Rev. bras. farmacogn. vol.25 número3

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RevistaBrasileiradeFarmacognosia25(2015)199–207

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Marine

organisms

as

source

of

extracts

to

disrupt

bacterial

communication:

bioguided

isolation

and

identification

of

quorum

sensing

inhibitors

from

Ircinia

felix

Jairo

Quintana

a

_,

_José

_{Brango-Vanegas}

a

_,

_Geison

_M.

_Costa

a

_,

_Leonardo

_Castellanos

a,∗

,

Catalina

Arévalo

a,b

_,

_Carmenza

_Duque

a

a_Grupo_de_Estudio_y_{Aprovechamiento}_de_Productos_Naturales_Marinos_y_Frutas_de_Colombia,_Departamento_de_Química,_Facultad_de_Ciencias,_Universidad_Nacional_de_Colombia,

Bogotá,Colombia

b_Grupo_de_{Comunicación}_y_Comunidades_Bacterianas,_Departamento_de_Biología,_Facultad_de_Ciencias,_Universidad_Nacional_de_Colombia,_Bogotá,_Colombia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14November2014 Accepted26March2015 Availableonline29April2015

Keywords:

Quorumsensing Marineproducts

Irciniafelix

Furanosesterterpenes Biofilm

a

b

s

t

r

a

c

t

Inthisstudy,39extractsfrommarineorganismswereevaluatedasquorumsensinginhibitors,collected intheColombianCaribbeanSeaandtheBrazilianCoastincluding26sponges,sevensoftcorals,five algaeandonezooanthid.TheresultsshowedthatcrudeextractsfromthesoftcoralEunicealaciniata,and thespongesSvenzeatubulosa,IrciniafelixandNeopetrosiacarbonariawerethemostpromisingsource ofquorumsensinginhibitorscompoundswithoutaffectingbacterialgrowth,unliketherawextracts ofAgelascitrina,Agelastubulata,Iotrochotaarenosa,Topsentiaophiraphidites,Niphatescaycedoi,Cliona tenuis,Ptilocauliswalpersi,Petrosiapellasarca,andthealgaeLaurenciacatarinensisandLaurenciaobtusa, whichdisplayedpotentantibacterialactivityagainstthebiosensorsemployed.Thecrudeextractfrom thespongeI.felixwasfractionated,obtainingfuranosesterterpeneswhichwereidentifiedandevaluated asquorumsensinginhibitors,showingamoderateactivitywithoutaffectingthebiosensor’sgrowth.

Introduction

Dueto thegreat chemical variability provided by biological diversityexistinginthemarineenvironment,researchinmarine naturalproductshasreceivedgreatinterest(Bluntetal.,2010). Manyofthesemoleculesareresponsibleforstabilityand adapt-abilityofmarineorganismstoachangingenvironment;besides theyareinvolvedinthesymbioticrelationshipbetweenmarine macro-organisms and associatedmicroorganisms thathasbeen thoroughlydocumented.Thisentirechemicalsignalingmakesthis multiorganismalconsortiumbehaveasabiochemicaland physio-logicalunitfundamentaltotheequilibriumofthewholeecosystem (Harvelletal.,2007).

Structure and relative density of microbial populations are associated with macro-organisms’ change in time and loca-tion. This alteration directly influencesthe organism health as wellasthe productionof bioactive substances.Therefore, mul-tiorganism consortium couldregulate settlement of exogenous bacteriausingcompoundstoavoidabiofilmwithanundesirable

∗ Correspondingauthor.

E-mail:lcastellanosh@unal.edu.co(L.Castellanos).

composition(TeplitskiandRitchie,2009).Coordinationbetween macro-organisms and associated bacteria is done, as well as betweenbacteria,bymeansofcommunicationsystemsregulated by chemicalmolecules. In bacteria,this phenomenon is known asquorumsensing(QS),andsignals[N-acylhomoserinelactones (AHL)forGramnegativebacteria]involvedinthismechanismare alsoresponsibleforinter-kingdomcommunication(Loweryetal., 2008).

DuetoQSsystems,bacteriabehaveinaverydifferentwaywhen theyareorganizedas acomplex community becauseinsucha way,theycoordinatedifferentphenotypesthatprovidethe com-munitywithmoreresistanceandadaptabilitytotheenvironment, likeantibioticproduction,sporulation,DNAexchange,expression andsecretionofvirulencefactors,bioluminescence,pigment pro-duction,andremarkablebiofilmmaturation(NgandBassler,2009; Greenberg,2003;BoyerandWisniewski-Dyé,2009).

Bacterial biofilms are sessile communities of differentiated cells associated with a surface, submerged in an extracellular matrixofexcretedpolysaccharides(alsotermed exopolysaccha-rides,EPS),proteinsandDNA.Specificstructureandcomposition ofbiofilmsvarywiththeresidentspeciesandenvironmental con-ditions(Dickschat,2010).Biofilmmaturationisalsocontrolledby QSthatregulatesmanyphenotypesasEPSproduction,adhesines

http://dx.doi.org/10.1016/j.bjp.2015.03.013

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200 J.Quintanaetal./RevistaBrasileiradeFarmacognosia25(2015)199–207

and otherfundamental requirementsfor theconsolidation of a strongmatrix(Dickschat,2010).Sincebiofilmsareknowntobe importantcuesforsettlementoflarvaeofmanymarine inverte-brates(Dobretsovetal.,2009),thecontrolofbiofilmformationon thesurfacebecomesatargetforbiofoulingcontrol.Furthermore, biofilmhasbeendisruptedoverunanimatedsurfacesusingquorum sensinginhibitors(QSI),leadingtothereductionofmacrofouling (Dobretsovetal.,2009).

Oneof themostpromisingalternativesforbiofilmcontrolis the use of marine natural products, which are extracted from someinvertebrateorganismswithamacro-foulingfreesurfacethat couldbeanindicatoroftheproductionofcompoundswith set-tlementdeterrenceproperties(Qianetal.,2007;Mesegueretal., 2004).Followingthenecessityofantifoulingcompoundswithout toxiceffectsoncolonizingspecies,quorumsensingdisruptionhas becomeapromisingalternative.QSImayactinfourdifferentways: first,inhibitingthesignalmoleculesbiosynthesis(i.e.AHL); sec-ond,inducingdegradationofthesignalmolecules;third,blocking specificbindsitesofAHLtoLuxRtypeproteins;and finally,by inhibitionof DNAtranscription (Dobretsovet al.,2009).A quo-rumsensing inhibitor suppressesspecific geneticexpression of bacteriawithoutcausingdeath. Sincesurvivablemechanismsof bacteriaarenotinducedbyQSIcompound,bacterialresistanceis notdeveloped;thusithasalsobeenconsideredforyearsbythe pharmaceuticalindustryasapromisingstrategyforthedesignand developmentofantipathogeniccompounds,usefulincontrolling microbialchronicinfections,duetotheobservedmicrobial devel-opmentofresistancetoantibiotics(RasmussenandGivskov,2006). Theseincreasingeffortsfocusedonnewalternativesintendedto preventandtreatsevereinfectionsinordertominimizethetotal antimicrobialexposure,andincludesseveralQS-modulating ther-apiessuchasmacrolideantibiotics,QSvaccines,andcompetitive QSinhibitorsthathavebeenprovedtobehelpfulindiminishing thetranslationofQS-directedtoxinsorbyprematurelyactivating theQSresponsetoalerttheimmunesystemtobacteriahidingina lowcelldensity(Martinetal.,2008).

ThisstudyisaimedtoextendknowledgeabouttheColombian andBrazilianmarinebiodiversityandtoevaluatetheQSIactivity ofmarineorganisms’extracts,whichhavenotbeenwidelystudied aroundtheworldand,asfarasweareaware,neverdonebefore attheSouth-EasternCaribbeanSeaandSouthBrazilianCoast.We presentevaluationsof theQSI activityof 39 marineorganisms’ extractsfromtheColombianCaribbeanSeaandtheBrazilianCoast (Santa Catarina State), using Chromobacterium violaceum ATCC 31532andEscherichiacolipSB401asbiosensorscoupledwith Pseu-domonasputidaIsoFbydiscdiffusionassays.Besides,weevaluated a furanosesterterpeneenriched-fraction fromtheactive sponge Irciniafelix,asanapproachtoidentifysomeofthepossibleactive compounds.

Materialsandmethods

Organismsandextractpreparation

OrganismswerecollectedbyscubadivinginSantaMartabay andIslasdelRosarioattheColombianCaribbeanSea,andinSanta Catarina State onthe Brazilian Atlantic Coast (Table 1; autho-rizationcode: DTC-C-33 11/7ofUAESPNN; permissionNo.4 of 10/02/2010).Organismsweredriedandkeptfrozenuntil extrac-tion.Driedorganismswerethencutintosmallpiecesandextracted three timeswith dichloromethane:methanol(1:1); solventwas thenevaporateduntilthecrudeextractswereobtained,andstored at4◦_C_before_using._The_extracts_of_Svenzea_tubulosa_and_Neopetrosia carbonaria were partitioned using Kupchan methodology (Anta

etal.,2002)andthefractionssolubleindichloromethane(DCM), butanol(WB)andwater(WW)weretested.

ExtractionandisolationoffuranosesterterpenesfromI.felix

Thecrudeextract(35.3g)ofI.felix,obtainedasdescribedabove from562.2goffreshmaterial,waspartitionedwithamixtureof DCM:H2O(1:1,v/v),yieldingthedichloromethanefractionthatwas

concentratedunderreducedpressure.Thisfraction(4.5g)was sub-mittedtovacuumcolumnchromatographyoversilicagel(Merck 60) using a gradient increasing in polarity, starting with 100% benzenetoethylacetate:methanol(1:1,v/v).Sevenfractions (IFF1-IFF7)wereyieldedandevaluatedintheQSIassay.FractionIFF3 (1.7g),elutedwithbenzene:ethylacetate(7:3,v/v),showedQSI activityandwasfurtherchromatographedagaininvacuum col-umnchromatographyoversilicagel(Merck60);andelutedwith hexane:ethylacetate(7:3,v/v),hexane:ethylacetate(1:1,v/v)and 100%ethylacetate,ledtoninefractions(FractionsIFF3-1to IFF3-9).FractionIFF3-3(600mg),elutedwithhexane:ethylacetate(7:3, v/v),containingamixtureofsesterterpenescompounds,showing QSIactivity,wasacetylated.Briefly,analiquot(300mg)offraction IFF3-3wassubmittedtoacetylationwith6mlofamixtureofacetic anhydride:pyridine(1:2,v/v)underagitationfor24h.The reac-tions’productsweremonitoredbythin-layerchromatography.The solventmixturewaspouredover10mlofhydrochloricacid(5%) coldsolutionandextractedwithethylacetate.Theorganicphase wasdriedwithanhydroussodiumsulfate,filtratedanddriedunder reducedpressure,yielding332.7mgofanacetylated furanosestert-erpenefraction.Thisfractionwassubmittedtohigh-performance liquid chromatography(HPLC) (forchromatographic conditions, see‘PreparativeHPLCseparations’section)toyieldcompounds1a and1b(2.0mg),2aand2b(2.5mg)and3(6.0mg),identifiedby1_H

and13_C_NMR,_and_by_comparison_with_the_literature_data₍_Martínez etal.,1995,1997).

FractionI(mixtureofthetwocompounds)

Colorlessoil.[␣]25D 23.0(c0.193,CH3OH).1a.(7Z,13Z,18R,20Z

)-felixininacetate.1_H_NMR₍₄₀₀_MHz,_CDCl

3)ı(ppm):7.35(1H,m,

H-1),7.21(1H,s,H-4),6.27(1H,s,H-2),5.15(1H,t,J=6.7Hz,H-7), 5.08(1H,t,J=6.8Hz,H-15),5.04(1H,d,J=10.2Hz,H-20),2.82(1H, m,H-18),2.43(2H,t,H-5aandH-5b),2.35(3H,s,H-27),2.22(2H,c, J=7.5Hz,H-6aandH-6b),1.96–2.06(m,H-16,H-12andH-10),1.82 (3H,s,H-25),1.65(3H,s,H-14andH-9),1.43–1.33(m,H-17and H-11),1.05(3H,d,J=6.8Hz,H-19).1b.(8Z,13Z,18R,20Z)-strobilinin acetate.1_H_NMR₍₄₀₀_MHz,_CDCl

3)ı(ppm):7.34(1H,m,H-1),7.21

(1H,s,H-4),6.27(1H,s,H-2),5.12(1H,t,J=6.7Hz,H-10),5.08(1H, t,J=6.8Hz,H-15),5.04(1H,d,J=10.2Hz,H-20),2.82(1H,m,H-18), 2.39(2H,t,H-5aandH-5b),2.34(3H,s,H-27),2.03(2H,m,H-7), 1.96–2.06(m,H-16andH-12),1.82(3H,s,H-25),1.67(3H,s,H-14 andH-9),1.62(m,H-6),1.43–1.33(m,H-17andH-11),1.05(3H,d, J=6.8Hz,H-19).

FractionII(mixtureofthetwocompounds)

Colorlessoil.[␣]25D 23.5(c0.20,CH3OH).2a.(7E,13Z,18R,20Z

)-felixinin acetate. 1_H _NMR ₍₄₀₀_MHz, _CDCl

3) ı (ppm): 7.34

(1H, m, H-1), 7.20 (1H, s, H-4), 6.27 (1H, s, H-2), 5.16 (1H, m, H-7), 5.08 (m, H-15), 5.05 (1H, d, J=10.3Hz, H-20), 2.82 (1H, m, H-18), 2.44 (2H, t, J=6.5Hz, H-5a and H-5b), 2.35 (3H, s, H-27), 2.25 (2H, t, J=6.5Hz, H-6a and H-6b), 1.95–2.09(m, H-11and H-10),1.88–1.95 (m,H-16,H-12), 1.81 (3H,s,H-25),1.65(3H,s,H-14),1.57(3H,s,H-9),1.30–1.50(m, H-17),1.06(3H,d,J=6.7Hz,H-19).2b.(8E,13Z,18R,20Z)-strobilinin acetate.1_H_NMR₍₄₀₀_MHz,_CDCl

3)ı(ppm):7.34(1H,m,H-1),7.20

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Table1

QSIandantibacterialresultsofmarineextractsfromColombianCaribbeanSeaandBrazilianSouthCoast.

Typeof collected sample

Marineorganism extractsource

Collection number

Collectiondate Geographic origin

EscherichiacolipSB401 ChromobacteriumviolaceumATCC31532

Growinginhibitiona _QS_inhibitiona _Growing_inhibitiona _QS_inhibitiona

200␮g 400␮g 200␮g 400␮g 100␮g 200␮g 400␮g 100␮g 200␮g 400␮g

Sponges Agelascitrina INV-POR969 05/04/2008 IslasdelRosario(Col) 7–9 7–9 5–7 >12 – – – – – 7–9

Agelastubulata ICN-MHN(Po) 154

07/12/2005 SantaMarta(Col) 7–9 7–9 – >12 – – NT – – NT

Agelasmarron ICN-MHN(Po) 0149

07/12/2005 IslasdelRosario(Col) – – – – – – – – – –

Akacachacrouense INV-POR-412 07/12/2005 SantaMarta(Col) – 5–7 – – – – NT 9–12 9–12 NT Biemnacribaria INV-POR-890 07/12/2005 SantaMarta(Col) 7–9 5–7 – 7–9 NT – – NT – –

Cinachyrella kuekenthalli

INV-POR-878 18/11/2009 SantaMarta(Col) – – 5–7 5–7 NT NT NT NT NT NT

Clionadelitrix INV-MHN(Po)-189

18/09/2005 SanAndrés(Col) – – – 7–9 NT – – NT – –

Clionatenuis INV-POR-669 06/04/2008 IslasdelRosario(Col) – 7–9 – – 9–12 9–12 NT – – NT Clionavarians INV-POR-339 07/09/2005 SantaMarta(Col) – – – – NT – – NT – –

Iotrochotaarenosa

(Iotrochotainminuta)b

INV-POR-883 15/11/2006 SantaMarta(Col) >12 – – 7–9 – – – – – –

Irciniafelix INV-POR-014 18/11/2009 SantaMarta(Col) NT NT NT NT – – – 7–9 7–9 7–9

Lissodendoryx colombiensis

ICN-MHN-PO 0246

06/04/2008 IslasdelRosario(Col) – – – – NT – – NT – –

Muriceopsissp. ICN-MHN-PO 0254

13/02/2006 SantaMarta(Col) – 5–7 – – – – NT - – NT

Neopetrosiacarbonaria

(DCM)(Xestospongia carbonaria)b

ICN-MHN-PO 0244

05/04/2008 IslasdelRosario(Col) – – – 7–9 – – – >12 – 9–12

(WB)

ICN-MHN-PO 0244

05/04/2008 IslasdelRosario(Col) – – 7–9 7–9 – – – – – >12

(WW)

ICN-MHN-PO 0244

Neopetrosiamuta

(Xestospongiamuta)b

ICN-MHN-PO 0247

Neopetrosiaproxima

(Xestospongia proxima)b

ICN-MHN-PO 0239

Neopetrosiarosariensis

(Xestospongia rosariensis)b

ICN-MHN-PO 0245

05/04/2008 IslasdelRosario(Col) – – – - NT – – NT – –

Neopetrosia subtriangularis

(Xestospongia subtriangularis)b

ICN-MHN-PO 0242

05/04/2008 IslasdelRosario(Col) – – – – – – – >12 7–9 7–9

Ptilocauliswalpersi ICN-MHN-PO 0248

06/04/2008 IslasdelRosario(Col) – – – 5–7 NT 9–12 9–12 NT – –

Svenzeatubulosa(crude extract)

ICN-MHN-PO 0249

06/04/2008 IslasdelRosario(Col) – – 9–12 >12 – – – – >12 9–12

Svenzeatubulosa(DCM) ICN-MHN-PO 0249

06/04/2008 IslasdelRosario(Col) NT NT NT NT – – – 7–9 9–12 9–12

Topsentiaophiraphidites ICN-MHN-PO 0243

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Table1(Continued)

Typeof collected sample

Marineorganism extractsource

Collection number

Collectiondate Geographic origin

EscherichiacolipSB401 ChromobacteriumviolaceumATCC31532

Growinginhibitiona _QS_inhibitiona _Growing_inhibitiona _QS_inhibitiona

200␮g 400␮g 200␮g 400␮g 100␮g 200␮g 400␮g 100␮g 200␮g 400␮g Softcorals Eunicealaciniata

ICN-MHN-CR-106

13/02/2006 SantaMarta(Col) – – 7–9 7–9 NT NT NT NT NT NT

Euniceasuccinea ICN-MHN-PO 0251

13/02/2006 SantaMarta(Col) – – – 5–7 NT – – NT – –

Euniceafusca ICN-MHN-PO 0252

13/02/2006 SantaMarta(Col) – – – 5–7 NT – – NT – –

Euniceasp1 – 13/02/2006 SantaMarta(Col) 5–7 5–7 – – NT – – NT – –

Erythropodium caribaeorum

INV-CNI-1193 07/09/2005 SantaMarta(Col) – – – – – – – – – 9–12

Pseudopterogorgia elisabethae

(Providencia)

INV-CNI-1612–1614

25/05/2008 Providenciaand SanAndrés(Col)

5–7 – – – – – NT – – NT

Pseudopterogorgia elisabethae(San Andrés)

INV-CNI-1615-1616

18/09/2005 Providenciaand SanAndrés(Col)

– – – 9–12 NT – – NT – –

Zoanthid Palythoacaribaeorum ICN-MHN-PO 0255

07/09/2005 SantaMarta(Col) – – – – – – – – 9–12 –

Algae Laurenciacatarinensis FLOR14516 02/2008 IlhadoArvoredo, Florianópolis(Bra)

– – – – 7–9 9–12 9–12 – – –

Laurenciaflagellifera FLOR14521 03/2006 PraiadaSepultura, Bombinhas(Bra)

– – – – – – – – – –

Laurenciamajuscule FLOR14524 11/2006 IlhadoFrancês, Florianópolis(Bra)

– – – – – 7–9 – – – –

Laurenciamicrocladia FLOR14520 03/2008 PraiadaSepultura, Bombinhas(Bra)

– – – – – – – – – –

Laurenciaobtusa FLOR14512 08/2009 PraiadeCanasvieiras, Florianópolis(Bra)

– – – – 7–9 9–12 >12 – – –

a_halo,_in_mm.

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H-10),5.04(1H,d,J=10.3Hz,H-20),2.82(1H,m,H-18),2.37(2H,t, H-5aandH-5b),2.34(3H,s,H-27),1.95–2.09(m,H-16,H-12,H-7), 1.81(3H,s,H-25),1.67(3H,s,H-14),1.58(3H,s,H-9),1.64(m, H-6),1.30–1.50(m,H-17andH-11),1.05(3H,d,J=6.7Hz,H-19).

FractionIII(3)

(7E,12E,18R,20Z)-variabilinacetate,colorlessoil. [˛]25D 26.1(c

0.167,CH3OH).1HNMR(400MHz,CDCl3)ı(ppm):7.33(1H,s,

H-1),7.20(1H,s,H-4),6.28(1H,s,H-2),5.16(1H,t,J=6.9Hz,H-7), 5.07(1H,t,J=6.9Hz,H-12),5.04(1H,d,J=10.2Hz,H-20),2.81(1H, m,H-18),2.44(2H,t,J=7.4Hz,H-5aandH-5b),2.35(3H,s,H-27), 2.23(2H,c,J=14.7and7.4Hz,H-6aandH-6b),2.05(2H,m,J=8.0 and7.6Hz,H-11aandH-11b),1.98(2H,t,J=7.8Hz,H-10aand H-10b),1.94(2H,t,J=7.0Hz,H-15aandH-15b),1.82(3H,s,H-25), 1.58(3H,s,H-9),1.55(3H,s,H-14),1.34(4H,m,H-16),1.34(4H, m,H-17),1.05(3H,d,J=6.7Hz,H-19).RMN13_C₍₁₀₀_MHz,_CDCl

3) ı(ppm):142.5(CH,C1),138.8(CH,C4),124.4(CH,C12),123.7(CH, C7),111.1(CH,C2),39.7(CH2,C10),39.5(CH2,C15),36.6(CH2,C17),

31.1(CH,C18),28.4(CH2,C6),26.6(CH2,C11),25.7(CH2,C16),25.0

(CH2,C5),20.6(CH3,C27),20.5(CH3,C19),16.1(CH3,C9),15.8(CH3,

C14),116.7(CH,C20),8.4(CH3,C26).

PreparativeHPLCseparations

HPLCseparationswere carriedout ona Merck-Hitachi6000 systemequippedwithaL-6000Abinarypump,Rheodyneinjector andaL-4250UV–Visdetector.Thestationaryphaseemployedwas ScharlauScienceNucleosil120C-18column,(300mm×8mm;i.d.;

10␮m),usingisocraticMeOH:H2O(95:5)asmobilephase,witha

flowrateat2ml/min,anddetectionat310nm.

Bacterialstrainsandcultureconditions

TwodifferentbiosensorswereusedtoevaluateQSIactivity:C. violaceumATCC31532andE.colipSB401coupledwithP.putidaisoF WTastheexogenoussourceofAHLtoinducebioluminescence pro-duction.E.coliandP.putidaisoFWTwereculturedinLuria–Bertani brothat37◦_C,_and_those_strains_were_kindly_provided_by_Prof._Dr. KathrinRiedel(DepartmentofMicrobiology,InstituteofPlant Biol-ogy,UniversityofZürich).C.violaceumwasculturedinCASOAgar® (Merck,Germany)atroomtemperature.

QSIassays

C.violaceumATCC31532wasinoculatedovernightatroom tem-perature in CASO brothand 100␮lof bacteria werespread on agarplates.Incontrast,E.colipSB401andP.putidaisoFWTwere inoculatedseparatelyat37◦_C_overnight_to_get_a_cellular_density equalto0.5MacFarlandstandard.AnequalamountofLBmedium withagarat1.6%waspreparedandmixedwithbacteriacultures withtheintentionof preparing0.8%agarplates,already inocu-latedwithboth,thebiosensorandtheAHLproducerstrain.Sterile paperdiscs(5mmdiameter)wereloadedwiththeappropriate vol-umeofcrudeextractssolutions,dissolvedinmethanol,inorder toobtain100, 200and 400␮gperdisc.Thesolventwasleftto evaporate.Methanolwasusedas blankcontroland methanolic fractionfromCecropiapachystachyawasusedaspositivecontrol. Discswereplacedonplatespreviouslyinoculatedwiththe appro-priatebiosensorandwereincubatedfor24hat37◦Cfortheassay withE.colipSB401andP.putidaisoFWT;andat28◦_C_for_C. vio-laceumATCC31532assay.AQSIpositiveresultwastheinhibitionof phenotypeexpression(bioluminescenceorviolacein,respectively) withoutaffectingthegrowingprocessofbacteria.Thepresenceofa growthinhibitionzonearoundthediscswastakenasantibacterial activity.Thisprocedurewasmadebyatriplicate.Theinhibitionof

quorumsensinginC.violaceumwasmeasuredbyvisualanalysisof violaceinproduction,andforthebioassaywithE.colipSB401and P.putidaisoFWTthebioluminescenceinhibitionwasregisteredby autoradiography.

Resultsanddiscussion

Manydifferentstudieshavedemonstratedtheabilityofnatural compoundstoblockQSsystems.Ornamental,medicinaland edi-bleplantshavebeenthemoststudiedsourcesofQSIcompounds (Dickschat,2010; Dobretsovet al., 2009);however, only a few examplesofsystematicscreeningstudieshavebeendocumented usingrawextractsfrommarinesources.Aninvestigationbased on78extractsof25speciesofmarineorganismscollectedfrom FloridawatersshowedthatcyanobacteriahavehighQSinhibitory potential(Dobretsovetal.,2011).Anotherstudyusedmarinecrude extractsoforganismsfromtheGreatBarrierReefinAustralia.The authorsidentifiedinthespongeLuffariellavariabilisthemanoalide asresponsiblefortheQSIactivityoftherawextract.Thiscompound isknownasananti-inflammatoryagent,anditsstructure hasa lactonicringwhichresemblesthestructureofAHL,andhasbeen foundtobeanactiveQSIcompoundinthefusionsystemlasB::gfp (ASV)ofPseudomonasaeruginosa(Skindersoeetal.,2008).Some othercompoundshavebeenidentifiedasQSIfrommarinesources, andthemostrepresentativeQSIcompoundsarethehalogenated furanones,isolatedfromtheredalgaDeliseapulchra,whichhave beendemonstratedtobeoneofthemostpromisingQSIcompounds andpotentantifoulingagents(BoyerandWisniewski-Dyé,2009). Ontheotherhand,threecompounds(floridoside,betonicineand isethionicacid)isolatedfromtheredalgaAhnfeltiopsisflabelliformis showedpromisingQSIactivitywiththeAgrobacteriumtumefaciens NTL4QSsystem(Kimetal.,2007).Anotherexampleisthe bromi-natedtriptamine-likealkaloidsthatwerefoundinbryozoanFlustra foliaceaandweretestedusingtheP.putida(pKR-C12),P.putida (pAS-C8)andE.coli(pSB403)QSsystemsasbiosensors(Petersetal., 2003).

Extractstestedinthepresentstudywereobtainedfrommarine organismsselectedspeciallybecauseoftheircleansurface;such organismshavemicroorganismcommunitiesinthesurfaceswhich mayhaveanecologicalcontrolthatavoidsettlementof macro-organisms. This control can be done by chemical compounds produced by the marine organism (the host) or by associated microorganismsasbacteria,orbyboth,theholobiont.As previ-ouslymentioned,inthisecologicalcontrol,QSplaysamajorrole duetoregulationofbiofilmmaturation,causingaselective settle-mentofbacteria,larvaeorothereukaryoticmicroorganisms(Boyer andWisniewski-Dyé,2009).

Discdiffusionassays wereusedtotestQSI andantibacterial activityof39marineorganismsextracts.Table1showstheresults usingdifferentconcentrationsofextractswithbothbiosensors.A promisingextractassourceofQSIcompoundsmustshowaQS inhi-bitionwithoutaffectingthebacterialgrowingprocesstoavoidlater developmentofbacterialresistance.

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Concentration

200 µg

400 µg

Petri dish pictures

A

1

2 ₃

1

2

3

2 ₃

2

3 C

D

B

X-ray plate

Fig.1.DiscdiffusionassayshowinginhibitionofbioluminescenceofEscherichiacolipSB401coupledwithP.putidaIsoFbymarineextracts.AandCarepicturesofPetri dishesandsensi-discs.BandDaretheX-rayplaquesofthesepetridishes.1:Svenzeatubulosa(crudeextract),2:Petrosiapellasarca(crudeextract)and3:Eunicealaciniata

(crudeextract).Theclearzonesinautoradiography(BandD)andtheabsenceofgrowthinhibitionzones(AandC)showQSinhibitoryactivityoftheextracts.

ofspongeN.carbonaria.AmildQSIactivitywasshownbycrude extractsfromthespongesClionadelitrix,dichloromethanefraction ofN.carbonariaandalowactivitywasshownbycrudeextracts fromtheoctocoralsEuniceasuccinea,Euniceafuscaandthesponge Ptilocauliswalpersi.

WhenC.violaceumATCC31532wasusedasabiosensor,nine (23%) of the39 extracts resultedactive. Of these,seven corre-sponded to sponges, one to an octocoral, and the other to a zoanthid. These results are in contrast withthe onesobtained by using the E. coli biosensor because with C. violaceum the most active extract belonged to sponges, 27% of the extracts were active (7 of 26) and so were just 14.3% of the octoco-ralextracts(1of7).Likewise,algaeextractsshowednoactivity withthisbiosensor.Fig.2showstheresultsfortherawextracts of the sponges S. tubulosa and I. felix in the assay with C. violaceum which, in conjunction with the crude extracts from sponges Neopetrosia subtriangularisand Aka cachacrouense, and as well as extracts from octocoral Erythropodium caribaeorum, dichloromethane extracts of S. tubulosa and N. carbonaria, and butanol fraction of N. carbonaria, showed high bacterial com-municationdisruption(QSI)activity.ExtractsfromthespongeE. caribaeorumandthezoanthidPalythoacaribaeorumshowed mod-erateactivity.

Withthesetestswecouldalsoseesomeextractswith antibacte-rialactivitiesagainstthreeGramnegativebacteria:E.coli,P.putida andC. violaceum.Extracts fromthesponges Agelas citrina, Age-lastubulata,IotrochotaarenosaandTopsentiaophiraphiditeswere themost potentantibacterial against E.coli and P.putidaIsoF, whileextractsfromthespongesNiphatescaycedoi,Clionatenuis, P.walpersi,PetrosiapellasarcaandthealgaeLaurenciacatarinensis andLaurenciaobtusashowedhighantibacterialactivityagainstC. violaceumATCC31532.Theseresultscouldbeexplainedduetothe factthatthespongesbelongingtothegenusAgelasarerecognized asasourceofbromopyrrolidinicalkaloids(Braekmanetal.,1992) withanimportantecologicalkeyroleasfishfeedingdeterrents (Assmannetal.,2000).Ithasalsobeenfoundthatsuchcompounds possessinteresting antiviral (Keifer et al.,1991), antihistaminic

(Cafierietal.,1997)andantimicrobialactivities(Shenetal.,1998). The sponges of the genus Iotrochota are known for producing brominatedalkaloids(Carlettietal.,2000)withmildantibacterial activity,andsulfatedpyrrolealkaloidswithpromising anti-HIV-1activity(Fan etal.,2010).Thesponges ofthegenusTopsentia arerecognizedassourceofsulfatedsteroidswithawidespread range of bioactivities, for example, ophirapstanol trisulfate – a sulfate steroidisolated fromT. ophiraphidites which hasshown activityasaninhibitorintheguanosinediphosphate/G-proteinRAS exchangeassay(GDPX),whichisdirectlyrelatedwithanticancer activity(Gunasekeraetal.,1994).Thespongesofthegenus Ptilo-caulishavebeenreportedbyproducingguanidinetypealkaloids withantimicrobialactivity,amongothers.In2003,acyclic guani-dine,withantibacterialactivityagainstStaphylococcusaureus,was identifiedfromthespongePtilocaulisspiculifer(Yangetal.,2003). AlgaeofthegenusLaurenciahaveshown sesquiterpenes, diter-penes,triterpenesandacetogeninsthatusuallyhaveoneormore halogenatoms.Thesetypesofcompoundswererecognizedfor var-iousactivities,suchascytotoxicandantibacterial(Lhullieetal., 2010).

Thoseresultsareinagreementwithapreviousworkdonein ourlaboratory, where many of theextracts used in this study weretestedagainstacollectionoftwelvemarinebacteriastrains, isolatedfrom colonizedsurfaces(Mora-Cristancho etal., 2011). Additionally,formanyofthesemarinestrains,presenceofQS sys-tems byidentificationof itsAHLproduction wasdemonstrated (Cuadrado,2010).Therefore, itcouldbeachallengetoevaluate thepropertiesoftheseQSIactiveextractsinthesemarinebacteria involvedinthebiofoulingprocessofQSsystems.

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A, C1 A, C2 B, C2 B, NC

PC A, C4 B, C4 A, NC

Fig.2. DiscdiffusionassayshowinginhibitionofviolaceinaproductioninC.violaceumATCC31532.Thepicture(A)isdichloromethanefractionfromSvenzeatubulosa

and(B)iscrudeextractfromIrciniafelix.C1=100␮g,C2=200␮g,C3=300␮g,andC4=400␮gperdisco.Positivecontrol,PC=100␮gofmethanolfractionfromCecropia pachystachya;negativecontrol,NC=20␮lofmethanolwithpriorevaporationbeforetoplacing.TheclearzonesurroundingthepaperdiscshowsQSinhibitoryactivityof theextracts.

abletocombatagainstantibioticresistantbacterialstrainsthat representaseriousthreattohumanhealth.

Inthissense,bioguidedisolationwasconductedwiththecrude extractofI.felix.Throughoutdifferentchromatographicsteps,we obtaineda furanosesterterpenes-enriched fraction (IFF3-3) that presentedamoderateQSIactivity.Duetotheknowninstability ofthesecompounds,thisfractionwasacetylatedandsubmitted toapreparativeHPLCtoyieldthreesub-fractions(FractionI–III). FractionIshowedamixtureof(7Z,13Z,18R,20Z)-felixininacetate

(1a)and(8Z,13Z,18R,20Z)-strobilininacetate(1b).Verysimilarly, fractionIIalsopresentedamixtureofthesefuranosesterterpenes, butwithadistinctconfiguration:(7E,13Z,18R,20Z)-felixininacetate (2a)and(8E,13Z,18R,20Z)-strobilininacetate(2b).InfractionIII, thecompound(7E,12E,18R,20Z)-variabilinacetate(3)wasisolated. Allthesefivecompounds,whichpresentsimilarstructuretoAHL (Dobretsovet al., 2009), have already been reportedfor I. felix (Martínezetal.,1995,1997).Besides,propertiessuchasanalgesic, antimicrobial, cytotoxicand anti-tumor of this furanosesterter-penesarewelldocumented(Martínezetal.,1997).

1

5

4

9 8

14 13

18 22 ₂₃

24 25

19

18

19 18

19

18

19 13

14 O

1

5

4 9

8 14

13

O

1

5

4 9

8 14

13

1

5

4 O

1

5

4 O

O O

22 23

24 25

O O

22 23

24 25

O O O O O

OAc 18 22 ₂₃

24 25 19 _O

O OAc

22 23

24 25 O

O OAc OAc

1a

2a

1b

2b

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The other two promising QSI sponges, S. tubulosa, formerly knownasPseudoaxinellatubulosa,havenoreportsaboutits chem-istry,butthespongesofthisgenusareknownassourceoflong chainfattyacids,steroidal,andpeptidecompounds,someofthem withantibacterial activities(Kong et al.,1992; Sjöstrand et al., 1981).InourpreliminaryQSIbioassay-guidedfractionationofthis sponge,wefoundthat organicfractionsareresponsible forthe activity,andthesefractionsarerichinepidioxysterolsandlipids, which couldbe theQS inhibitors in thecrude extract (unpub-lishedresults)consideringthat,forsomefattyacids,QSIactivity isreported(Osornoetal.,2012).ThegenusNeopetrosiaisknownas asourceofquinolinictypealkaloidsandothernitrogencompounds thatexhibitcytotoxic,antimicrobialactivity,amongothers(Nakao etal.,2004;DeAlmeidaetal.,2008).ParticularlyfromN.carbonaria, previouslyknownasXestospongiacarbonaria,itwaspossibleto isolateacridinetypepolaralkaloids(Thaleetal.,2002)with recog-nizedanticanceractivity.Inapreviousstudy,thesealkaloidswere presentinthebutanolicfractionwhich,inthepresentstudy,isalso activedisruptingQSbiosensorsystems.Consequently,itcouldbe assumedthatthesealkaloidsareresponsiblefortheQSIactivityin ourextract,butithasnotbeendemonstratedyet.

In conclusion, we were able to identify the extracts of the spongesN.carbonaria,S.tubulosaandI.felixasthemost promis-ingQuorumsensinginhibitorsdisruptingtheexpressionofboth biosensors’phenotypeswithoutaffectingtheirgrowingprocess. Differentfromtheseextracts,theextractsofthespongesA.citrina, A.tubulata,I.arenosa,andT.ophiraphiditesshowedantibacterial activityagainstE.coliandP.putida,andtheextractsofthesponges N.caycedoi, C. tenuis, Ptilocaulis wallpersi, P.pellasarca and the algaeL.catarinensisandLaurenciaobtusewerehighlyantibacterial againstC.violaceum.FromthespongeI.felix,afuranosesterterpenes enriched-fractionshowedamoderateQSIactivitywithoutaffecting thebiosensor’sgrowth.Ingeneral,thesefuranosesterterpenesare structurallyrelatedwithAHLandotherQSI,whichcouldexplain theobservedactivityagainstC.violaceum,additionallypossessing asimilarmechanismofaction.Notwithstanding,otherstudiesare requiredtoimprovetherealpotentialofthesecompoundsasQSI exploitablebyman.

Authors’contributions

JQandJBV(PhDstudents)contributedbyrunningthe labora-torywork,biologicalstudies,analysisofthedataanddraftingthe paper.GMC(Postdoctoral researcher) contributedto chromato-graphicproceduresandtocriticalreadingofthemanuscript.CA contributed bycollecting samples,identification,and biological studies.LCandCDdesignedthestudy,supervisedthelaboratory workandcontributedtocriticalreadingofthemanuscript.Allthe authorshavereadthefinalmanuscriptandapprovedits submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisworkwasconductedaspartofajointprojectfinancially supportedbyCNPq/MCT/Brazil(grantnumber490151/2007-8)and COLCIENCIAS/Colombia(grantnumber358-2007).Theworkwas alsofinanced by other grants fromColciencias and “Fundación paralaPromocióndelaInvestigaciónylaTecnologíadelBanco delaRepública”.TheColombianauthorsgreatlyacknowledgeProf. Dr.KathrinRiedel(DepartmentofMicrobiology,InstituteofPlant Biology,University of Zürich) and Prof. Dr. Sven Zea from the

UniversidadNacionaldeColombiafortheirinterestandassistance inthiswork,andProf.Dr.PauloHorta,Dr.CintiaLlhulierandProf. Dr.EloirP.Schenkelforthecollectionandidentificationofthealgae.

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