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The
Vibrio
campbellii
quorum
sensing
signals
have
a
different
impact
on
virulence
of
the
bacterium
towards
different
crustacean
hosts
Gde
Sasmita
Julyantoro
Pande
a,b,
Fatin
Mohd
Ikhsan
Natrah
c,d,
Patrick
Sorgeloos
a,
Peter
Bossier
a,
Tom
Defoirdt
a,e,*
aLaboratoryofAquaculture&ArtemiaReferenceCenter,GhentUniversity,Rozier44,9000Gent,Belgium
bDepartmentofAquaticResourcesManagement,FacultyofMarineScienceandFisheries,UdayanaUniversity,BukitJimbaranCampus,
80361Bali,Indonesia
cDepartmentofAquaculture,FacultyofAgriculture,UniversityPutraMalaysia,43400Serdang,Selangor,Malaysia dLaboratoryofMarineBiotechnology,InstituteofBioscience,UniversityPutraMalaysia,Malaysia
eLaboratoryofMicrobialEcologyandTechnology,GhentUniversity,Belgium
1. Introduction
Pathogenic bacteria are often capable of infecting multiple host species (Faruque et al., 1998; Salanoubat etal.,2002;Heetal.,2004;Seshadrietal.,2006;Defoirdt et al., 2007).Thiswillrequire somelevelof flexibilityin sensing of and responding to the environment sincethe bacteriawillexperiencedifferentenvironmentalconditions in different hosts (especially when these are distantly related). One of the regulatory mechanisms involved in interacting with the environment is quorum sensing, a mechanisminwhichbacteriacoordinatetheexpressionof
certaingenes inresponsetothe presenceofsmallsignal molecules(Hense et al., 2007). Quorumsensing systems havebeenreportedtoregulatetheexpressionofvirulence genesinmanypathogenicbacteria,andmanyofthemuse different signal molecules to regulate virulence gene expression(Jayaramanand Wood, 2008;Ng and Bassler, 2009).However,itisoftennotclearwhydifferentsignalsare used, especially when they are controlling the same virulencefactorsandwhentheyareusingacommonsignal transductioncascade,asisthecaseinvibrios(Milton,2006).
Vibrio campbelliiBB120 (=ATCC BAA-1116; previously designated Vibrio harveyi (Lin et al., 2010)) and closely relatedbacteriaareamongstthemostimportantpathogens of aquatic organisms, causing significant losses in the aquaculture industry worldwide (Ruwandeepika et al., 2012).Thesepathogenscaninfectmultiplehostsbelonging
ARTICLE INFO
Articlehistory: Received3July2013
Receivedinrevisedform21August2013 Accepted23August2013
Keywords: Quorumsensing Vibriosis
Host–microbeinteraction Host–pathogeninteraction
ABSTRAC T
Pathogenicbacteriacommunicatewithsmallsignalmoleculesinaprocesscalledquorum sensing, and they often use different signal molecules to regulate virulence gene expression.Vibriocampbellii,oneofthemajorpathogensofaquaticorganisms,regulates virulencegeneexpressionbyathreechannelquorumsensingsystem.Hereweshowthat althoughtheyusea commonsignaltransductioncascade,the signalmoleculeshavea differentimpactonthevirulenceofthebacteriumtowardsdifferenthosts,i.e.thebrine shrimp Artemia franciscana and the commercially important giant freshwater prawn
Macrobrachiumrosenbergii.Theseresultssuggestthattheuseofmultipletypesofsignal
moleculestoregulatevirulencegeneexpressionisoneofthefeaturesthatallowbacteria toinfectdifferenthosts.Ourfindingsemphasizethatitishighlyimportanttostudythe efficacyofquorumsensinginhibitorsasnovelbiocontrolagentsunderconditionsthatare ascloseaspossibletotheclinicalsituation.
ß2013ElsevierB.V.Allrightsreserved.
*Correspondingauthor.Tel.:+32092643754;fax:+32092644193. E-mailaddress:Tom.Defoirdt@UGent.be(T.Defoirdt).
ContentslistsavailableatScienceDirect
Veterinary
Microbiology
j o urn a l hom e pa ge : ww w. e l se v i e r. c om / l oca t e / v e t mi c
to distantly related taxa, ranging from mollusks over crustaceans tofish (Defoirdt et al., 2007). The species is also one of the model organisms in studies on quorum sensing in bacteria (Ng and Bassler, 2009). V. campbellii
BB120 contains athree-channel quorumsensingsystem, withthreedifferenttypesofsignalmolecules(HAI-1,AI-2 andCAI-1,respectively)feedingacommonsignal transduc-tion cascade (Fig. 1). HAI-1, harveyi autoinducer-1, is 3-hydroxybutanoyl-L-homoserinelactone;AI-2,
autoinducer-2, isthefuranosyl boratediester 3A-methyl-5,6-dihydro-furo(2,3-D)(1,3,2)diox-aborole-2,2,6,6A-tetraol;andCAI-1, cholerae autoinducer-1, is (Z)-3-aminoundec-2-en-4-one (NgandBassler,2009;Ngetal.,2011).V.campbelliiquorum sensinghasbeendocumentedtocontroltheexpressionof different virulence genes, including vhp metalloprotease (Moketal.,2003;Ruwandeepikaetal.,2011),asiderophore (Lilley and Bassler, 2000), type III secretion system components (Henke and Bassler, 2004a), chitinase A (Defoirdt et al., 2010) and three phospholipase genes (Natrah et al., 2011). We previously reported that AI-2 andCAI-1,butnotHAI-1,areneededforfullvirulenceofthis bacterium towards brine shrimp larvae (Defoirdt et al., 2005;DefoirdtandSorgeloos,2012).Here,wereportthat HAI-1andAI-2,butnotCAI-1,areneededforfullvirulence towards the commercially important giant freshwater prawnMacrobrachiumrosenbergii.Thesedatasuggestthat the useof differenttypesof signalmolecules toregulate virulencegeneexpressionisoneofthefeaturesthatallow bacteriatoinfectdifferenthosts.
2. Materialsandmethods
2.1. Bacterialstrainsandgrowthconditions
V.campbelliistrainsusedinthisstudyaredescribedin Table1.Allstrainswerestoredat 808Cin40%glycerol
and the stocks were streaked onto Luria-Bertani agar containing 12gl 1 Instant Ocean synthetic sea salt (Aquarium Systems Inc., Sarrebourg, France) (LB12) or
35gl 1InstantOcean(LB
35)forusein giantfreshwater
prawn and brine shrimp, respectively. After 24h of incubationat 288C, asinglecolonywasinoculatedinto 5ml fresh LB broth with appropriate salinity and incubated overnight at 288C under constant agitation (100min 1).
2.2. SelectionofrifampicinresistantmutantsofV.campbellii BB120andMM77
100
m
lofdenselygrownculturesofBB120andMM77(OD600of1)wereinoculatedintotubescontaining5mlof
freshLB12brothsupplementedwith50mgl 1rifampicin
(Sigma)andincubatedfor5daysat288Cunderconstant agitation(100min 1).Thegrowncultureswereinoculated
into fresh LB12 broth with 50mgl 1 rifampicin and
incubatedovernight. The grown cultureswere stored at 808Cin40%glyceroluntiluse.
2.3. Signalmolecules
HAI-1, N-3-hydroxybutanoyl-L-homoserine lactone
(Sigma) was dissolvedin distilled water at 1000mgl 1.
AI-2 precursor (S)-4,5-dihydroxy-2,3-pentadione (DPD) was obtained from OMM Scientific Inc. (Dallas, Texas, USA).
2.4. Axenichatchingofbrineshrimp
Decapsulationandhatchingofaxenicbrineshrimpwas performedasdescribedpreviously(Defoirdtetal.,2005), with some modifications. Briefly, 200mg cysts (Ocean NutritionEurope,Essen,Belgium)werehydratedina50ml tubecontaining18mldistilledwaterfor1h.Sterilecysts wereobtainedviadecapsulationusing660
m
lNaOH(32%)and10mlNaOCl(50%).Thesuspensionwasgentlyshaken underalaminarflowhood.Thereactionwasstoppedafter 2min by adding 14ml Na2S2O3 (10gl 1). The
decapsu-latedcystswerewashedwithfresh autoclavedsynthetic seawater(35gl 1InstantOcean)overa100
m
msieveandtransferred to two sterile 50ml tubes, each containing
Fig.1. QuorumsensinginVibriocampbellii.TheLuxM,LuxSand CqsA enzymessynthesizetheautoinducersHAI-1,AI-2andCAI-1,respectively. TheseautoinducersaredetectedatthecellsurfacebytheLuxN,LuxQand CqsStwo-componentreceptor proteins,respectively.DetectionofAI-2 by LuxQ requiresthe periplasmicprotein LuxP. The receptors feed a commonphosphorylation/dephosphorylationsignaltrandusctioncascade regulatingtheexpressionoftargetgenes.‘‘P’’denotesb,cphosphotransfer.
Table1
Vibriocampbelliistrainsusedinthisstudy.
Strain Relevantinformation Reference
BB120 Wildtypefromwhichall otherstrainsarederived
Bassleret al.(1997)
BB120RifR Spontaneousrifampicin resistantmutantofBB120
Thisstudy
BB152 MutationinluxM (HAI-1synthase)
Bassleret al.(1994)
MM30 MutationinluxS (AI-2synthase)
Suretteet al.(1999)
MM77 MutationinluxMandluxS Suretteetal.(1999) MM77RifR Spontaneousrifampicin
resistantmutantofMM77
Thisstudy
JMH603 MutationincqsA (CAI-1synthase)
HenkeandBassler (2004b)
30ml sterile synthetic sea water. The tubes with dec-apsulatedcystswereincubatedat288Cfor24honarotor underconstantlight.Forbrineshrimplarvaetobeusedas feedforgiantriverprawnlarvae,brackishwater(12gl 1
InstantOcean)wasused.
2.5. Brineshrimpchallengetest
The brine shrimp challenge test was performed as described previously (Defoirdt et al., 2006). Briefly, the shrimpwereculturedingroupsof20larvaeinglasstubes containing 10ml synthetic sea water (35gl 1 Instant Ocean).Thelarvaewerefedanautoclaved suspensionof
Aeromonas sp. LVS3 bacteria at 107cellsml 1 and V.
campbelliistrainswereaddedat105CFUml 1.
2.6. Giantriverprawnchallengetests
Twodifferentprawnbroodstocksweremaintainedat our laboratory (further denoted broodstock 1 and 2, respectively). Prawn broodstock maintenance was per-formed according to Cavalli et al. (2001) and water quality parameters were adjusted according to New (2003).Thelarvaewereobtainedfromasingleoviparous female breeder. A matured female which had just completed its pre-mating moult was mated with a hard-shelled male as described before (Baruah et al., 2009). The female with fertilized eggs was then maintained for 20–25 days to undergo embryonic development. When theeggswerefully ripe(indicated by dark grey colour), the female was transferred to a hatching tank (30l) containing slightly brackish water (6gl 1 Instant Ocean). The water temperature was
maintained at 288C bya thermostatheater. The newly hatched larvae with yolk were left for 24h in the hatchingtank.Thenextday,prawnlarvaewithabsorbed yolk were distributed in groupsof 25 larvae in 200ml glassconescontaining100mlfreshautoclavedbrackish water (12gl 1 Instant Ocean). The glass cones were
placed in a water bath maintained at 288C and was providedwithaeration.Thelarvaewerefeddailywith5 brineshrimplarvae/prawnlarvaandacclimatizedtothe experimental conditions for 24h. During the experi-ments,waterqualityparameterswerekeptatminimum 5mgl 1 dissolvedoxygen, maximum0.5mgl 1
ammo-nium-N and maximum 0.05mgl 1 nitrite-N. Prawn
larvae were challenged with V. campbellii by adding the strainsat 106CFUml 1 totheculturewater on the dayafterfirstfeeding.Signalmoleculeswerealsoadded to the culture water on the day after first feeding. Survivalwas counteddailyin thetreatmentchallenged to wild type V. campbellii and the challenge test was stoppedwhenmorethan50%mortalitywasachievedin thistreatment(inordertohaveenoughlarvaeremaining for the growth measurement). At this time point, larval survival was determined in all treatments by consideringthatonlythoselarvaepresentingmovement of appendageswerealive.The growth parameterlarval stage index(LSI) wasdetermined according toMaddox and Manzi(1976) on 5 randomly sampled larvae from each cone.
2.7. DeterminationofV.campbelliilevelsintheprawnlarval gutandtheculturewater
In the challenge test using the rifampicin resistant mutants,larvaeandwatersampleswerecollectedtwice, onday5andday8.Threesamplesof10larvaeweretaken atrandom and were collected onsterile 500
m
m sieves.The animals were anaesthetized in a 0.1% benzocaine solution(Sigma)for10s. Surfacebacteriawereremoved accordingtoNhanetal.(2010)byimmersingthesamples ina0.1%benzalkoniumchloridesolution(Sigma)for10s and thenrinsingthem three timeswith sterile brackish water.Thelarvaewerethentransferredintosterileplastic bagscontaining4mlofbrackishwaterandhomogenized for60susingastomachermachine,asdescribedbyNhan et al. (2010). Subsequently, the samples were serially diluted,platedonLB12agarcontaining100mgl 1
rifam-picinandincubatedfor48hat288C.
2.8. Statisticaldataanalysis
Statistical analyses were performed using the SPSS software,version20.Thegiantfreshwaterprawnsurvival datawere arcsin transformed in ordertosatisfy normal distributionandhomoscedasticityrequirements.
3. Results
3.1. TheimpactofV.campbelliiquorumsensingonsurvivalof brineshrimplarvae
Inafirstexperiment,wechallengedbrineshrimplarvae with V. campbellii wild type strain BB120 and different quorum sensing mutants defective for signal molecule production.Consistentwithourpreviousreports(Defoirdt etal.,2005; Defoirdtand Sorgeloos,2012),the AI-2and CAI-1 deficient mutants were significantly less virulent thanthe wildtype, whereasthe HAI-1deficientmutant wasasvirulentasthewildtype(Fig.2A).
3.2. TheimpactofV.campbelliiquorumsensingonsurvival andgrowthofgiantfreshwaterprawnlarvae
723%and7414%forHAI-1andAI-2,respectively,versus 6011%inuntreatedlarvae).IncontrasttotheHAI-1and AI-2 deficient mutants, significant mortality (similar to that caused by the wild type) was observed in prawn larvae challengedwiththeCAI-1deficientmutant(Fig.2BandC). Finally,noneofthestrainsaffectedgrowthofthesurviving larvae(datanotshown).
3.3. ImpactofquorumsensingonV.campbelliidensityinthe giantfreshwaterprawnlarvalgutandculturewater
Inordertobeable todistinguishV. campbelliistrains addedtotheprawnculturesfromthenaturalmicrobiota associatedwiththecultures,weusedrifampicinresistant mutantsofthewildtypeandtheHAI-1andAI-2synthase double mutant MM77. These strains were used to challenge larvae originating from broodstock 2 and showed a similar virulence as observed for their non-resistant counterparts. Mortality of larvae challenged to the wild type reached the 50% level after 8 days of challengeandwassignificantlyhigherthanthatoflarvae challenged to the HAI-1 and AI-2 deficient mutant. Additionofthesignalmoleculestogetherwiththedouble
mutant restored its virulence (Fig. 2D). Although the V. campbellii density showed a gradual increase as the challengeperiod progressed,the levelsinside the larval gut or culture water were not significantly different between the wild type and the HAI-1 and AI-2 double mutantMM77(Table2).
4. Discussion
V. campbellii, one of the major pathogens of aquatic animals,usesathree-channelquorumsensingsystemto regulatevirulencegeneexpression(Defoirdtetal.,2007; Ruwandeepika et al., 2012). The data presented in this paper show that although they feed a common signal transductioncascade,thethreedifferentquorumsensing signal molecules produced by the bacterium have a different impact on its virulence towards different crustacean hosts. AI-2 and CAI-1 are needed for full virulence of the pathogen towards brine shrimp larvae, whereasHAI-1hasnoeffect.Incontrast,HAI-1andAI-2are neededforfullvirulencetowardsgiantfreshwaterprawn larvae, whereas CAI-1 has no effect in this host. We proposethreedifferent mechanismsthat eitheraloneor
A
B
C
D
0 20 40 60 80 100
Control WT HAI-1- AI-2- CAI
-1-Mortality (%)
0 20 40 60 80 100
Control WT HAI-1- AI-2-
CAI-1-Mortality (%)
no AI added + 1 mg/l AI
0 20 40 60 80 100
Control WT HAI-1- AI-2- CAI
-1-Mortality (%)
no AI added + 1 mg/l AI
0 20 40 60 80 100
Control WT RifR HAI-1-AI-2- RifR
Control WT RifR HAI-1-AI-2- RifR
Mortality (%)
No AI added + 1 mg/l AI
a
b
d c
e a
b
a
c
a b
a b
d a,b
c b
c a
x
y
z
z
Day 5 Day 8
a a
a
b,c
Fig.2.ImpactofthethreequorumsensingsignalsonvirulenceofVibriocampbelliitowardstwodifferenthosts.(A)Percentagemortalityofbrineshrimp larvaeafter2daysofchallengewithVibriocampbelliiwildtypeandsignalmoleculesynthasemutants.Errorbarsrepresentthestandarddeviationofthree replicatebrineshrimpcultures.(B)Percentagemortalityofgiantfreshwaterprawnlarvaeoriginatingfrombroodstock1after5daysofchallengewith Vibriocampbelliiwildtypeandsignalmoleculesynthasemutants.TheHAI-1-andtheAI-2-negativemutantweretestedwithoutandwith(greybars) 1mgl 1ofthecorrespondingsignal.Errorbarsrepresentthestandarddeviationoffivereplicateprawncultures.(C)Percentagemortalityofgiant
freshwaterprawnlarvaeoriginatingfrombroodstock2after8daysofchallengewithVibriocampbelliiwildtypeandsignalmoleculesynthasemutants.The HAI-1-andtheAI-2-negativemutantweretestedwithoutandwith(greybars)1mgl 1ofthecorrespondingsignal.Errorbarsrepresentthestandard
deviationoffivereplicateprawncultures.(D)Percentagemortalityofgiantfreshwaterprawnlarvaeoriginatingfrombroodstock2after8daysofchallenge withrifampicinresistantmutantsofVibriocampbelliiwildtypeandtheHAI-1AI-2synthasedoublemutantMM77(withoutandwith1mgl 1HAI-1and
1mgl 1AI-2).ErrorbarsrepresentthestandarddeviationoffivereplicateprawnForeachpanel,barswithadifferentletteraresignificantlydifferentfrom
eachother(ANOVAwithTukey’spost-hoctest;p<0.05).‘‘Control’’referstounchallengedanimalsthatwereotherwisetreatedinthesamewayasthe challengedanimalscultures.
togethermightexplaintheapparentdiscrepancybetween the fact that the receptorsofthe three signalmolecules havebeenreportedtofeedacommonsignaltransduction cascade and the fact that the signals have a different impact on virulence towards different hosts. First, the molecules can have a different stability in different environments (andthusin differenthosts). Forinstance, short side chain acylhomoserine lactones (such as V. campbelliiHAI-1)havebeenreportedtohavelowstability atpHvaluesabove8(Byersetal.,2002;Yatesetal.,2002). AlkalinehydrolysisofthelactoneringoftheHAI-1signal mightexplainitsinactivityinthebrineshrimphostwhich isculturedinseawater(pH8).Anotherpossibilitymight bethatthehostsproduceenzymesthatcaninactivate(one of) the signal molecules.Acylhomoserine lactone inacti-vatingenzymeactivityforinstance,hasbeenreportedin different higherorganisms(Dong etal.,2007).A second possible explanation is that the expression of signal molecule synthases and/or receptors might be different in different environments. Indeed, Teng et al. (2011) recently reported that V. campbellii can alter the ratio between the signalmolecule receptors,allowingcells to pay more attention to one of the signals under certain conditions.Athirdpossibilityisthattheremightbenotyet identified signal transduction cascades that are not affected by all three signals. We recently reported that the expressionofaV. campbelliihemolysingene(vhh)is decreasedinanAI-2deficientmutantandanAI-2receptor (luxP)mutant,whereasitisnotinamutantinwhichthe knownsignaltransductioncascadeisinactivated (Ruwan-deepikaetal.,2011).Thissuggeststhattheremightbean additionalsignaltransductioncascadethatisonlyaffected byAI-2.
Manypathogenicbacteriausedifferentquorumsensing signal molecules to regulate virulence gene expression (Jayaraman and Wood, 2008; Ng and Bassler, 2009). However, it is often not clear why different signals are used, especially when they are using a common signal transductioncascade (asisthecaseinvibrios). Ourdata suggestthattheuseofdifferenttypesofsignalmolecules might beoneof thefeaturesthat enableV. campbellii to infectdifferent host organisms.Indeed, ifthe bacterium wouldrelyonlyonHAI-1signalling,thenitwouldfailto infectbrineshrimp,whereasifitwouldrelyonlyonCAI-1 signalling, then it would fail to infect giant freshwater prawn.Similarmechanismsmightapplytoother patho-gensusingmultipletypesofsignalmoleculestoregulate virulence gene expression and specifically other Vibrio
species, includinganimal and humanpathogens suchas
Vibrio alginolyticus and Vibrio parahaemolyticus (both of
whichalsobelong tothe Harveyicladeofvibrios),Vibrio choleraeandVibriovulnificus.
Becausequorumsensingsystemsregulatethe expres-sion of virulence genes in many bacteria that are pathogenictoplants,animalsand/orhumans,quitesome research effort currently is being directed towards the disruption of quorum sensing as a novel biocontrol strategy(Rasmussenand Givskov,2006; Janssens et al., 2008;Njoroge and Sperandio,2009;Pan andRen,2009; Gallowayetal.,2012;Kalia,2013).Ourresultsindicatethat forspeciesthatusedifferentsignalmoleculestoregulate virulencegeneexpressionitishighlyimportanttostudy theimpactofquorumsensinginhibitorsunderconditions thatareascloseaspossibletotheclinicalsituationwhere the inhibitors will be used. Indeed, compounds that specificallytargetoneofthesignals(e.g.signalmolecule receptorinhibitors)mightturnouttohavenoeffectinthe hostforwhichtheyareintendedalthoughtheywerefound towork wellin a modelsystem (e.g.animal modelsfor humanpathogens).
Acknowledgements
We thankBonnieBasslerforprovidingus withtheV. campbellii wild type and quorum sensing mutants. This workwasfundedbytheResearchFoundationofFlanders (FWO-Vlaanderen; project no 1.5.013.12N) and by the
Directorate General of Higher Education of Indonesia throughadoctoralscholarshiptoGdeSasmitaJulyantoro Pande. Tom Defoirdt is a postdoctoral fellow of FWO-Vlaanderen.
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Day5 Day8 Day5 Day8
Control NotDetected Notdetected Notdetected NotDetected
WTRifR 3.90.2 4.80.2 5.70.1 6.10.0
HAI-1 AI-2 RifR 3.80.2 4.70.2 5.60.1 6.10.0
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