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---.----- / Available online at www.sciencedirect.com SCIENCE @DIRECTO Aquaculture ELSEVIER Aquaculture240 (2004) 69-88 www.elsevier.comllocate/aqua-online Disruption of bacterial quorum sensing: an unexplored strategy to fight infections in aquaculture Tom Defoirdta.b, Nico Boona, Peter Bossierb, Willy Verstraetea,* aLaboratory of Microbial Ecology and Technology (LabMET), Ghent University. Coupure Links 653, 9000 Gent, Belgium b Laboratory of Aquaculture and Artemia Reference Center; Ghent University. Rozier 44, 9000 Gent, Belgium Received 14 April 2004; accepted 18 June 2004 Abstract Disease outbreaks-some of them caused by pathogenic bacteria-are considered to be one of the largest constraints to development of the aquaculturesector.So far, antibiotics and disinfectants have only had limited success in the prevention or cure of aquatic disease. Moreover, the frequent use of biocides, especiallyin subtherapeuticdoses, is leadingto the rapid developmentof resistance. Therefore, there is an urgent need to develop alternative ways to control infections caused by bacterial pathogens in aquaculture. Many of these pathogens are found to control virulence factor expression by a cell-to-<:ell communicationsystem. Hence, disruption of bacterial quorum sensing has been proposed as a new anti-infective strategy and several techniques that could be used to disrupt quorum sensing have been investigated. These techniques comprise (1) the inhibition of signal molecule biosynthesis, (2) the application of quorum sensing antagonists (including natural occurring as well as synthetic halogenated furanones, antagonistic quorum sensing molecules and undefined exudates of higher plants and algae), (3) the chemical inactivation of quorum sensing signals by oxidised halogen antimicrobials, (4) signal molecule biodegradation by bacterial lactonases and by bacterial and eukaryotic acylases and (5) the application of quorum sensing agonists. The few reports that deal with disruptionof quorumsensing of aquaticpathogens, together with the results obtained with human and plant pathogens, indicate that this new approach has Abbreviations: AHL, acylated homoserine lactone; AI, autoinducer. · Correspondingauthor.Te1.:+329264 59 76; fax: +32 9 264 62 48. E-mail address:[email protected] (W. Verstraete). 0044-8486/$ - see tront matter ~ 2004 Elsevier B.V. All rights reserved. doi: 10.10 16/j.aquaculture.2004.06.031 -- -- -- -----

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Page 1: Disruption of bacterial quorum sensing: an unexplored ... · Hence, disruption of bacterial quorum sensing has been proposed as a new anti-infective strategy and several techniques

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Available online at www.sciencedirect.com

SCIENCE @DIRECTO Aquaculture

ELSEVIER Aquaculture240 (2004)69-88www.elsevier.comllocate/aqua-online

Disruption of bacterial quorum sensing:an unexplored strategy to fight

infections in aquaculture

Tom Defoirdta.b,Nico Boona, Peter Bossierb, Willy Verstraetea,*aLaboratory of Microbial Ecology and Technology (LabMET), Ghent University. Coupure Links 653,

9000 Gent, BelgiumbLaboratory of Aquaculture and Artemia Reference Center; Ghent University. Rozier 44, 9000 Gent, Belgium

Received 14 April 2004; accepted 18 June 2004

Abstract

Disease outbreaks-some of them caused by pathogenicbacteria-are considered to be one ofthe largest constraints to developmentof the aquaculturesector.So far, antibioticsand disinfectantshave only had limited success in the prevention or cure of aquatic disease. Moreover, the frequentuse of biocides, especiallyin subtherapeuticdoses, is leadingto the rapid developmentof resistance.Therefore, there is an urgent need to develop alternative ways to control infections caused bybacterial pathogens in aquaculture.Many of these pathogens are found to control virulence factorexpression by a cell-to-<:ellcommunicationsystem. Hence, disruption of bacterial quorum sensinghas been proposed as a new anti-infective strategy and several techniques that could be used todisrupt quorum sensing have been investigated.These techniques comprise (1) the inhibition ofsignal molecule biosynthesis, (2) the application of quorum sensing antagonists (including naturaloccurring as well as synthetic halogenated furanones, antagonisticquorum sensing molecules andundefined exudates of higher plants and algae), (3) the chemical inactivation of quorum sensingsignals by oxidised halogen antimicrobials, (4) signal molecule biodegradation by bacteriallactonases and by bacterial and eukaryotic acylases and (5) the application of quorum sensingagonists. The few reports that deal with disruptionof quorumsensingof aquaticpathogens, togetherwith the results obtained with human and plant pathogens, indicate that this new approach has

Abbreviations: AHL, acylated homoserine lactone; AI, autoinducer.· Correspondingauthor.Te1.:+329264 59 76; fax:+32 9 264 62 48.E-mail address:[email protected] (W. Verstraete).

0044-8486/$ - see tront matter ~ 2004 Elsevier B.V. All rights reserved.doi: 10.1016/j.aquaculture.2004.06.031

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70 T. Defoirdt et al. / Aquaculture 240 (2004) 69-88

potential in fighting infections in aquaculture.However,before this new strategy can be applied inaquaculture, the impact of quorum sensingdisruptionon the virulenceof aquatic pathogens and theimpact of the proposed quorum sensing disruptingtechniqueson the aquaculture system of interestshould be studied in more depth.@ 2004 Elsevier RV. All rights reserved.

Keywords: Pathogen; Infection; Quorum sensing; Quorum quenching; Biodegradation; Antagonism

1. Introduction

According to FAO statistics, aquaculture is one of the fastest-growing food-producing industries. Worldwide, the sector has increased at an average rate of 9.2%per year since 1970. As a comparison, capture fisheries and terrestrial fanned meatproduction increased at a rate of 1.4% and 2.8%, respectively (FAO, 2002). FAOreports consider disease outbreaks as a significant constraint to development of thesector worldwide, with annual losses of billions of dollars (Subasinghe, 1997). Diseasescaused by pathogenic or opportunistic bacteria such as Vibrio spp. and Aeromonas spp.can be problematic in the intensive rearing of molluscs, fish and shrimp as they cancause losses up to 100% (Moriarty, 1999; Zhang and Austin, 2000; Muroga, 2001;Soto-Rodriguez et aI., 2003).

So far, conventional approaches such as the use of antibiotics and disinfectantshave only had limited success in the prevention or cure of aquatic disease(Subasinghe, 1997). Moreover, their frequent use is leading to the rapid developmentof (multiple) resistance (Dias et al., 1995; Molina-Aja et aI., 2002; Vivekanandhan etaI., 2002; Vattanaviboon et aI., 2003). Therefore, according to FAO, there is an urgentneed for alternative control techniques in aquaculture, with the emphasis onprevention, which is likely to be more cost-effective than cure (Subasinghe, 1997).Disease prevention can be achieved by improved management including theprevention of the transmission of pathogens between farms (e.g. by quarantine;Subasinghe, 1997), the improvement of the water quality (e.g. by bioaugmentation;Grommen and Verstraete, 2002; Moriarty, 1998), the avoidance of stress (e.g. due tohigh stocking densities, handling and temperature and salinity changes; Brock andBullis, 2001) and a good hygiene (e.g. the disinfection of culture tanks, culture waterand eggs; Brock and Bullis, 2001). Alternative strategies to the use of antimicrobialsthat have been applied successfully in aquaculture include the so-called microbialmatured water (Skjermo and Vadstein, 1999), the greenwater system (Tendencia anddela Peiia, 2003), bacteriophage therapy (Nakai and Park, 2002), the application ofprobiotics (for reviews see Gatesoupe, 1999; Verschuere et al., 2000) and theapplication of immunostimulants (for reviews see Sakai, 1999; Smith et aI., 2003b)and vaccines (for reviews see Newman, 1993; Gudding et aI., 1999; Heppell andDavis, 2000). More alternatives are very welcome since no anti-infective techniqueseems to be able to solve every problem alone. This paper deals with an alternativestrategy that has not received much attention in aquaculture so far: disruption ofbacterial quorum sensing.

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T. Defoirdt et al. / Aquaculture 240 (2004) 69-88 71

2. The new target: bacterial cell-to-cell communication

Quorum sensing is a mechanism in which bacteria coordinate the expression of certaingenes in response to their population density by producing, releasing and detecting smallsignal molecules. This mechanism was fIrst discovered in the marine bacterium VibrioflScheri (Nealsol1et aI., 1970) and was thought to be restricted to only a limited series ofspecies. Later on, similar systems were found to be present in many other Gram-negativebacteria. These Gram-negativebacteriause acylatedhomoserine lactones (ARLs) as signalmolecules (Fig. lA, for reviews see Fuqua et aI., 2001; Miller and Bassler, 2001;

A.....

I protein

B -- -

c. .

~AI-2

Fig. I. Three major quorum sensing systems. (A) AHL-mediated quorum sensing. The I protein is the AHLsynthase enzyme. The AHL molecules diffuse treely through the plasma membrane. As population densityincreases, the AHL concentration increases as well and once a critical concentration has been reached, AHL bindsto the R protein, a response regulator. The AHL-R protein complex activates or inactivates transcription of thetarget genes. (B) Peptide-mediated quorum sensing in Gram-positive bacteria. A peptide signal (PS) precursorprotein is cleaved, releasing the actual signal molecule. The peptide signal is transported out of the cell by an ATPbinding cassette (ABC) transporter. Once a critical extracellular peptide signal concentration is reached, a sensor

kinase (SK) protein is activated to phosphorylate the response regulator (RR). The phosphorylated responseregulator activates transcription of the target genes. (C) Quorum sensing in V.harveyi. In this system, there aretwo types of signal molecules. AI-I is an AHL and its biosynthesis is catalysed by the luxLM enzyme. AI-2 is afuranosyl borate diester; its biosynthesis is catalysed by the LuxS enzyme. AI-I and AI-2 are detected at the cell

surface by the LuxN and LuxP-LuxQ receptor proteins, respectively. At low cell density, LuxN and LuxQautophosphorylate and transfer phosphate to LuxO via LuxU. The phosphorylated LuxO is an active repressor forthe target genes. At high cell density, LuxN and LuxQ interact with their autoinducers and change trom kinases tophosphatases that drain phosphate away trom LuxO via LuxU. The dephosphorylated LuxO is inactive.Subsequently, transcription of the target genes is activated by LuxR. Redrawn after Miller and Bassler (200 I).

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72 T. Defoirdt et al. / Aquaculture 240 (2004) 69-88

Whitehead et aI., 2001). AHLs of different species differ in the acyl side chain, whichusually contains between 4 and 14 carbons and which can have an oxo or a hydroxylsubstitution at the third position. In addition to the AHL-mediated systems in Gram-negative bacteria, some Gram-positive bacteria were also found to regulate a variety ofprocesses in response to their population density. The quorum sensing systems ofStreptococcus pneumoniae, Bacillus subtilis and Staphylococcus aureus, for instance,have been extensively studied (for reviews see Dunny and Leonard, 1997; Miller andBassler, 2001). In contrast to the Gram-negative bacteria, these bacteria use secretedpeptides as signal molecules (Fig. 1B). A third major quorum sensing system was recentlyunravelled in the luminescent bacterium Vibrioharveyi (Chen et aI., 2002). The V.harveyisystem has two components: autoinducer 1 (AI-I) and autoinducer 2 (AI-2). Both canactivate bioluminescence by a phosphorylation and dephosphorylation cascade (Fig. 1C,for reviews see Miller and Bassler, 2001; McNab and Lamont, 2003; Xavier and Bassler,2003). AI-I is an AHL and because of its specificity, it is probably used for intraspeciescommunication. AI-2, on the other hand, is a furanosy1borate diester. Because AI-2activity has been detected in many other species (Gram-negativeas well as Gram-positive)and because AI-2 biosynthesis is closely linked to the methyl cycle, this type of quorumsensing is believed to be used for interspeciescommunicationand might be involved in thedetection of the growth phase and the growth potential of the bacterial community (Xavierand Bassier, 2003).

Table 1The quorum sensingsystemsof differentaquaticpathogensand the link betweenquorumsensingand virulencefactor expressionand/or virulenceas such

Species Signal Quorum sensing-regulatedvirulence (factors)

References

Yersinia ruckeri

Aeromonas

hydrophila

Aeromonassalmonicida

Vibrio

anguillarumVibrio harveyi

Vibrio

parahaemolyticusVibrio vulnificus

a BHL: N-butanoyl-L-homoserine lactone.b HHL: N-hexanoyl-L-homoserine lactone.c ODHL: N-(3-oxodecanoyl)-L-homoserine lactone.d OHBHL: N-(3-hydroxybutanoyl)-L-homoserine lactone.

BHLa,HHLb biofilm formation, Swift et at (1997),exoproteaseproduction Swift et al. (1999),

Lynch et al. (2002)BHLa,HHLb serineproteaseproduction Swift et 81.(1997)

ODHLc unknown Milton et al. (1997)

OHBHLd, siderophoreproduction, Bassler et al. (1993),AI-2 productionof type III LiIleyand Bassler (2000),

secretionsystemcomponents, Manefieldet a!. (2000),extracellulartoxin production Mok et al. (2003)

unknown opacity McCarter(1998)

AI-2 proteaseand haemolysin McDougaldet aI. (2000),production,lethalityto mice Kim et aI. (2003)

unidentified unknown Temperanoet a!. (2001)AHL

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T. Defoirdt et al. / Aquaculture 240 (2004) 69-88 73

Phenotypes that are controlled by a quorum sensing system include luminescence,conjugation, nodulation, swanning, sporulation, biocorrosion, antibiotic production andmost importantly biofilm fonnation and the expression of virulence factors such as lyticenzymes, toxins, siderophores and adhesion molecules (De Kievit and Iglewski, 2000;Miller and Bassler, 2001; Whitehead et aI., 2001; De Wmdt et aI., 2003). Quorum sensingsystems are found in a still growing list of bacteria that are pathogenic to plants, animalsand humans (for reviews see De Kievit and Iglewski,2000; Williams et aI., 2000). The listincludes (but is not restricted to) the aquaticpathogensAeromonas hydrophila,Aeromonassalmonicida, Vibrioanguillarum, V.harveyi, Vibrioparahaemolyticus, Vibrio vulnificusand Yersinia ruckeri (Table I). For most of them, a link between quorum sensing andvirulence factor expression has been demonstrated (Table I). These quorum sensingpathogens probably increase their chances to infect their host successfully by delayingvirulence factor production until the population density is large enough to overwhelm thehost's immune system (Donabedian, 2003). It has been shown that inactivating the quorumsensing system of quorum sensing pathogens can result in a significant decrease invirulence factor expression (Jones et aI., 1993; Swift et aI., 1999) and in a decrease ofvirulence as such (WUet al., 200I). As the importance of quorum sensing in virulencedevelopment of pathogenic bacteria became clear, disruption of quorum sensing wassuggested as a new anti-infective strategy (Finch et aI., 1998). Subsequently, severalresearch groups started to investigate different techniques that could disrupt the quorumsensing systems of pathogens. The techniques that have been developed so far will bediscussed throughout the following paragraphs.

o o oo o o

o o oo

o

signalsynthase

responseregulator

(~o

target ge~e(s) >

o

target ge~e(s) )~ virulence

Fig. 2. Schematic overview of different strategies that have been developed to disrupt bacterial quorum sensing.(A) Inhibition of signal molecule biosynthesis by the application of substrate analogues. (B) Blocking signal

transduction by the application of quorum sensing antagonists. (C) Chemical inactivation and biodegradation of

signal molecules. (D) Application of quorum sensing agonists to evoke virulence factor expression at lowpopulation density. See text for details.

00

0 . 00

00 0 . (C) .

0 0 A- A. 0 0

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74 T. Defoirdt et al. / Aquaculture 240 (2004) 69-88

3. Inhibition of signal molecule biosynthesis

A first quorum sensing disrupting technique aims at inhibiting signal moleculebiosynthesis (Fig. 2A). In many cases,homologues of the V.fischeri Lux! protein catalysethe biosynthesis of Gram-negativeAHL signal molecules, using acyl-acyl carrier proteins(for the acyl chain) and S-adenosylmethionine (for the homoserine lactone moiety) assubstrates (Whitehead et aI., 2001). In an attempt to block AHL biosynthesis, Parsek et al.(1999) found that analogues of S-adenosylmethionine(such as S-adenosylcysteine)couldinhibit activity of the Pseudomonas aeruginosa Lux! homologue RhlI by up to 97%.Database research revealed that no AHL synthase sequence motifs were present in otherenzymes with S-adenosylmethionine binding sites. Therefore, it might be possible to usethe S-adenosylmethionine analogues as specific quorum sensing inhibitors, withoutaffecting other vital processes in prokaryotic and eukaryotic organisms. However, furtherresearch is necessary to elucidate whetherAHL biosynthesis inhibitorswould be useful tocombat infections in aquaculture since the paper of Parsek et al. (1999) is the only onedealing with this type of compounds so far.

4. Application of quorum sensingantagonists

4.1. Antagonists for AHL-mediated quorum sensing

4.1.1. De/isea pulchra halogenatedfuranonesAs quorum sensing-mediated processes are often involved in the interaction with plant

and animal hosts, it might not be surprising that these higher organisms have developedmechanisms to disrupt quorum sensing. One of these mechanisms is the production ofquorum sensing antagonists: molecules that can bind to quorum sensing responseregulators, but fail to activate them (Fig. 2B). The red marine alga D. pulchra hasdeveloped such a defense mechanismto protect itself from extensivebacterial colonisation(Givskov et aI., 1996). The alga produces halogenated furanones as antagonists for AHL-mediated quorum sensing. Because of their structural similarity with AHLs (Fig. 3), the

HBr

o oDelisea pulchra furanoneAHL

Fig. 3. Structuml similarity between AHL and the D. pulchra halogenated furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(Compound2). The R in the AHL molecule is usually an alkylgroup consistingof between 3 and 13 carbons,which can have an oxo or hydroxylsubstitutionat the secondcarbon.

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T. Defoirdt et al. / Aquaculture 240 (2004) 69-88 75

halogenated furanones most probably bind to LuxR type proteins without activating them(Manefield et aI., 1999; Rasmussen et aI., 2000). Givskov et al. (1996) showed thatswarming of the pathogen Serratia liquefaciens on agar plates could be inhibitedcompletely by adding 100 mg/1 of (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (Compound 2). This furanone could also suppress the expression of bio-luminescence genes, located on a reporterplasmid in S. liquefaciens,without affecting thegrowth rate of the bacterium. Interestingly,Manefield et al. (2000) found that the furanoneinhibited extracellular toxin production in a pathogenic V.harveyi strain. Mortality in theshrimp Penaeus monodon was reduced to 50% after intramuscular injection with dilutedcell supematant extracts from V.harveyi cultures grown in the presence of the halogenatedfuranone, compared to extracts from untreated cultures.

4.1.2. Antagonistic AHL molecules

Apart from the D. pulchra furanones, it was shown that some naturally occurringAHL molecules could stimulate quorum sensing-regulated pigment production in theGram-negative bacterium Chromobacterium violaceum, while other AHLs completelyinhibited this phenotype (McClean et aI., 1997). The stimulatory or inhibitory effectwas linked to the structure of the acyl side chain of the molecules: AHLs with an acylside chain containing up to eight carbons were stimulatory, acting as quorum sensingagonists. AHLs with an acyl chain containing 10 carbons or more, on the other hand,were inhibitory and acted as quorum sensing antagonists. Apart from that, research byZhu et al. (1998) indicated that heterologous AHLs are potent antagonists for AHL-mediated quorum sensing in bacteria that express the LuxR-type proteins at nativelevels. Overexpression of these proteins abolished the repression. Interestingly, researchby Swift et al. (1997) and Swift et al. (1999) indicated that heterologous AHLs areable to reduce virulence factor production by the aquatic pathogens A. hydrophila andA. salmonicida. In the first report, Swift et al. (1997) showed that serine proteaseproduction by A. salmonicida was delayed and that the final concentration wasreduced to 50% by applying N-(3-oxododecanoyl)-L-homoserine lactone at aconcentration of 10 JJ.M.In a second research, Swift et al. (1999) demonstrated thatN-(3-oxodecanoyl)-L-homoserine lactone, at a concentration of 10 JJ.M, inhibitedquorum sensing-regulated exoprotease production by the Gram-negative pathogen A.hydrophila as well. Two other 3-oxo substituted long-acyl AHLs, N-(3-oxododeca-noyl)-L-homoserine lactone and N-(3-oxotetradecanoyl)-L-homoserinelactone, had asimilar effect.

4.1.3. Synthetic AHL-mediated quorum sensing antagonistsBased on the fmdings mentioned above, several research groups started to investigate

the activities of different synthetic AHL and furanone analogues with respect to quorumsensing (Reverchon et aI., 2002; Smith et aI., 2003a; Hentzer et aI., 2002). As far as weknow, a synthetic derivative of the D. pulchra halogenated furanones, (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone, is the most active AHL antagonist mentioned inliterature thus far. This furanone, dosed in a concentration of 10 JJ.M,could almostcompletely reduce virulence factor expression in pure cultures of P. aeruginosa PAOI(Hentzer et aI., 2003). Interestingly, the furanone was equally active on biofilm bacteria

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76 T. Defoirdt et al. / Aquaculture 240 (2004) 69-88

compared to planktonic cells, making them susceptible to sodium dodecyl sulphate andantibiotics. In the absence of the furanone,on the contrary, 100-to 1000-foldhigher dosesof antibiotics are required to eradicate biofilm bacteria compared to their planktoniccounterparts (Anwar and Costerton, 1990).Furthennore, Hentzer et at. (2003) showed thatthe furanone was also active in vivo in mouse lungs after infection with a P. aeruginosastrain harboring a fluorescent AHL reporter plasmid. The fluorescence signal from the P.aeruginosa strain was significantly reduced after subcutaneous injection of the furanone.After 8 h, however, the signal reappeared, indicating that the furanone had cleared fromthe mouse blood.

4.1.4. Antagonists produced by higherplants and micro-algaeIn an attempt to learn whether eukaryotes can interact with bacterial quorum sensing,

Teplitski et al. (2000) found that exudates from higher plants, such as pea, rice, soybean,tomato, crown vetch and Medicago truncatula also influence AHL-mediated quorumsensing. Reverse phase high-perfonnance liquid chromatography revealed that there areseveral different AHL mimicking substances present in extracts from pea and Mtruncatula seedlings (Teplitski et aI., 2000; Gao et aI., 2003). In contrast to D. pulchra,these plants secrete substances that stimulate AHL-dependent quorum sensing as well assubstances that inhibit such responses. Interestingly,similar results were recently obtainedfor micro-algae (Teplitski et aI., 2004). The algae Chlamydomonas reinhardtii,Chlamydomonas mutablis, Chlorella vulgaris and Chlorellafusca all stimulated quorumsensing-regulated luminescence in wildtype V. harveyi. In contrast, colonies of C.reinhardtii inhibited AHL-mediated luminescence in several different Escherichia coliAHL reporter strains. The inhibition of luminescencewas shown not to be due to toxicityor to an inhibition of luminescence as such since the luminescence of a constitutivelyluminescent derivative of E. coli was not inhibited.Apart from that, culture age as well asgrowth conditions were shown to be important factors in the production of AHLmimicking substances by C. reinhardtii. The levels of these substanceswere considerablyhigher if the alga was cultured phototrophically compared to culturing on a mediumcontaining acetate as a carbon source. Unfortunately, the chemical nature of the quorumsensing mimic compounds secreted by higher plants and micro-algae still has to beelucidated.

4.2. Antagonists for Al-2-mediated quorum sensing

The AI-2 structure has only recentlybeen elucidated (Chen et aI., 2002). Therefore, notmuch effort has been done to disrupt AI-2-mediatedquorum sensing so far. In one report,however, Ren et at. (2001) found that the halogenated D. pulchra furanone Compound 2,previously described as an AHL antagonistic analogue, could completely inhibit AI-2-regulated swarming of E. coli. Moreover, the furanone decreased thickness of E. colibiofilms by 55% and the percentage of live cells in the biofilms by 87%. Finally, thefuranone also inhibited AHL-mediated as well as AI-2-mediated luminescence in V.harveyi. The furanone might also attenuate virulence of this aquatic pathogen since theexpression of some virulence factorswas also found to be regulatedby its quorum sensingsystem (Table I).

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T. Defoirdt et al. / Aquaculture 240 (2004) 69-88 77

4.3. Significance for aquaculture

Altogether, the results mentioned above indicate that the application of quorum sensingantagonists in aquaculture might constitute an alternative approach for preventing orcombatting infections caused by pathogens that regulate virulence factor expression by anAHL-mediated quorum sensing system (such as A. hydrophila, A. salmonicida and V.harveyi) as well as by an AI-2-mediated system (such as V. harveyi and V. vulnificus).Apart ftom that, they also indicate that the positive effect of the so-called microbialmatured water might partly be due to the presence of antagonistic AHLs. Indeed, theproduction of microbial matured water takes place on a biofilter with a previouslyestablished biofilm (Skjenno and Vadstein,1999)and very high AHL concentrations werereported in biofilms before (Charlton et aI., 2000). Finally, the results obtained in theresearches with the macro-algaD. pulchra and with the unicellular algae Chlamydomonasand Chlorella indicate that algae might be useful to control infections in aquaculture asthey disrupt the quorum sensing systems of pathogenic bacteria.

5. Chemical inactivation of quorum sensing molecules

It has been established for a long time that AHLs are chemically inactivated via alkalinehydrolysis, yielding the cognate acyl-homoserine (Voelkert and Grant, 1970). To ourknowledge, the only other chemical inactivationthat has been studied so far is the reactionwith oxidised halogen antimicrobials(Fig. 2C). These antimicrobials,at a concentration ofapproximately 0.14 mM, were found to decrease the concentration of 3-oxo-substitutedAHLs to about one-fourth after 1 min incubation, but had no effect on unsubstituted ones(Borchardt et aI., 2001). Moreover, the inactivationof 3-oxo AHLs was shown to proceedin the presence of polysaccharide biofilm compounds despite the much higherconcentration of the latter compared to the AHL concentration. In a further study,Michels et al. (2003) unravelled the inactivation mechanism by liquid chromatography-mass spectrometry. Apparently, the reaction kinetics are largely influenced by the pH ofthe reaction mixture. At pH 6, a 3-oxo AHL molecule reacts quickly with two moleculesof hypobromous or hypochlorous acid, yielding a 2,2-dihalo-3-oxo AHL molecule.Subsequently, the acyl chain is hydrolysed, yielding a fatty acid and 2,2-dihalo-N-ethanoyl-L-homoserinelactone (Fig. 4). At pH 3, the reaction stops after the halogenationsteps, whereas at pH 8, the lactone ring of 2,2-dihalo-N-ethanoyl-L-homoserinelactone ishydrolysed, yielding 2,2-dihalo-N-ethanoyl-L-homoserine. These data indicate thattreating culture water with low concentrations of strong oxidising agents, such as ozone(Summerfelt, 2003), might be useful as an anti-infective therapy in aquaculture byremoving the quorum sensing molecules of pathogens.

6. Enzymatic inactivation and biodegradationof quorum sensing molecules

The ability to degrade AHLs seems to be widely distributed in the bacterial kingdom(Fig. 2C). Enzymes that are able to inactivate AHLs have been discovered in species

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78 T. Defoirdt et al. / Aquaculture 240 (2004) 69-88

o

JlR OH

Fig. 4. Reaction between a 3-oxo AHL and halogen antimicrobials (HOX; hypobromous or hypochlorous acid) atpH 6. First, two a-halogenation reactions occur, yielding 2,2-dihalo-3-oxo AHL. Subsequently, the acyl chain ishydrolised, yielding a fatty acid and 2,2-dihalo-N-ethanoyl-L-homoserine lactone. The R is an alkyl groupconsisting of between 3 and 13 carbons, which can have an oxo or hydroxyl substitution at the second carbon.Redrawn after Michels et at (2000).

belonging to the ~-Proteobacteria (Zhang et aI., 2002), the a-Proteobacteria (Leadbetterand Greenberg, 2000; Lin et aI., 2003; Uroz et aI., 2003) and the ')'-Proteobacteria(Uroz etaI., 2003) as well as in some Gram-positivespecies (Dong et aI., 2002; Lee et aI., 2002;Uroz et aI., 2003). These bacteria might block the quorum sensing systems of theirbacterial competitors to obtain a selectiveadvantage over them. This could be the case, forinstance, for those microbes living in proximity of bacteria that regulate the production ofantibiotics via quorum sensing (pierson et aI., 1998).The actual inactivationof the signalcompound can be mediated by two types of enzymes: AHL lactonases and AHL acylases(Fig. 5). Moreover, eukaryotic acylases might be able to inactivate AHLs as well (Xu etaI., 2003).

6.1. Bacterial AHL lactonases

Dong et al. (2000) screened more than 500 field and laboratory bacterial isolates forAHL-inactivating activity. Of these, 24 isolates showed different levels of enzymaticactivity in eliminating AHLs. The enzyme responsible for the AHL-inactivating activity(AiiA) was isolated from the strain that showed the strongest activity,Bacillus sp. strain240B1. The purified enzyme, at a concentrationof 50 mgll, reduced the concentration ofN-(3-oxohexanoyl)-L-homoserine lactone from 20 IlM to about 5 IlM after 10 min.

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RAN i\ ~H/lf' ~AHL lactonase

fatty acid

+ H,N~oo

homoserine lactone

o

AR OH

A ~g~R N

Ho

N- acyl homoserine

Fig. 5. Enzymatic inactivation of AHLs. Cleavageof the amidebond by an AHL acylaseenzymeyields a fattyacid and homoserine lactone. Cleavage of the lactone ring by an AHL lactonase enzyme yields the correspondingacylated homoserine. The R is an alkyl group consisting of between 3 and 13 carbons, which can have an oxo orhydroxyl substitution at the second carbon.

Electrospray ionisation-mass spectrometryof the hydrolysisproduct revealed that the AiiAenzyme opens the lactone ring to produce N-(3-oxohexanoyl)-L-homoserine(Dong et aI.,2001). Further research demonstratedthat genes encoding AHL-degrading lactonases arewidespread in many Bacillus species (Dong et aI., 2002; Lee et aI., 2002; Dong et aI.,2004). These AiiA homologues showed about 90% sequence homology at the amino acidlevel.

The fIrst evidence indicating that enzymatic AHL inactivation could be used as abiocontrol strategy was provided in the study of Dong et a1. (2000). In this study,expression of the AiiA enzyme in transformed Erwinia carotovora decreased theproduction of cell wall degrading enzymes by the pathogen to about 10% and inhibitedsoft rot disease symptoms in susceptible plants almost completely. In a further in vivostudy, Molina et a1. (2003) tested the efficacy of using an AHL-degrading Bacillus sp.strain for the biocontrol of plant diseases.The Bacillus sp. strain could reduce potato tubersoft rot caused by E. carotovora to about 15% and crown gall in tomato caused byAgrobacterium tumefaciens to about 10%. AHL degradation by the Bacillus sp. strainoffered a protection as effective as or better than antibioticproduction by a Pseudomonaschlororaphis biocontrol strain. Moreover,degradation of AHLs had not only a preventive,but also a curative biocontrol activity.Recently,similarresults were obtained with Bacillusthuringiensis (Dong et aI., 2004).

6.2. Bacterial AHL acylases

6.2.1. AHL acylases in Variovoraxparadoxus and RalstoniaAt the same time as Dong et a1. (2000) discovered an AHL-degrading Bacillus sp.

strain, Leadbetter and Greenberg (2000) isolated a strain that could use AHLs as the solesource of carbon and nitrogen. This strain, V.paradoxus VAI-C, was enriched on amedium with 500 mg/1 N-(3-oxohexanoyl)-L-homoserinelactone as the sole source ofcarbon and nitrogen. The researchers deduced trom experiments with radiolabeled AHL

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that V.paradoxus cleaves the AHL by an AHL acylase enzyme, releasing homoserinelactone and a fatty acid. Subsequently,the fatty acid is used as carbon source via the ~-oxidation pathway. Further research is needed to elucidate how the bacteriwn obtains thenitrogen from the homoserine lactonemoiety.More recently,Flagan et a1.(2003) isolated abacterium, Arthrobacter sp. strain VAl-A, capable of degrading and utilising thenitrogenous breakdown products of AHL signal molecules. Interestingly, the AHL-dependent growth rate and yield of a coculture of the Arthrobacter sp. strain and V.paradoxus VAl-C were superior to those of monocultures of these bacteria, indicating thatconsortia may have a synergistic effect in quorum sensing signal turnover andmineralisation.

Recently, Lin et al. (2003) isolated an AHL-inactivatingbacterium,Ralstonia sp. strainXJI2B, from a mixed-species biofilm. The enzyme responsible for the AHL-inactivatingactivity (AiiD) was purified and subsequently,N-(3-oxodecanoyl)-L-homoserinelactonewas incubated with the purified enzyme. Electrospray ionisation-massspectrometry of thehydrolysis product demonstrated that the AiiD enzyme hydrolyses the amide bond ofAHLs. Expression of the AiiD enzyme in transformed P. aeruginosa PAO1 inhibitedswarming of the pathogen and reduced mortality of the nematode Caenorhabditis elegansto about 15%, compared to wildtype PAOl.

6.2.2. Specific AHL inactivation by AHL acylasesproduced by pseudomonadsAnother recent research, by Huang et al. (2003), indicated that some substrate

specificity of AHL-inactivating enzymes can exist. The researchers isolated a soilpseudomonad, strain PAl-A, that inactivated AHLs by means of an AHL acylaseenzyme. In contrast to V.paradoxus and Ralstonia sp. strain XJ12B, the pseudomonadcould only utilise AHLs with acyl side chains longer than eight carbons. Since the 16SrRNA gene from strain PAl-A showed high similarity to the one from the pathogen P.aeruginosa PAOl, the ability of the pathogen to degrade AHLs was investigated aswell. In accordance with the results obtained for the strain PAl-A, the pathogen startedto grow on long-acyl AHLs but not on short-acyl AHLs. The investigators presumedthat degradation of its own long-acyl AHL, N-(3-oxododecanoyl)-L-homoserinelactone,may play a role in the regulation of the quorum sensing system of P. aeruginosa. Sucha signal turnover control mechanism had already been found in A. tumefaciens (Zhanget al., 2002).

6.3. Eukaryotic acylases

Since several bacterial AHL acylases had been found to inactivate AHLs bydeacylation, Xu et al. (2003) investigated the ability of an eukaryotic counterpart ofthese bacterial enzymes to inactivate AHL molecules. Different AHLs were shown to beinactivated by the porcine kidney acylase I enzyme. Since the inactivationwas best at highpH, it could have been due to simple alkalinehydrolysis of the lactonering. However, theacylase could reduce a model biofilm, made mainly by a Pseudomonas and aMicrobacterium species, indicating that the enzyme might be able to inactivate AHLs atenvironmentally relevant concentrations. Nevertheless, further research is needed toinvestigate to what extent eukaryotic acylases can indeed inactivate AHLs and whether

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these enzymes have any significancein the in vivo inhibitionof quorum sensing-mediatedgene expreSSIOn.

6.4. Enzyme activity and substrate threshold levels

Several bacteria were shown to be able to degrade AHLs if these molecules werepresent at J.LMto mM concentrations (Dong et al., 2000; Leadbetter and Greenberg,2000). Unfortunately, no enzyme activity studies have been conducted so far. Moreover,previous research indicated that the biodegradation abilities of micro-organisms can belimited to above certain threshold substrate levels (Janssens et aI., 1997; Kovar et al.,2002). As transcription of quorum sensing-regulated genes can be activated at AHLconcentrations as low as 1 nM (Andersen et al., 2001), it still has to be establishedwhether substrate specificity of the AHL-degrading enzymes is sufficiently high todecrease the AHL concentration to below this threshold. However, mixed substrategrowth experiments indicate that threshold concentrations might be lower or even non-existent if other carbon sources are present (as in most natural ecosystems) than if thereis only one single carbon source (Egli, 2004). Indeed, several in vivo studies showedthat the degradation of AHLs might continue until below the level needed for activationof quorum sensing-regulated virulence genes (Dong et aI., 2000; Molina et al., 2003;Uroz et al., 2003; Dong et al., 2004).

6.5. Significancefor aquaculture

The data mentioned above indicate that bacteria that are able to degrade quorumsensing molecules might be useful as biocontrol agents in aquaculture. Hence, it is ofinterest to investigate whether quorum sensing molecule degraders would be goodprobionts. Apart from that, the reportsdealing with AHL-degradingBacillus spp. (Dong etal., 2000; Molina et al., 2003; Dong et al., 2004) suggest that the positive effect of Bacillusspp., used as probionts in aquaculture (Moriarty, 1998; Rengpipat et al., 2003), mightpartly be due to inactivation of quorum sensing molecules-apart from the production ofgrowth-inhibiting substances.

7. The opposite way: application of quorum sensing agonistic analogues

All techniques discussed so far aim to inactivate quorum sensing-regulated virulencefactor expression. Mae et al. (2001), however, tested an opposite strategy: they activatedquorum sensing-regulated virulence factor expression by using quorum sensing agonists.The idea behind this strategy was that by adding the signal molecule of a pathogen,virulence factor expression would be activated at low population density (Fig. 2D).Subsequently, the virulence factors could trigger the activation of the host's defensesystem allowing resistance to develop. In the research of Mae et al. (2001), disease intobacco plants caused by E. carotovorawas reduced to 10%by applying a 5 mM solutionof the pathogen's own AHL. Furthermore, the ability of E. carotovora to cause diseaseafter local inoculation with 106 pathogens per plant was decreased to about half in

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transgenic tobacco plants producing the pathogen's AHL compared to wildtype lines. Ifthe infection was already establishedor if the inoculum size was increasedby a factor 4 ormore, however, there was no significant difference between AHL-producing and controlplants. Apparently, the pathogen was present in sufficiently large numbers to overwhelmthe defense system of the plant in these cases. Taken together, these results are inaccordance with the original hypothesis that quorum sensing is used to avoid prematurevirulence factor production and subsequent activation of plant defense responses. As thisresearch was conducted with plants, it would be very interesting to conduct similar testswith (aquatic) animals to investigate whether their immune systems-specific ornonspecific-could be activated successfully if a quorum sensing pathogen is presentby the application of the microbe's signal molecule.

8. Limitations to disruption of bacterial quorum sensing

8.1. Resistance development

A first limitation to the use of quorum sensing disrupting techniques as a new anti-infective therapy might be resistance development. Indeed, research by Zhu et al. (1998)indicates that bacteria could simply circumvent quorum sensing blockade by over-expressing quorum sensing genes. These researchers found that many synthetic AHLanalogues were potent inhibitors of quorum sensing responses in wildtype A. tumefaciens,whereas in a transformed strain that overexpressed the A. tumefaciens LuxR homologueTraR, inhibition was not detected for any of the analogues. However, as disruption ofquorum sensing is less likely to pose a selective pressure for development of resistancethan conventional antibacterial compounds do (Hentzer et aI., 2003), the chance that aquorum sensing mutant will arise might be rather small.

8.2. Lack of specificity

Apart from resistance development,the lack of specificitycould also confme the use ofquorum sensing disrupting techniques to control pathogens. Most quorum sensingdisrupting techniques developed so far are not specifically blocking the quorum sensingsystem of one or more pathogens. Most AHL degrading bacteria, for instance, inactivate awide range of AHL molecules. However, not all bacteria found to contain a quorumsensing system are pathogens. Quorum sensingwas shown, for example, to be involved innodule formation by Rhizobium species and in antibiotic production by fluorescentpseudomonads (pierson et aI., 1997; Pierson et aI., 1998). As could be the case for thesegrowth promoting and biocontrol activitiesof plant-associatedbacteria, gross disruption ofquorum sensing might adversely affectyet unknown favourablequorum sensing-regulatedprocesses in aquatic ecosystems. On the other hand, it will probably be difficult to developtechniques that only disrupt the quorum sensing systemof a single pathogenic species. It isclear that much more knowledge about the occurrence and function of quorum sensing inaquaculture systems will be necessary in order to be able to develop a new anti-infectivestrategy.

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9. Conclusions and further perspectives

The results obtained by using techniques that disrupt quorum sensing systems ofpathogenic bacteria indicate that it is a promising alternative for antibiotics in fightingbacterial infections. This new approach might also have value in aquaculture since a linkbetween quorum sensing and virulence factor expression in several aquatic pathogens hasbeen demonstrated. Unfortunately, data about the impact of quorum sensing on virulence(i.e. the net result of all virulence factors) of aquatic pathogens are still lacking.Fundamental research in the quorum sensing domain will undoubtedly provide moreprecise insights into the mechanismby which the expressionof quorum sensing-regulatedgenes is activated or inhibited.This research will make it possible, for example, to conducta more directed search for antagonists. So far, most of the research has been done on theAHL-mediated quorum sensing systems of Gram-negative human and plant pathogens.However, it can be expected that more techniques to disrupt quorum sensing systems ofother pathogens will be developed in the future.

Before this new strategy can be applied in aquaculture,there are some important topicsthat should be faced. First of all, the impact of disrupting the quorum sensing system ofseveral aquatic pathogens on their virulence should be studied in full depth in order toelucidate whether it is a valid anti-infectivestrategy in aquaculture.Secondly,the differentquorum sensing disrupting techniques should be investigatedfurther in order to determinewhich technique(s) suits best for the aquaculturesystemof interest.In this view, one shouldtry to get an idea about the impactof the techniqueson the healthof the final consumer of theaquaculture product and also on the aquaculture system itself since gross disruption ofquorum sensing might adversely affectyet unknown favourablequorum sensing-regulatedprocesses. Moreover, some practicalproblems-such as the cost of a treatment and how todeliver the quorum sensing disruptingcompound or organism to the site of action-shouldbe considered. Finally, the problem of eventual resistance development should not beneglected although disruptionof quorumsensing is less likelyto pose a selectivepressure fordevelopment of resistance than conventionalantibacterialcompounds do.

Acknowledgements

The authors wish to acknowledge financial support from the "Instituut voor deaanmoediging van Innovatie door Wetenschapen Technologiein Vlaanderen"(IWT grantno. 31205) and the "Fonds voor WetenschappelijkOnderzoek" (project no. 3G0230.02).Special thanks go to Wim De Windt, Roeland Grommen, Fernando Morgan (posdoctoralFellow supported by the Francqui Foundation of Belgium) and Klara Van Driessche forcritically reading the manuscript.

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