipes
immunizetheworkers,agroupof3gofsurface-sterilizedtermites(8groupspertreatment)
wasintroducedintoPetriplates(15cm×2.5cm)provisionedwithsterilefilterpapers(12.5
cmindiameter,Whatman#1;1filterpaperperPetridish)moistenedwith3mlofheat-killed
P.aeruginosaorMRSAsuspension,respectively.Sterilefilterpapersweremoisturizedwiththe
sameamountofMQwaterandwereusedasnegativecontrols.Thetermiteswereallowedto
feedinPetriplatesfor24hoursandthenharvestedforanalysis.
Preparationofwholebodyandsize-fractionatedCFE
Surfacesterilizednaïveandheat-killedpathogenchallengedtermiteworkers(24geach)were
suspended,separately,in120mlof20mMTris-HCl,20mMNaCl(pH=7.5)bufferand
homogenizedaspreviouslydescribed[12].ThecrudeCFEwasquicklyfrozeninliquidnitro-
genandlyophilized(HetoLyolab3000,ThermoFisherScientific,Pittsburgh,PA)at-57°C
overnightbeforebeingdissolvedinMQwatertoachievethefinalconcentrationofapproxi-
mately20mg/mlproteinconcentrationasdeterminedbytheBradfordassay(Bio-Rad,Hercu-
les,CA)[21].
Additionally,asampleofeachcrudeCFE(15ml)wassequentiallysizefractionatedwith
Microsep™AdvanceCentrifugalDevices(PallCorporation,PortWashington, NY)withthe
molecularweightcut-offs(MWCO)of100K,30K,10K,and3K.ThisseparatedtheCFEinto
fivefractionscontainingproteinswithapproximatemolecularweightof>300kDa,90–180
kDa,30–90kDa,10–20kDa,and<10kDa,respectively.Thefractionatedsolutionswere
lyophilizedanddissolvedinMQwatertoachievethefinalproteinconcentrationofapproxi-
mately20mg/mltomatchthatofthecrudeextract.Allsampleswerestoredat-80°Cuntilthe
antibacterialassays.
Proteindenaturation
To denatureproteins,5mlofthelyophilizedcrudeextractofeachtreatmentwasheatedto
100°Cforfiveminutesaspreviouslydescribed[12].Trypsindigestionwasperformedasfol-
lows.To 100μloflyophilizedcrudeextractforeachtreatment,5μlof200mMdithiothreitol
(DTT)in100mMNH HCO wasaddedandincubatedfor30minatroomtemperature.
4 3
Then,4μlofthe1Miodoacetamidealkylatingreagentwasaddedtothesample,mixed,and
incubatedfor45minatroomtemperature.Finally,50μloftrypsin(0.2μg/μlin100mM
NH HCO )wasaddedtothesampleandincubatedovernightat37°C.
4 3
Termite hemolymphcollectionandhindgutextraction
Hemolymphwasimmediatelydrawn(~0.05–0.1μl/individual)fromsurfacesterilizedtermites
byinsertingasterileinsectneedleintothedorsalintersegmentalmembraneofcold-immobi-
lizedinsects.Anysamplecontaminatedwiththegutorfatwasdiscarded.Theextractedhemo-
lymph(~200–400μl)wastransferredtoamicrocentrifugetubecontaining1mlof20mM
Tris-HCl,20mMNaCl(pH=7.5)bufferandkeptonice.Hindgutsforthesameindividuals
wereseparatedandrinsedin5mlof20mMTris-HCl,20mMNaCl(pH=7.5)bufferbefore
beinghomogenizedin1mlofbufferonice.Thehemolymphandgutextractswerecentrifuged
at12,000rpmat4°Cfor5mintoacquirethecell-freesamples.Theproteinconcentrationof
eachsamplewasmeasuredandwasadjustedtothefinalconcentrationof25mg/mlbyeither
dilutionorconcentrationfollowinglyophilization.
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ProductionofAntibioticsbyReticulitermesflavipes
Table1. Listofthetestedbacteria.
Bacterium GramStain Multi-DrugResistant SourceorReference
Staphylococcusaureus + No ATCC12600viaRobertMiller
Methicillin-resistantStaphylococcusaureus(MRSA) + Yes JamesBarbaree
Streptococcuspyogenes + No ATCC19615viaRobertMiller
Pseudomonasaeruginosa(PAO1) - Yes [22]
EscherichiacoliO157:H7(CDCB1409-C1) - No ATCC43889
E.coliK-12(MG1655) - No ATCC700926
SalmonellaentericaserovarTyphimurium(LT2) - No [23]viaJorgeEscalante-Semerena
Acinetobacterbaumannii(AYE) - Yes ATCCBAA-1710[24]
doi:10.1371/journal.pone.0162249.t001
Antibacterialassay
Antibacterialactivityofeachextractagainstapanelofselectedbacteria(Table 1)wasdeter-
minedusingamodifiedinhibitionzoneassayaspreviouslydescribed[12].Foreveryassay,
eachbacteriumwasfreshlygrownfromafrozenstock.Briefly,acolonyfromafreshlystreaked
platewasinoculatedintoLBmedium,grownovernightat37°Cwithshakingat~220rpm,sub-
culturedthenextdayintofreshLBmediumandgrownat37°Cwithshakingat~220rpmto
earlylog-phaseofgrowth(OD =0.3±0.05),anddilutedto~2.5×107CFU/ml.Theantibac-
600
terialactivitiesofcrudeextractswereexaminedbyplacingeightsterilizedfilterpaperdisks
(5×5mm)uniformlyonthebacteriallawnineachplate.Thepaperdisksweretreatedwith
oneofthefollowingeightsamples,respectively:20μlofthreecrudeextracts(naïve,P.aerugi-
nosa-challenged,andMRSA-challenged)ataconcentrationofapproximately20mg/ml
(=400μg/disk),20μlofthreeheat-treatedcrudeextracts(naïve,P.aeruginosa-challenged,
andMRSA-challenged),1μlofampicillin(=25μg/disk)asthepositivecontrol,and20μlof
80mMTris-HCl,80mMNaClbufferasthenegativecontrol.Theantibacterialactivitiesof
size-fractionatedextracts,hemolymph,andgutextractweredeterminedusingthesameassay.
Allplateswereincubatedat37°Cfor24htoallowbacterialgrowthandthezonesofclearing
weremeasured.Everyassaywasrepeatedthreetimes,eachwith3replicates,toacquireanNof
9foreachtreatment.
Statisticalanalysis
Thediameters(D;mm)ofinhibitionzoneswerecomparedusingtheANOVA andTukey’s
method(PROCGLM;α=0.05;SAS9.2)todetermineallpossiblepairwisedifferencesamong
treatments.TheANOVA (PROCGLM;α=0.05;SAS9.2)wasusedtodeterminethedifference
inactivitiesofthesametreatmentoneachtestedbacterium.
Gelelectrophoreticanalysisofproteins
ProteinprofilesoftheMWCOof30Kand100KfractionatedsamplesoftermiteCFEwereana-
lyzedbypolyacrylamidegelelectrophoreses(PAGE) includingbothnon-denaturingNative
PAGE anddenaturingSDS-PAGE. Approximately20μgofproteinsampleswereloadedper
lane.TheproteinladdersforNativePAGE (14–500kDa)andSDS-PAGE (10–250kDa)were
purchasedfromSigma-Aldrich(StLouis,MI)andLifeTechnologies(GreenIsland,NY),
respectively.Inaddition,non-sizefractionatedhemolymphsampleswereanalyzedon
SDS-PAGE. TheproteinsonPAGE werestainedwithCoomassiebrilliantblueR-250(Bio-
Rad,Hercules,CA)forvisualization.
Two-dimensionalgelelectrophoreticanalysisoftheproteinsinhemolymphwasperformed
bytheKendrickLabs,Inc.(Madison,WI).Approximately200μgoftotalproteinpersample
PLOSONE|DOI:10.1371/journal.pone.0162249 September9,2016 4/17
ProductionofAntibioticsbyReticulitermesflavipes
wasusedtoseparatetheproteinsbetweenpIof3to10forthefirstdimensionandmolecular
weightof14–220kDafortheseconddimension.Duplicategelswererunforeachsample,
stainedwithSypro1Ruby(Bio-Rad),andscannedonaTyphoonFLA9000scanner(GE
Healthcare,Piscataway, NJ).Atotalof493spotswereanalyzedusingProgenesisSameSpots
software(version4.5,2011,TotalLab, UK)andProgenesisPG240software(version2006,
TotalLab, UK).Thequantityofeachspotwascalculatedasspotpercentages(individualspot
densitydividedbytotaldensityofallmeasuredspots).Thequantitydifferencesbetween
MRSA-challengedversusnaïveandP.aeruginosa-challengedversusnaïvewereanalyzedusing
two-samplet-test.
Results
Broad-spectrumconstitutiveantibacterialactivityofR.flavipes
InordertounderstandthebiologicalrangeofantibacterialactivityofR.flavipesCFE,wetested
apanelofnon-MDRandMDRhumanbacterialpathogensfortheirsusceptibility.Table 1
showsthepanelofbacteriausedinthisstudywhichincludesbothgram-positiveandgram-
negativebacteria.TheCFEpreparedfromthewholebodyofnaïvetermitedisplayedsignificant
inhibitoryactivityagainstthefivenon-MDRbacteria(Staphylococcusaureus,Streptococcus
pyogenes,EscherichiacoliO157:H7,SalmonellaentericaserovarTyphimurium,andE.coliK-
12)butitwasinactiveagainstthethreeMDRpathogens(methicillinresistantS.aureusor
MRSA,Pseudomonasaeruginosa,andAcinetobacterbaumannii)(Table2).Ofthefivesuscepti-
blebacteria,thestrongestinhibitoryeffectwasagainstS.aureus,followedbyE.coliO157:H7
andS.Typhimurium.TheweakestactivitywasagainstS.pyogenes.Asexpected,ampicillin
inhibitedthegrowthofallbutthethreeMDRpathogens.We observednoobviouscorrelation
betweenGram-stainingandtheeffectivenessofthetermiteCFEongrowthinhibition.The
antibacterialactivityoftermiteCFEdisappearedcompletelywhenitwasheat-denatured
(Table 2)ortreatedwithtrypsin(datanotshown).Thesedatasuggestthattheantibacterial
activityofR.flavipesislikelytobeproteinaceousinnature.
MDR-inducedalterationinantibacterialactivitiesofR.flavipes
Inadditiontotheconstitutiveantibacterialactivity,wedeterminedthatR.flavipespossesses
inducibleantibacterialactivities.Feedingtermiteswithheat-killedP.aeruginosaorMRSA
Table2. AntibacterialactivityofCFEofnaïvetermites.
Bacteria Termitecrudeextract(400μg) Ampicillin(25μg) Heat-treatedcrudeextract Control*
Non-infectious E.coli 9.41±0.37d 8.49±0.33e 0 0
Infectious Non-MDR S.aureus 16.16±0.73a 27.99±0.4a 0 0
S.pyogenes 9.14±0.30d 16.41±0.36b 0 0
E.coliO157:H7 14.51±0.57b 11.28±0.24d 0 0
S.Typhimurium 10.42±0.50c 12.39±0.28c 0 0
MDR MRSA 0 0 0 0
P.aeruginosa 0 0 0 0
A.baumannii 0 0 0 0
Differentletterswithinacolumnindicatesignific antdifferenceininhibitionzonediametersatsignifican celevelofα=0.05(ANOVA,F =
4,40(CrudeExtract)
1854.68,P<0.0001;F =2960.78,P<0.0001).TheexperimentwasperformedthreeindependenttimeswithtriplicatesamplesforatotalNof9
6,56(Ampicillin)
foreachtreatment.Thediameterofzoneofinhibitionwasmeasuredfollowing24hincubationat37°C.
*Control:80mMTris-HCl,80mMNaClbuffer.
doi:10.1371/journal.pone.0162249.t002
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ProductionofAntibioticsbyReticulitermesflavipes
alteredtheirantibacterialactivitiesandstimulatedspecificanti-MDRactivityasillustratedin
Fig1.Inbothcases,thetermitesproducednewantibacterialactivitythatwaseffectiveagainst
theinducerbacterium.Specifically,termitechallengedbyheat-killedP.aeruginosaproduced
activityagainstbothP.aeruginosaandMRSAwhilemaintainingorslightlyincreasingantibac-
terialactivityagainstbacterialistedinTable 1.Termiteschallengedbyheat-killedMRSApro-
ducedanti-MRSAactivitywhilemaintainingactivityagainsttwonon-MDRGram-positive
pathogens.Interestingly,antibacterialactivityagainstGram-negativebacterialistedinTable 1
wascompletelyabolishedinMRSA-challengedtermites(Fig1).NeitherP.aeruginosanor
MRSAinducedantibacterialactivityagainstA.baumannii inR.flavipes(datanotshown).
Sizefractionationofantibacterialactivities
We previouslydemonstratedthatmultiplefractionsofsize-fractionatedR.flavipesCFEhad
antibacterialactivityagainstthesoil-borneentomopathogenicB.subtilis[12].Thisindicated
thatR.flavipesproducedmultipleproteinswithanti-B.subtilisactivity.Incontinuingourchar-
acterization,wetestedsize-fractionatedCFEfromthenaïve,P.aeruginosachallenged,and
MRSAchallengedR.flavipesagainstthepanelofbacterialistedinTable 1.We determinedthat
Fig1.AntibacterialactivitiesofcellfreeextractsofR.flavipes.Approximately400μgofCFEofthenaïve,P.aeruginosa-challenged,andMRSA-
challengedR.flavipeswasappliedrespectivelytoeachfilterdiskonabacteriallawn.Thezoneofinhibitionofgrowthwasmeasuredfollowingincubation
for24hoursat37°C.Ampicillinwasusedasthepositivecontrolat25μgperfilterdiskand20μlofbufferwasusedasthenegativecontrol.Thedata
shownareacompilationofthreeindependentexperimentsdoneintriplicateforatotalNof9persample(ANOVA,F ,P<0.0001).Datafor
27,224=2635.47
A.baumanniiarenotshownbecausethetermiteextractswereineffectiveagainstthebacterium.
doi:10.1371/journal.pone.0162249.g001
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ProductionofAntibioticsbyReticulitermesflavipes
allantibacterialactivitiesofthreegroupsoftermitesagainstthispanelofbacteriawerecon-
tainedinfractionswithproteinslargerthan90kDainmolecularweight(MWCOfiltersof30K
and100K;S1Fig).However,manyoftheselargeproteinsappearedtobecomposedofsmaller
subunitsbecausetheymigratedasproteinsof25–90kDaondenaturingSDS-PAGE (S2Fig).
Inallthreegroupsoftermites,theMWCO100Kfractionscontainingproteinsof>300kDa
demonstratedgreateractivityagainstthesusceptiblebacteriathantheMWCOof30Kfractions
with90–180kDaproteins(Fig2).ThesedatasupportourpreviousfindingthatR.flavipespos-
sessesmultipleproteinswithantibacterialactivity.
TheP.aeruginosa-inducedantibacterialactivitiesagainstthesevensusceptiblebacteria
wereallduetotheproteinfractionfromtheMWCO100Kfilterexceptagainsttheinducerbac-
terium.Theanti-P.aeruginosaactivitywaspresentinboththeMWCO100Kand30Kfrac-
tions,indicatingthepossibilityofinductionofmultipleanti-P.aeruginosaproteins.In
addition,theP.aeruginosa-challengedtermitesexhibitedmoreeffectiveantibacterialactivityin
theMWCO100Kproteinfractionagainstthosenon-MDRbacteriathandidthenaïvetermites.
TheincreasedactivityintheMWCO100KfractionwasespeciallyevidentagainstE.coliK-12
andS.aureus,indicatingthattheinductionofantibacterialactivitybyP.aeruginosawasinde-
pendentoftheGramstaining-basedclassificationofbacteria.Interestingly,P.aeruginosa
inducedgreateranti-MRSAactivityintheMWCO100KfractionthandidMRSA.
SimilartoP.aeruginosainduction,theMRSA-inducedantibacterialactivitywascontained
intheMWCO100Kfractionwiththeexceptionofanti-MRSAactivitywhichwaspresentin
boththeMWCO30Kand100Kfractions.
OriginofR.flavipesantibacterialactivity
Inordertodeterminewhethertheantibacterialactivitieswereoftermiteoriginorofthegut
microbiota,wepreparedextractsfromthehindgutandcomparedtheactivitytothehemo-
lymph.AsillustratedinFig3,theantibacterialactivityagainstourpanelofbacteriawereonly
observedinthehemolymphofthenaïve,P.aeruginosa-challenged,orMRSA-challenged.
Hindgutextractsfromthesametermitesshowednoperceivedantibacterialactivities(datanot
shown).Theantibacterialactivityprofileofthehemolymphresembledthatofthewhole-body
extractshowninFig1.Ourdatasuggestthatbothconstitutiveandinducibleantibacterial
activitiesofR.flavipesarelikelyoftermiteoriginandresideinhemolymph.
Proteinprofilesoftermitehemolymph
We analyzedthehemolymphproteinprofilesofnaïve,P.aeruginosa-challenged,andMRSA-
challengedtermitesviabothone-dimensionalandtwo-dimensionalgelelectrophoresestoiden-
tifydifferentiallyexpressedproteins.Onone-dimensional8%NativePAGE, weobservedvery
littledifferencebetweenthenaïve,MRSA-challenged,andP.aeruginosa-challengedtermitesfor
both100Kand30KMWCOfractionatedsamples(S1Fig).Ondenaturing8%SDS-PAGE, we
observedsubtledifferencesbetweenthethreesamplesandbetweenthe100Kand30KMWCO
fractionatedsamples(S2Fig).Specifically,therewasaslightupregulationof~50kDa,~110
kDaand~150–200kDaproteins,andaslightdownregulationof~35kDa,~55kDaproteinsin
MRSA-challengedtermites.ForP.aeruginosa-challengedtermites,weobservedaslightupregu-
lationof~80kDa,~50kDa,and~35kDaproteinsandaslightdownregulationof>250kDa
and~60kDaproteins.Whenthehemolymphsampleswereanalyzedon8%SDS-PAGE, we
observedtwoabundantproteinsbetween~60–85kDathatappearedtobeconservedamong
thenaïve,P.aeruginosa-challenged,andMRSA-challengedtermites.We alsoobservedsome
differencesbetweenthreesamplesbutespeciallyforP.aeruginosa-challengedtermitesinwhich
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Fig2.Antibacterialactivityofsize-fractionatedcellfreeextractsofR.flavipes.Antibioticactivitywas
measuredasdiameterofinhibitionzonescausedbyrespectiveapplicationofapproximately400μgofsize-
fractionatedCFEfromthenaïve,P.aeruginosa-challenged,andMRSA-challengedR.flavipesonabacteriallawn.
ThedatashownareacompilationofthreeindependentexperimentsdoneintriplicateforatotalNof9persample
(ANOVA,F ,P<0.0001).(A)MWCO30K(90–180kDa)fraction.(B)MWCO100K(>300kDa)
27,224=2635.47
fraction.Dataforfractionsof<10kDa,10–20kDa,30–90kDaarenotshownbecausetheydidnotdemonstrate
antibacterialactivities.
doi:10.1371/journal.pone.0162249.g002
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ProductionofAntibioticsbyReticulitermesflavipes
Fig3.AntibacterialactivityofR.flavipeshemolymph.Approximately400μgofhemolymphextractfromthenaïve,P.aeruginosa-challenged,and
MRSA-challengedR.flavipeswasappliedrespectivelyonabacteriallawn.Thezoneofinhibitionwasmeasured.Ampicillinwasusedasthepositive
controlat25μgperfilterdisk.ThedatashownareacompilationofthreeindependentexperimentsdoneintriplicateforatotalNof9persample
(ANOVA,F =9762.44,P<0.0001).
41,336
doi:10.1371/journal.pone.0162249.g003
wesawdisappearanceofaprominentbandof~250kDaandappearanceof~35kDa,~50kDa,
and~65kDa(S3Fig).
Itwasclearfromouranalysesthattermitehemolymphwastoocomplextobeaccurately
analyzedviaone-dimensionalPAGE. Thus,weperformedtwo-dimensionalelectrophoretic
analysesofthenaïve,P.aeruginosa-challenged,andMRSA-challengedtermitehemolymphs.
Basedonourdata,thetermitehemolymphcontainsapproximately493proteinsthatarevisible
ontwo-dimensionalgelstainedwithSypro1Ruby. Acomparisonofthehemolymphproteins
betweenthenaïve(Fig4A)andtheP.aeruginosa-challengedtermites(Fig4B)indicatedthat
38proteinsweredifferentiallyexpressedatleast2.5-fold(P<0.05).Ofthese,18proteinswere
upregulatedand20proteinsweredownregulated(S1Table).Acomparisonofthenaïveter-
mites(Fig5A)andtheMRSA-challengedtermites(Fig5B)showedthat65proteinsweredif-
ferentiallyexpressedatleast2.5-fold(P<0.05).Ofthese,11proteinswereupregulatedand54
proteinsweredownregulated(S2Table).
InP.aeruginosa-challengedtermites,thehighestupregulatedprotein(approximately
11-foldincrease)hadMWofapproximately37kDa.The20downregulatedproteinsdisplayed
nodiscernablepatterninsize.InMRSA-challengedtermites,themajorityofupregulatedpro-
teins(11spots)hadMWofapproximately18to58kDawhiledownregulatedproteins(53
PLOSONE|DOI:10.1371/journal.pone.0162249 September9,2016 9/17
ProductionofAntibioticsbyReticulitermesflavipes
Fig4.Two-dimensionalelectrophoreticanalysisofhemolymphproteinsfromnaïveandP.aeruginosa-challengedR.flavipes.Approximately
200μgofhemolymphproteinswereseparatedbytwo-dimensionalgelelectrophoresisandvisualizedwithSypro1Ruby.Bluecirclesindicateprotein
spotsthatareupregulatedinP.aeruginosachallengedtermitewhileredcirclesindicateproteinspotsthataredownregulated.(A)Naïvetermites.(B)P.
aeruginosa-challengedtermites.
doi:10.1371/journal.pone.0162249.g004
Fig5.Two-dimensionalelectrophoreticanalysisofhemolymphproteinsfromnaïveandMRSA-challengedR.flavipes.Approximately200μgof
hemolymphproteinswereseparatedbytwo-dimensionalgelelectrophoresisandvisualizedwithSypro1Ruby.Bluecirclesindicateproteinspotsthatare
upregulatedinMRSAchallengedtermiteandredcirclesindicateproteinspotsthataredownregulated.(A)Naïvetermites.(B)MRSA-challenged
termites.
doi:10.1371/journal.pone.0162249.g005
PLOSONE|DOI:10.1371/journal.pone.0162249 September9,2016 10/17