Quorum quenching enzymes

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Quorum quenching enzymes Susanne Fetzner ∗ Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstrasse 3, D-48149 Muenster, Germany

a r t i c l e

i n f o

Article history: Received 9 June 2014 Received in revised form 29 August 2014 Accepted 4 September 2014 Available online xxx Keywords: Quorum sensing Quorum quenching Lactonases Acylases Oxidoreductases

a b s t r a c t Bacteria use cell-to-cell communication systems based on chemical signal molecules to coordinate their behavior within the population. These quorum sensing systems are potential targets for antivirulence therapies, because many bacterial pathogens control the expression of virulence factors via quorum sensing networks. Since biofilm maturation is also usually influenced by quorum sensing, quenching these systems may contribute to combat biofouling. One possibility to interfere with quorum sensing is signal inactivation by enzymatic degradation or modification. Such quorum quenching enzymes are wide-spread in the bacterial world and have also been found in eukaryotes. Lactonases and acylases that hydrolyze N-acyl homoserine lactone (AHL) signaling molecules have been investigated most intensively, however, different oxidoreductases active toward AHLs or 2-alkyl-4(1H)-quinolone signals as well as other signal-converting enzymes have been described. Several approaches have been assessed which aim at alleviating virulence, or biofilm formation, by reducing the signal concentration in the bacterial environment. These involve the application or stimulation of signal-degrading bacteria as biocontrol agents in the protection of crop plants against soft-rot disease, the use of signal-degrading bacteria as probiotics in aquaculture, and the immobilization or entrapment of quorum quenching enzymes or bacteria to control biofouling in membrane bioreactors. While most approaches to use quorum quenching as antivirulence strategy are still in the research phase, the growing number of organisms and enzymes known to interfere with quorum sensing opens up new perspectives for the development of innovative antibacterial strategies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cell-to-cell communication in bacteria, or quorum sensing (QS), relies on the production, secretion, and detection of diffusible signaling molecules (Fig. 1). Bacteria can modulate their gene expression patterns in a growth- and cell-density dependent manner in response to the concentration of chemical signals in their local environment, affording a coordinated, synchronized behavior

Abbreviations: AHL, N-acyl homoserine lactone; AI-2, autoinducer-2; AQ, 2-alkyl-4(1H)-quinolone; Cx-HSL, N-acyl homoserine lactone with x denominating the acyl chain length; DPD, 4,5-dihydroxy-2,3-pentanedione; DSF, diffusible signal factor; HDL, high-density lipoprotein; HHQ, 2-heptyl-4(1H)-quinolone; HSL, homoserine lactone; IQS, 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde; MBR, membrane bioreactor; 3OCx-HSL, 3-oxo-N-acyl homoserine lactone with x denominating the acyl chain length; 3OHCx-HSL, 3-hydroxy-N-acyl homoserine lactone with x denominating the acyl chain length; 3-OH-PAME, 3-hydroxypalmitic acid methyl ester; PG, 2-phosphoglycolic acid; PLL, phosphotriesterase-like lactonase; PON, paraoxonase; PQS, Pseudomonas quinolone signal (2-heptyl-3hydroxy-4(1H)-quinolone); QQ, quorum quenching; QS, quorum sensing; THMF, 2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran. ∗ Tel.: +49 251 8339824; fax: +49 251 8338388. E-mail address: [email protected]

of the population (reviewed by, e.g., Heeb et al., 2011; Ng and Bassler, 2009; Schuster et al., 2013; Williams, 2007). Because the signal molecules also trigger their own biosynthesis (Fig. 1), they have been termed autoinducers. QS systems regulate diverse functions which are thought to require the concerted action of numerous cells to be productive, such as bioluminescence, production of secondary metabolites, biofilm maturation, sporulation, and competence for DNA uptake. Many bacterial pathogens, for example, Pectobacterium spp. causing soft rot diseases in important crop plants, or Pseudomonas aeruginosa which is one of the leading opportunistic pathogens, control the expression of virulence factors via QS networks (Crépin et al., 2012; Nadal Jimenez et al., 2012). Gram-negative bacteria predominantly employ N-acyl homoserine lactones (AHL; Fig. 2A), which cross membranes and are detected in the cytoplasm by LuxR-type proteins that upon ligand binding act as transcriptional activators of QS-controlled genes (Fig. 1). In Gram-positive bacteria, QS signaling mainly relies on small cyclic or linear peptides, which either are detected by a membrane-bound sensor kinase that transduces the signal by a phosphorylation cascade, or after import into the cell directly interact with a cognate regulator that modulates gene expression (Cook and Federle, 2014; Thoendel and Horswill, 2010). However,

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Fetzner,

S.,

Quorum

quenching

enzymes.

J.

Biotechnol.

(2014),

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2

QS system at high signal concentration

Strategies to interfere with QS

Inactivation of the signal molecules

R

R

Inhibition of signal detection

I

Inhibition of signal biosynthesis

I

R

I targets

Fig. 1. Simplified general scheme of a quorum sensing system of Gram-negative bacteria, and targets and strategies to interfere with QS. A signal synthase (I), or a set of biosynthetic enzymes, produce chemical signal molecules (triangles), which reach the extracellular environment by diffusion or transport. At high signal molecule concentrations, the signal receptor (R) forms a complex with the signal. The signalreceptor complex activates expression of the signal synthase gene(s) (autoinduction loop), and directly or indirectly modulates the transcription of sets of target genes.

Streptomyces spp. and some other actinobacteria use ␥butyrolactones for QS signaling to regulate differentiation and antibiotic production (Takano, 2006). Some Gram-negative bacteria respond to lipid signals such as cis-11-methyl-2-dodecenoic acid, the so-called diffusible signal factor (DSF; Fig. 2B) (Wang et al., 2004a), or 3-hydroxypalmitic acid methyl ester (3-OHPAME; Fig. 2C) which is involved in regulating the virulence of

Ralstonia solanacearum (Flavier et al., 1997). The signaling molecule autoinducer-2 (AI-2; Fig. 2D), which actually is a mixture of several interconvertible furanones derived from 4,5-dihydroxy-2,3pentanedione (DPD), is thought to be a “universal” signal involved in intra- as well as interspecies communication (Xavier and Bassler, 2003). Multiple QS systems can be integrated in one bacterial species. P. aeruginosa, for example, uses a complex hierarchical network that integrates N-3-oxododecanoylhomoserine lactone (3OC12-HSL)-dependent, N-butanoylhomoserine lactone (C4-HSL)-dependent, and 2-alkyl-4(1H)-quinolone-dependent QS circuits (the las, rhl, and pqs systems) (Heeb et al., 2011; Nadal Jimenez et al., 2012). The pqs system uses 2-heptyl-3-hydroxy4(1H)-quinolone (the “Pseudomonas quinolone signal”, PQS) as well as its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) as signal molecules (Fig. 2E). The las system moreover controls the synthesis of the thiazole signal IQS (Fig. 2F), which links las to phosphate stress response (Lee et al., 2013). P. aeruginosa additionally uses pyoverdine and DSF-like unsaturated fatty acid signals to coordinate gene expression (Nadal Jimenez et al., 2012). There is considerable interest in agents that selectively interfere with the QS systems of pathogenic bacteria, in order to target bacterial virulence and to develop new anti-infective therapies. QS interference (or “quorum quenching”, QQ) may be achieved by inhibiting the biosynthesis of the signal molecules, by inhibiting signal detection by blocking the signal receptor, or by enzymecatalyzed degradation or modification of the signal molecules (Fig. 1). Compared with bactericidal or bacteriostatic strategies, QS interference is generally believed to less likely select for resistance, because it usually does not directly affect growth (Defoirdt et al., 2010). However, it has been shown that under certain

E

A R1 R2

O R

O

O N H

N H

O

B

F

OH

C

H

N

COOH S

O

OH O O

HO HO

D

CH3 OH O

HO

OH O

CH3

R-THMF

OH O DPD

OH

HO

spontaneous HO HO

OH CH3

B(OH)4 - 2 H 2O

O

HO

S-THMF

B O HO

O CH3

HO

O

Fig. 2. Chemical structures of representative quorum sensing signal molecules of Gram-negative bacteria. (A) N-Acyl homoserine lactone (AHL); R1 , H or O; R2 , nalkyl. (B) cis-11-Methyl-2-dodecenoic acid (diffusible signal factor, DSF). (C) 3-Hydroxypalmitic acid methyl ester (3-OH-PAME). (D) The autoinducer-2 (AI-2) family: the biosynthetic product 4,5-dihydroxy-2,3-pentanedione (DPD) spontaneously undergoes cyclization and hydration to form R-THMF and S-THMF (R- and S-2-methyl-2,3,3,4tetrahydroxytetrahydrofuran); the latter in the presence of boron reacts to S-THMF-borate. (E) R, H: 2-Heptyl-4(1H)-quinolone (HHQ); R, OH: 2-heptyl-3-hydroxy-4(1H)quinolone (PQS, the Pseudomonas quinolone signal). (F) 2-(2-Hydroxyphenyl)-thiazole-4-carbaldehyde (IQS).

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conditions, disruption or quenching of QS can indeed influence bacterial growth (reviewed by Defoirdt et al., 2010; García-Contreras et al., 2013). Natural heritable variation in QS genes and their expression level, if resulting in differences in fitness under QQ conditions, can lead to the selection of bacteria that are insensitive to QQ. One example of a reported resistance mechanism in P. aeruginosa is the development of mutations that result in the upregulation of an efflux system that exports the QQ compound (5Z)-4bromo-5-(bromomethylene)-2(5H)-furanone (Maeda et al., 2012). Nature has evolved different tools to interfere with QS. Quorum quenching allows a prokaryotic or eukaryotic species to modulate the behavior of their microbial community. The red macroalga Delisea pulchra, for example, produces halogenated furanones that show structural similarity to AHLs and act as quorum sensing inhibitors (Givskov et al., 1996). Indole, a metabolite produced by Escherichia coli and other bacteria, and related compounds were reported to repress the biosynthesis of PQS, albeit at comparatively high concentrations (Tashiro et al., 2010). Many secondary metabolites may act as small-molecule inhibitors or modulators of QS (for a recent review, see LaSarre and Federle, 2013). With respect to signal inactivation, enzyme-catalyzed modification or degradation of bacterial signal molecules is a QQ strategy that seems to be wide-spread among microorganisms, but can also be found in the eukaryotic host. It is not yet known whether bacteria can evolve resistance to signal-degrading enzymes. An increase of signal production, or an increased affinity of the signal receptor, could outcompete the effect of enzymatic signal inactivation. Bacteria could also evolve modified signal molecules that are not attacked by the QQ enzyme. However, QQ tools that address an extracellular target may exert less selective pressure to evolve resistance than QQ compounds that act intracellularly (Defoirdt et al., 2010; García-Contreras et al., 2013). This review will give an overview on QQ enzymes, and discuss potential biotechnological applications of enzyme-based QQ. Most of the research on QQ has focused on the QS systems of Gram-negative bacteria. With respect to the peptide-mediated QS systems employed by Gram-positive bacteria, recent publications suggest that natural interference with these systems is rather based on inhibitory secondary metabolites, or bacterially produced inhibitor peptides that act competitively, rather than QQ enzymes (reviewed by Cook and Federle, 2014; LaSarre and Federle, 2013). In this context, it may be interesting that reactive oxygen and nitrogen compounds such as hypochlorous acid and peroxynitrite, which are produced by phagocyte NADPH oxidase, myeloperoxidase, and NO synthase, were reported to oxidize and thus inactivate a virulence-inducing QS peptide of Staphylococcus aureus, indicating an important (but indirect) role of host enzymes in interference with the QS signals of Gram-positive pathogens (Rothfork et al., 2004). However, since QQ enzymes that specifically target peptide signals have not been described so far, these systems will not be discussed further. The majority of QQ enzymes characterized to date have been isolated from bacteria with the ability to degrade AHL signal molecules (Table 1), these will be specified in Sections 2–4. Only a few enzymes active toward other signal molecules have been described, and examples will be given in Sections 5 and 6. An extracellular esterase produced by an Ideonella sp. strain was reported to hydrolyze the 3-OH-PAME signal, which in the phytopathogen R. solanacearum regulates the production of virulence-associated extracellular polysaccharides and proteins, however, the enzyme has a relaxed specificity to a series of 3-hydroxylated long-chain fatty acid esters (Shinohara et al., 2007). Several bacterial strains able to degrade the DSF fatty acid signal molecule produced by Xanthomonas spp. and Xylella fastidiosa have been isolated, which upon coinoculation with X. campestris into model plants reduced Please cite this article in press as: http://dx.doi.org/10.1016/j.jbiotec.2014.09.001

Fetzner,

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the severity of disease, however, the biochemistry involved is not fully understood (Newman et al., 2008). 2. AHL lactonases Lactonases active toward AHL signal molecules (Fig. 3A) have been found in a wide variety of organisms and among different protein families (Table 1). The first enzyme identified as a QQ lactonase, AiiA from Bacillus sp. strain 240B1 (Dong et al., 2000), belongs to the metallo-␤-lactamase superfamily; others are similar to phosphotriesterases or are members of the paraoxonase family (Table 1). However, recent identification of AiiM and AidH from the leaf-associated isolate Microbacterium testaceum StLB037 and from Ochrobactrum sp. T63, respectively, indicated that the scaffold of the ␣/␤-hydrolase fold can also support the lactonolysis of AHLs (Mei et al., 2010; Wang et al., 2010). Some lactonases derived from soil metagenomes appear to belong to yet other protein families (Bijtenhoorn et al., 2011b; Schipper et al., 2009). The AHL lactonase QsdH from the marine bacterium Pseudoalteromonas byunsanensis 1A01261 was identified as an inner membrane protein with an N-terminal catalytic GDSL hydrolase domain predicted to be located at a periplasmic extension, and a C-terminal RND-type efflux pump domain (Huang et al., 2012). A putatively extracellular lactonase DlhR with a conserved GSD(L) motif and a predicted dienelactone hydrolase domain was isolated from Rhizobium sp. strain NGR234, which has several other predicted QQ enzymes, among them at least one other AHL lactonase (QsdR1) that belongs to the metallo-␤-lactamase family (Krysciak et al., 2011). Most AHL lactonases identified so far are of bacterial origin. However, root-associated fungi of the Ascomycota and Basidiomycota with the ability to degrade AHLs also were proposed to possess lactonases (Uroz and Heinonsalo, 2008). Expression of an aiiA gene from B. cereus A24 in the opportunistic pathogen P. aeruginosa reduced the content of 3OC12-HSL, prevented accumulation of C4-HSL, decreased the production of extracellular virulence factors, and reduced swarming motility (Reimmann et al., 2002). Several other QQ enzymes when expressed in P. aeruginosa PAO1 were also observed to affect motility and biofilm formation (Bijtenhoorn et al., 2011b; Krysciak et al., 2011; Schipper et al., 2009). Expression of Bacillus sp. AiiA lactonases in Burkholderia thailandensis reduced AHL concentrations and altered swarming and twitching motility (Ulrich, 2004). For several AHL lactonases, it was shown that heterologous expression of the respective gene in the plant pathogen Pectobacterium carotovorum (formerly, Erwinia carotovora) reduced the AHL levels, decreased the levels of extracellular pectolytic enzymes and attenuated the virulence of this pathogen in tobacco, potato, or other plants (Carlier et al., 2003; Dong et al., 2000; Mei et al., 2010; Morohoshi et al., 2012; Riaz et al., 2008; Wang et al., 2010). These observations suggest that enzymatic quenching of AHL signals by lactone hydrolysis is a feasible approach for interfering with bacterial infections. 2.1. Metallo-ˇ-lactamase-like lactonases QQ lactonases belonging to the metallo-␤-lactamase superfamily, which have a characteristic Zn2+ -binding HXHXDH motif, besides AiiA of Bacillus sp. 204B1 (Dong et al., 2000) comprise AiiA homologues from Bacillus sp. A24 (Reimmann et al., 2002), Bacillus thuringiensis strains (Lee et al., 2002; Liu et al., 2005) and other bacilli of the B. cereus group (Dong et al., 2002), as well as AttM/AiiB of Agrobacterium tumefaciens (Carlier et al., 2003; Liu et al., 2007; Zhang et al., 2002), AhlD from Arthrobacter sp. IBN110 (Park et al., 2003), AhlS from Solibacillus silvestris StLB046 (Morohoshi et al., 2012), AidC from Chryseobacterium sp. StRB126 (Wang et al., 2012), Quorum

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4 Table 1 Enzymes active on QS signaling molecules. Protein AHL lactonases AiiA

AhlS AhlD

Source

Protein family

Localization/signal peptidea

Substrates

References

Bacillus spp. (B. cereus group)

Metallo-␤-lactamase superfamily

Cytoplasmic

C4-, C6-, C8-, C10-HSL; 3OC4-, 3OC6-, 3OC8-, 3OC12-HSL; 3-OH-C4-HSL

Solibacillus silvestris StLB046 Arthrobacter sp. IBN110

Metallo-␤-lactamase superfamily Metallo-␤-lactamase superfamily Metallo-␤-lactamase superfamily

No SP

C10-HSL

Dong et al. (2000), Dong et al. (2002), Lee et al. (2002), Liu et al. (2005), Reimmann et al. (2002), Wang et al. (2004b) Morohoshi et al. (2012)

No SP

C6-, C8-, C10-HSL, 3OC6-, 3OC12-HSL C4-, C6-, C7-, C8-, C10-HSL; 3OC6-, 3OC8-HSL

AttM (AiiB)

Agrobacterium tumefaciens C58, M103

QsdR1

Rhizobium sp. NGR234

AhlK

Klebsiella pneumoniae KCTC2241 Chryseobacterium sp. StRB126 Soil metagenome (acidobacterial origin) Mycobacterium tuberculosis

AidC QlcA PPH, identical to Php of strain H37Rv MCP (MAP3668c) QsdA (AhlA)

SsoPox SisLac PON1

M. avium ssp. paratuberculosis K-10 Rhodococcus erythropolis W2, SQ1, Mic1, MP50, CECT3008; Rhodococcus sp. BH4 Sulfolobus solfataricus P2 (ATCC 35092) Sulfolobus islandicus M.16.4 Mammalian liver, serum

3OC8-HSL

No SP

C6-HSL, 3OC6-HSL

Park et al. (2003)

SP

Wang et al. (2012)

No SP

C6-, C8-, C10-, C12-HSL; 3OC6-, 3OC8-, 3OC10-, 3OC12-HSL C6-HSL

No SP

C4-, C10-HSL; 3OC8-HSL

Afriat et al. (2006), Chow et al. (2009)

No SP

C6-, C7-, C8-, C10-, C12-HSL; 3-oxo-C8-HSL C4-HSL, C6- to C14-HSLs, with or without substitution at C3

Chow et al. (2009)

PLL

No SP; cell-associated

PLL

n.d.b

PLL

n.d.

Paraoxonase (PON) family

Secreted protein, associated with high-density lipoprotein (HDL) in serum Nonsecreted transmembrane protein, extracellular active site Secreted protein, associated with HDL in serum Membrane-associated

All mammalian tissues

PON family

PON3

Mammalian liver (and kidney), serum

PON family

Bacterial PON

Oceanicaulis alexandrii HTCC2633 Microbacterium testaceum StLB037 Ochrobactrum sp. T63

PON family

AidH

No SP

Carlier et al. (2003), Liu et al. (2007), Zhang et al. (2002) Krysciak et al. (2011)

Metallo-␤-lactamase superfamily Metallo-␤-lactamase superfamily Metallo-␤-lactamase superfamily Metallo-␤-lactamase superfamily Phosphotriesteraselike lactonase (PLL); amidohydrolase superfamily PLL

PON2

AiiM

No SP

␣/␤-Hydrolase superfamily ␣/␤-Hydrolase superfamily Dienelactone hydrolase Dienelactone hydrolase GDSL (SGNH) hydrolase family (N-terminal domain)

No SP No SP

DlhR BpiB07 QsdH

Rhizobium sp. NGR234 Soil metagenome Pseudoalteromonas byunsanensis 1A01261

BpiB04 BpiB01 BpiB05

Soil metagenome Soil metagenome Soil metagenome

Glycosyl hydrolase Hypothetical Hypothetical

SP No SP GDSL hydrolase domain facing the periplasm, RND-type efflux transporter transmembrane domain No SP SP No SP

Variovorax paradoxus VAI-C Streptomyces sp. M664

n.i.

n.i.

N-terminal nucleophile (Ntn) hydrolase family Ntn hydrolase

Extracellular

AHL acylases n.i.c AhlM AibP

Brucella melitensis 16M (ATCC 23456)

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Fetzner,

Park et al. (2003)

No SP

S.,

Quorum

C4-, C6-, 8-, C12-HSL; 3OC6-, 3OC8-, 3OC10-, 3OC12-HSL C4-, C8-, C12-HSL; 3OC8-, 3OC10-, 3OC12-HSL C7-, C12, C14-HSL 3OC6-, 3OC10-, 3OC12-HSL

C7-, C12-, C14-HSL; 3OC6-, 3OC10-, 3OC12-HSL

C7-, C12-, C14-HSL 3OC12-HSL

C12-HSL, 3OC10-, 3OC12-HSL C6-, C8-, C10, C12-HSL; 3OC6-, 3OC8-, 3OC10, 3OC12-HSL C4-, C6-, C10-HSL; 3OCC6-, 3OC8-HSL; 3-OH-C6-HSL 3OC8-HSL 3OC8-HSL C4-, C6-, C8-, C10-, C12-, C14-HSLs; 3OC6-, 3OC8-HSL

Riaz et al. (2008)

Afriat et al. (2006), Oh et al. (2013), Uroz et al. (2008)

Afriat et al. (2006), Hiblot et al. (2013) Hiblot et al. (2012) Bar-Rogovsky et al. (2013), Draganov et al. (2005)

Bar-Rogovsky et al. (2013), Draganov et al. (2005), Hagmann et al. (2014) Bar-Rogovsky et al. (2013), Draganov et al. (2005) Bar-Rogovsky et al. (2013) Wang et al. (2010) Mei et al. (2010) Krysciak et al. (2011) Schipper et al. (2009) Huang et al. (2012)

3OC8-HSL 3OC8-HSL 3OC6-, 3OC8-, 3OC12-HSL

Schipper et al. (2009) Schipper et al. (2009) Bijtenhoorn et al. (2011b)

C4-, C6-, C8-, C10-, C12-, C14-HSL; 3OC6-HSL C6-, C8-, C10-HSL; 3OC6-, 3OC8-, 3OC12-HSL C12-HSL; 3OC12-HSL

Leadbetter and Greenberg (2000) Park et al. (2005)

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Terwagne et al. (2013)

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5

Table 1 (Continued) Protein

Source

Protein family

Localization/signal peptidea

Substrates

References

AiiD

Ralstonia sp. XJ12B

Ntn hydrolase

SP

Lin et al. (2003)

Aac

Ntn hydrolase

Cell-associated; SP

PvdQ (PA2385)

Ralstonia solanacearum GMI1000 P. aeruginosa PAO1

3OC6-, 3OC8-, 3OC10-, 3OC12-HSL, C7-, C8-, C10-HSL; 3OC8-HSL

Ntn hydrolase

SP

QuiP (PA1032)

P. aeruginosa PAO1

Ntn hydrolase

SP

Bokhove et al. (2010), Huang et al. (2003), Koch et al. (2010), Sio et al. (2006) Huang et al. (2006)

HacB (PA0305)

P. aeruginosa PAO1

Ntn hydrolase

SP

HacB (Psyr 4858)

P. syringae B728a

Ntn hydrolase

Cell-associated

HacA (Psyr 1971)

P. syringae B728a

Ntn hydrolase

Extracellular

C7-, C8-, C10-, C11-, C12-, C14-HSL; 3OC10-, 3OC12-, 3OC14-HSL; 3OH-C12-, 3OH-C14-HSL C7-, C8-, C10-, C12-, C14-HSL; 3OC12-HSL C6-, C7-, C8-, C10-, C12-, C14-HSL; 3OC10-, 3OC12-, 3OC14-HSL C4-, C6-, C8-, C10-, C12-HSL; 3OC6-, 3OC8-HSL C8-, C10-, C12-HSL; 3OC8-HSL

Aac AiiC

Shewanella sp. MIB015 Anabaena sp. PCC7120

Ntn hydrolase Ntn hydrolase

SP Cell-associated; SP

AiiO

Ochrobactrum sp. A44

␣/␤-Hydrolase

Cytoplasmic

QsdB

Soil metagenome

No SP

n.i.

Rhodococcus erythropolis W2 Comamonas sp. D1

Amidase signature (AS) family n.i.

Cell-associated

n.i.

Cell-associated

n.i.

Cell-associated

Oxidoreductases active toward AHLs CYP102A1 Bacillus megaterium (P450BM-3) n.i. Rhodococcus erythropolis W2

Cytochrome P450 monooxygenase n.i.

Cytoplasmic Cell-associated

n.i.

Burkholderia sp. GG4

n.i.

n.i.

BpiB09

Soil metagenome

Short-chain dehydrogenase/reductase (SDR) family

Cytoplasmic

␣/␤-Hydrolase

FGGY family of carbohydrate kinases

n.i.

n.i.

Tenacibaculum maritimum NCIMB2154

Enzymes acting on AQs 2-Alkyl-3-hydroxyArthrobacter sp. Rue61a 4(1H)-quinolone 2,4-dioxygenase (Hod) Enzymes acting on AI-2 E. coli, other enteric AI-2 kinase (LsrK) bacteria a b c

C8-, C12-, C12-HSL C4-, C6-, C8-, C10-, C12-, C14-HSL; 3OC4-, 3OC6-, 3OC8-, 3OC10-, 3OC12-, 3OC14-HSL and corresponding 3OH-Cx -HSLs C4-, C6-, C10-, C12-, C14-HSL; 3OC4, 3OC6, 3OC8-, 3OC10-, 3OC12-, 3OC14-HSL and corresponding 3OH-Cx -HSLs C6HSL; 3OC8-HSL

Chen et al. (2009)

Wahjudi et al. (2011)

Shepherd and Lindow (2009) Shepherd and Lindow (2009) Morohoshi et al. (2008) Romero et al. (2008)

Czajkowski et al. (2011)

Tannières et al. (2013)

3OC6-, 3OC10-HSL; 3-OH-C10HSL C6-, C12-, C16-HSL; 3OC6-, 3OC8-, 3OC10-, 3OC12-, 3OC14-HSL; 3OH-C12-HSLto C16 AHLs, with or without substitution at C3 C10-HSL

Uroz et al. (2005)

C12- to C16-HSL; 3OC12-HSL (␻-1, ␻-2, ␻-3-hydroxylation) 3OC8-, 3OC10-, 3OC12-, 3OC14-HSL (reduction to 3OH-HSL) 3OC4-, 3OC6-, 3OC8-HSL (reduction to 3OH-HSL) 3OC12-HSL (NADPH-dependent reduction to 3OH-C12-HSL)

Chowdhary et al. (2007, 2008) Uroz et al. (2005)

Cytoplasmic

C1- to C9-n-alkyl-3-hydroxy4(1H)-quinolones

Pustelny et al. (2009), Thierbach et al. (2014)

Cytoplasmic

Linear form of AI-2 (4,5dihydroxy-2,3-pentanedione)

Roy et al. (2010)

Uroz et al. (2007)

Romero et al. (2010)

Chan et al. (2011) Bijtenhoorn et al. (2011a)

SP: predicted signal peptide; No SP: no signal peptide predicted from amino acid sequence (SignalP/LipoP Server). n.d., not determined. n.i., not identified.

and QlcA from a metagenomic library (Riaz et al., 2008). As summarized in Table 1, these lactonases usually have a broad substrate specificity and hydrolyze AHLs with or without C3-substitution, with a preference for medium- to long-chain AHLs. 2.2. Phosphotriesterase-like lactonases (PLLs) Phosphotriesterase-like lactonases (PLLs) are members of the amidohydrolase superfamily, possessing a binuclear metal center within a (␤/␣)8 -barrel structural scaffold (the so-called Please cite this article in press as: http://dx.doi.org/10.1016/j.jbiotec.2014.09.001

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TIM-barrel). PLLs generally have a broad substrate spectrum with preference for hydrophobic lactones. Some PLLs were originally isolated by virtue of their activity toward the organophosphate pesticide paraoxon and described as paraoxonases, however, biochemical studies revealed that these enzymes are lactonases with promiscuous organophosphate hydrolase activity (Afriat et al., 2006). While many PLLs have been proposed to be natural AHL lactonases, there are also members of the family whose substrates are lactones other than AHLs (Xiang et al., 2009). PLLs active toward AHLs comprise QsdA (also termed AhlA (Afriat et al.,

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R1

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A R1

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Fig. 3. Reactions catalyzed by quorum-quenching enzymes. (A) Hydrolytic cleavage of AHLs by AHL lactonases and AHL acylases. (B) Hydroxylation of AHLs by the cytochrome P450 monooxygenase CYP102A (n = 5–9). (C) Reduction of 3-oxo-substituted AHLs by AHL oxidoreductases (R, C5 to C11 n-alkyl). (D) Sequestration of AI-2 by the AI-2 kinase LsrK and subsequent decomposition to 2-phosphoglycolic acid (PG). (E) Dioxygenolytic cleavage of PQS by 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase (Hod).

2006)) from Rhodococcus erythropolis W2 and related Rhodococcus strains, which can hydrolyze C6- to C14-HSLs with or without substitution on C-3 (Afriat et al., 2006, Uroz et al., 2008), PPH from Mycobacterium tuberculosis (Afriat et al., 2006), MCP from M. avium ssp. paratuberculosis K-10 (Chow et al., 2009), and the thermostable lactonases GKL and GsP which have been isolated from Geobacillus kaustophilus HTA426 (Chow et al., 2010) and G. stearothermophilus strain 10 (Hawwa et al., 2009), respectively. Hyperthermophilic enzymes termed SsoPox and SisLac which hydrolyze AHLs have been found in the archaea Sulfolobus solfataricus MT4 and S. islandicus, respectively (Afriat et al., 2006; Hiblot et al., 2012, Ng et al., 2011). The proficient activity of the latter enzymes suggests a potential biological role as quorum quenching enzyme in Sulfolobus. Due to their broad substrate spectrum, PLL

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lactonases – especially the thermostable ones – are promising candidates for the development of quorum-quenching enzymes as antivirulence therapeutic biomolecules. Hence, some PLLs have been subjected to structure-based mutagenesis or in vitro evolution in order to improve their catalytic efficiency and alter their substrate range (Chow et al., 2009, 2010; Hiblot et al., 2013). 2.3. Paraoxonases (PONs) In contrast to the enzyme families described above, the paraoxonase (PON) family comprises primarily mammalian lactonases. PONs have a six-bladed ␤ propeller fold with a structural and a catalytic Ca2+ ion (Harel et al., 2004). The name paraoxonase derives from the ability of mammalian PON1 to hydrolyze paraoxon, the Quorum

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active metabolite of the organophosphate pesticide parathion. PONs have been proposed to play a key role in organophosphate detoxification, however, PONs are in fact lactonases (Draganov et al., 2005). In 2004, Chun et al. reported that human airway epithelial cells can inactivate 3OC12-HSL by a cell-associated rather than a secreted factor, and hypothesized that an enzymatic activity is responsible. Later studies revealed that PON2 of human keratinocytes inactivates 3OC12-HSL (Simanski et al., 2012), and that PON2 deficiency of murine airway epithelial cells impairs 3OC12HSL degradation and enhances P. aeruginosa QS (Stoltz et al., 2007). All three mammalian PONs are able to hydrolyze 3OC12-HSL (Draganov et al., 2005; Ozer et al., 2005; Yang et al., 2005), indicating that mammalian tissues possess QQ activity. Whereas PON1 and PON3 are localized primarily in serum, associated with high density lipoprotein (HDL), and in the liver, PON2 is expressed in many tissues as intracellular enzyme. Thus, PON2, which is the more ancient among the mammalian PONs (Draganov et al., 2005), was proposed to represent an important defense against pathogens which use AHL signals (Teiber et al., 2008). PON1, which can hydrolyze a broad variety of substrates, was capable of protecting transgenic Drosophila from P. aeruginosa-induced lethality (Stoltz et al., 2008). Recently, bacterial PONs were identified in a bioinformatics approach, and the PON-like protein from Oceanicaulis alexandrii HTCC2633 was biochemically characterized. It efficiently hydrolyzes 3OC12-HSL, and like mammalian PON2 it lacks the lactonase activity against lipophilic ␥- and ␦-lactones which is characteristic for mammalian PON1 and PON3 (Bar-Rogovsky et al., 2013). 3. AHL acylases Shortly after the discovery of the QQ lactonase AiiA, a strain of Variovorax paradoxus was isolated which is able to utilize AHLs as source of energy and nitrogen. Degradation required an AHL acylase, which forms HSL and the corresponding fatty acid (Fig. 3A). The fatty acid supported growth, whereas the HSL only served as nitrogen source (Leadbetter and Greenberg, 2000). AHL acylases were later also identified in P. aeruginosa (Huang et al., 2003, 2006; Sio et al., 2006; Wahjudi et al., 2011), Ralstonia strains (Chen et al., 2009; Lin et al., 2003), Comamonas sp. (Uroz et al., 2007), P. syringae (Shepherd and Lindow, 2009), Shewanella sp. (Morohoshi et al., 2008), Ochrobactrum sp. (Czajkowski et al., 2011), the cyanobacterium Anabaena sp. PCC7120 (Romero et al., 2008), and the actinobacteria R. erythropolis W2 (Uroz et al., 2005) and Streptomyces sp. M664 (Park et al., 2005) (Table 1). The pathogen Brucella melitensis, which produces long-chain AHLs in vitro and during macrophage infection, was proposed to possess a cytoplasmic AHL acylase (AibP) which is thought to control the intrinsic AHL autoinducer levels (Terwagne et al., 2013). In contrast to the AHL lactonases, which have been identified in various protein families, the majority of the AHL acylases characterized to date are members of a single superfamily, exhibiting an N-terminal nucleophile (Ntn) hydrolase fold (Table 1). A notable exception is AiiO from Ochrobactrum sp. A44, which has an ␣/␤hydrolase fold (Czajkowski et al., 2011). The Ntn hydrolase fold is characteristic for ␤-lactam acylases such as penicillin G acylase. Interestingly, the penicillin acylase of Kluyvera citrophila is able to hydrolyze C6- to C-8 AHLs with or without a 3-oxo substituent, albeit with comparably low catalytic efficiency (Mukherji et al., 2014). Most AHL acylases exhibit a preference for long-chain AHLs (with or without a substituent at C-3 of the acyl chain). However, AiiC from Anabaena sp. PCC7120 (Romero et al., 2008) hydrolyzes a broad range of AHLs including C4-HSL, the signal of the rhl system of P. aeruginosa. QQ activity of AHL acylases has been demonstrated in Please cite this article in press as: http://dx.doi.org/10.1016/j.jbiotec.2014.09.001

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various systems, indicating that these enzymes could be applied to the control of AHL-mediated pathogenicity. Heterologous expression of AHL acylase genes in P. aeruginosa affected the production of virulence factors and other QS-dependent traits, albeit to different extents (Lin et al., 2003; Sio et al., 2006; Wahjudi et al., 2011). Compared to AHL lactonases, AHL acylases are considered as advantageous for biotechnological applications, because in contrast to the lactonase product N-acyl homoserine, which can re-circularize to the AHL at acidic pH, the acylase reaction products cannot spontaneously regenerate a functional QS signal. Moreover, the fatty acid generated by the acylase usually is readily metabolized. 4. AHL oxidoreductases The cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium, also known as P450BM-3, catalyzes the hydroxylation of AHLs as well as of their lactonolysis products, the corresponding N-acyl homoserines, at the ␻-1, ␻-2 and ␻-3 carbons of the acyl chain (Fig. 3B). The AHL oxidation products still act as QS autoinducers, but are significantly less active than the parent compounds (Chowdhary et al., 2007). The wild-type CYP102A1 enzyme showed similar affinity for AHLs and N-acyl homoserines. Using a structurebased rational approach, Chowdhary et al. (2008) replaced the R47 residue, which contributes to binding of the negatively charged carboxylate of N-acyl amino acids and fatty acids, by serine. This indeed resulted in a 125-fold increase in the N-acyl homoserine lactone to N-acyl homoserine selectivity. Besides the lactonase QsdA and an AHL acylase, R. erythropolis W2 has an oxidoreductase which catalyzes the reduction of 3oxo-C(8-14)-HSLs to the corresponding 3-hydroxy-HSLs, thereby inactivating the signal molecules (Uroz et al., 2005) (Fig. 3C). Burkholderia sp. strain GG4, isolated from ginger rhizosphere, is also capable of reducing 3-oxo-AHLs (Chan et al., 2011), however, the enzymes from these strains were not identified. A metagenome-derived oxidoreductase, termed BpiB09, which catalyzes the reduction of 3-oxo-C12-HSL to 3-hydroxy-C12-HSL, was characterized as a member of the short-chain dehydrogenase/reductase (SDR) family which probably uses NADPH as cosubstrate. Its expression in P. aeruginosa affected the transcription of QS regulated genes, reduced pyocyanin production, motility, and biofilm formation, and attenuated virulence on Caenorhabditis elegans (Bijtenhoorn et al., 2011a). A function-based identification of QQ loci involved in AHL degradation by Rhizobium sp. strain NGR234 besides several genes of QQ hydrolases (among them QsdR and DhlR, see Table 1) revealed the gene of a predicted aldehyde dehydrogenase AldR. When expressed in P. aeruginosa PAO1, aldR reduced motility and biofilm formation (Krysciak et al., 2011). Oxidized halogen compounds like hypobromous and hypochlorous acid, which can be formed by the activity of haloperoxidases, have biocidal activity, but can also react with 3-oxo-AHLs. It has been proposed that a number of marine macroalgae control biofilm formation on their surface by using haloperoxidase systems. The HOBr produced by the alga Laminaria digitata deactivated 3-oxo-AHL molecules (but not AHLs), suggesting that signal inactivation by enzymatically produced halogen antimicrobials could contribute to biofilm control in natural communities (Borchardt et al., 2001). 5. Enzymes active toward AI-2 The signaling molecule autoinducer-2 (AI-2) is thought to be a “universal” signal involved in intra- as well as interspecies communication. Its signal synthase LuxS is conserved in a multitude of bacterial species. In bacteria with the Lsr system of AI-2 signal perception such as E. coli, AI-2 after transporter-mediated uptake is phosphorylated by the kinase LsrK to produce phospho-AI-2. Quorum

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Binding of phospho-AI-2 to the transcriptional repressor LsrR leads to the de-repression of lsr genes (Pereira et al., 2013). Recently, Bentley and colleagues (Roy et al., 2010) assessed the potential of LsrK as a QQ enzyme. When purified E. coli LsrK kinase and ATP were added exogenously to E. coli, Salmonella typhimurium, and Vibrio harveyi cultures, their QS response was significantly reduced, both in pure cultures as well as in co-cultures of the three species. Addition of LsrK and ATP to co-cultures of the signal-generating E. coli and the AI-2-detecting S. typhimurium reduced “cross-talk” between the species. Presumably, the negative charge conferred onto the signal molecule by the phosphorylation restricted or prevented its transport into the cell. Moreover, phospho-AI-2 is unstable and breaks down over time to 2-phosphoglycolic acid (Fig. 3D). An advantage of quenching AI-2 signaling by chemical modification of the signal is that this strategy is effective irrespective of the transport/sensing mechanisms utilized by different bacteria. Possible biomedical applications of this system were suggested to involve encapsulation of the kinase together with ATP for the treatment of infections or to interfere with biofilm formation (Roy et al., 2010). However, the requirement for ATP, which decomposes at acidic pH or at elevated temperatures, may limit the applicability of this enzyme. 6. Enzymes active toward 2-alkyl-4(1H)-quinolone-type signal molecules AHL signaling is wide-spread among Gram-negative bacteria, whereas QS systems that use 2-alkyl-4(1H)-quinolones (AQs) as autoinducers appear to be limited to Pseudomonas and Burkholderia spp. This offers the potential to develop antivirulence tools that are more specific than those targeting AHL signals. Since AQ signaling plays an important role in the control of virulence gene expression and biofilm development in P. aeruginosa (Déziel et al., 2005; Diggle et al., 2003; Gallagher et al., 2002), the signal molecules HHQ and PQS constitute attractive antibacterial targets. Whereas numerous bacterial strains with the ability to utilize or inactivate AHLs and a wealth of enzymes acting on AHLs have been described in the literature, the biodegradation of AQ signaling molecules has not been demonstrated so far. The only enzyme described that is able to inactivate an AQ-type QS signal is the dioxygenase Hod (for 1H-3-hydroxy-4-oxoquinaldine 2,4dioxygenase) from Arthrobacter sp. Rue61a, which catalyzes the cleavage of the heterocyclic ring of PQS to N-octanoylanthranilic acid and carbon monoxide (Pustelny et al., 2009) (Fig. 3E). Hod is a cytoplasmic enzyme involved in the quinaldine degradation pathway of strain Rue61a, and its activity toward PQS is considered as fortuitous. Nevertheless, Hod exogenously added to P. aeruginosa cultures was capable of reducing the expression of key virulence factors (Pustelny et al., 2009). AQ-type secondary metabolites and related quinoline and quinolone alkaloids are also produced by a variety of higher organisms, especially by higher plants of the family Rutaceae (Heeb et al., 2011; Michael, 2007, 2008). Therefore, it is well conceivable that soil microorganisms have evolved enzymes which may have roles in the detoxification of AQs, or in AQ degradation to access the carbon and/or the nitrogen, or which even may have evolved as “natural” QQ enzymes to modulate the behavior of bacterial communities. We recently observed the degradation of PQS and related AQs by a new bacterial isolate from soil (unpublished data), however, the pathway and the enzymes involved remain to be characterized. 7. Applications of enzyme-based quorum quenching Different biocontrol strategies have been assessed which aim at reducing the QS signal concentration in the bacterial environment, Please cite this article in press as: http://dx.doi.org/10.1016/j.jbiotec.2014.09.001

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in order to control infections by plant or animal pathogens, or to mitigate biofouling. However, since bacteria use QS to control many functions that are essential for their competitiveness or for the establishment of beneficial interactions, possible negative effects of QQ cannot be excluded. For example, AHL signaling contributes to the symbiotic relationship between N2 -fixing rhizobacteria and leguminous plants. Expression of an aiiA lactonase in Sinorhizobium meliloti indeed was observed to reduce its efficiency in initiating root nodule formation on Medicago truncatula host plants (Gao et al., 2007). Another study showed that quenching of AHL signaling may interfere with other biocontrol strategies: The biocontrol strain Pseudomonas chlororaphis relies on phenazine production for protection of plants against Fusarium oxysporum. Recombinant Pseudomonas fluorescens producing an AiiA lactonase, when coinoculated together with P. chlororaphis into tomato plants, severely impaired the protective activity of the biocontrol strain (Molina et al., 2003). These examples illustrate that QQ approaches directed toward “general” signals such as AHLs that are used by many bacteria must be assessed with great care, especially when addressing complex communities in their natural environment. Strategies aiming at inhibiting a limited group of bacteria, or even individual pathogens, should be less prone to affecting beneficial organismic interactions and should be promising tools for selective disease control. Therefore, QS systems that are less wide-spread, such as the AQ-based systems of P. aeruginosa or Burkholderia pseudomallei, are attractive targets for interference. Another approach toward more selective QS interference involves the engineering of AHL hydrolases, which often have a broad substrate range, to narrow down their specificity. To this end, the acylase PvdQ from P. aeruginosa was recently engineered in a structure-based approach to switch its substrate specificity toward efficient hydrolysis of C8-HSL, a major signaling molecule of Burkholderia spp. PvdQ is an enzyme involved in the biosynthesis of the peptide siderophore pyoverdine, but the wild-type enzyme also has been shown to hydrolyze AHLs, with a preference for longchain congeners and only weak activity toward C8-HSL compounds (Koch et al., 2010; Table 1). Contrary to wild-type PvdQ, the mutant enzyme very efficiently attenuated the virulence of Burkholderia cenocepacia in larvae of the great wax moth Galleria mellonella (Koch et al., 2014). 7.1. Transgenic plants with quorum quenching ability The development of transgenic plants that express bacterial quorum-sensing genes is one possibility to interfere with bacterial phytopathogens. In a pioneering study on enzymatic QQ by Dong et al. (2001), expression of the gene encoding AiiA lactonase of Bacillus sp. 240B1 in tobacco and potato plants was demonstrated to increase the resistance of the plants toward P. carotovorum infection, and to reduce soft rot disease symptoms, illustrating the potential of this approach to control bacterial infections. Expression of the aiiA gene also conferred enhanced resistance against P. carotovorum to the Konjac plant (Amorphophallus konjac), which is grown in China, Korea, Taiwan, Japan and southeast Asia for its corm (Ban et al., 2009). A transgenic Chinese cabbage line producing Bacillus lactonase, which was fused to a signal peptide to target the enzyme to the intercellular space where P. carotovorum initiates infection, showed significant tolerance to soft rot disease (Vanjildorj et al., 2009). In order to assess whether genetic modification of the plant affects the bacterial community in the rhizosphere, D’AngeloPicard et al. (2011) compared the bacterial populations associated with the roots of a transgenic tobacco line expressing the attM lactonase gene with those of the wild-type parent line. While their study suggested that the genetic modification of the plant did not induce detectable changes in the composition of the Quorum

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root-associated bacterial community, possible modulations of functional properties of the rhizosphere community cannot be excluded. 7.2. Quorum quenching bacteria as biocontrol agents in plant protection In plant disease management, another strategy besides expression of genes encoding signal-inactivating enzymes in the plant itself is to employ signal molecule-degrading bacteria for protection against pathogens such as P. carotovorum. Engineered nonpathogenic bacteria that produce AHL inactivating enzymes have been tested as potential biocontrol strains. For example, Burkholderia sp. KJ006 transformed with the lactonase gene aiiA degraded the QS signal of pathogenic Burkholderia glumae and reduced the incidence of rice seedling rot when coinoculated (Cho et al., 2007). A recombinant B. thuringiensis strain expressing the AHL lactonase AiiA extracellularly on the cell surface increased the ability to resist soft rot disease in a potato tuber model infection system (Zhang et al., 2007). While these examples highlight the potential of this approach in plant protection, the release of genetically modified organisms (as well as the cultivation of transgenic plants) is not universally accepted. More practicable approaches could be based upon utilization of bacteria that are naturally able to inactivate signal molecules, either by using wild-type isolates or consortia that degrade signal molecules, or by the stimulation of the natural AHL-degrading microflora. A number of bacterial strains with the ability to transform AHLs have been identified, such as Arthrobacter sp. IBN110 (Park et al., 2003), several Rhodococcus strains (Jafra et al., 2006; Park et al., 2006; Uroz et al., 2003), Ochrobactrum, Delftia, and Bacillus isolates (Czajkowski et al., 2011; Jafra et al., 2006), Comamonas sp. D1 (Uroz et al., 2007), and Acinetobacter and Burkholderia strains (Chan et al., 2011). Co-cultures of AHL degrading bacteria with P. carotovorum were shown to reduce the amount of AHLs and to attenuate QS-dependent functions. Among these bacterial genera, especially the genus Rhodococcus is well known for its metabolic versatility and biodegradation capacities. Rhodococci, similar as Arthrobacter spp., are virtually ubiquitous bacteria residing in soil and water environments which show high resistance to harsh environmental conditions such as desiccation. Therefore, members of these genera capable of interfering with QS are attractive candidates for agricultural applications. Since rhodococci have also been used for bioremediation, formulations have already been developed for trapping or fixation of the cells, e.g., in alginate-based beads, which have been tested for their suitability for release of rhodococci into the soil (Latour et al., 2013). Interestingly, the AHL lactonase QsdA of R. erythropolis W2, R. erythropolis R138 and related strains is the key enzyme of a catabolic pathway to assimilate various ␥-lactone substrates (Barbey et al., 2012; Uroz et al., 2008). The pathway disrupts Pectobacterium communication and prevents plant soft-rot. Since in strain R138 the pathway is induced by pathogen communication, it is thought to comprise a “natural” biocontrol pathway (Barbey et al., 2013). Based on the finding that QsdA is part of a ␥-lactone utilization pathway, ␥-lactones were tested as substrates to stimulate AHL-degrading microbial populations. Amendment of bacterial consortia recovered from soil with ␥-caprolactone as well as addition of ␥-caprolactone to hydroponic cultures of Solanum tuberosum was observed to increase the ratio of AHL degrading bacteria. Caprolactone-treated consortia moreover showed biocontrol activity against P. atrosepticum in potato soft rot assays. The bacteria that were stimulated by the caprolactone amendment were found to belong to the genera Rhodococcus and Delftia (Cirou et al., 2007, 2011). However, in another study, a metagenomics approach suggested that ␥-caprolactone treatment of potato rhizosphere Please cite this article in press as: http://dx.doi.org/10.1016/j.jbiotec.2014.09.001

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biased the structure of the bacterial community toward the genus Azospirillum, which assimilates ␥-caprolactone but is thought to be incapable of AHL inactivation. Besides the qsdA gene, attM and qsdB (cf. Table 1) had a higher abundance in the caprolactone-treated rhizosphere (Tannières et al., 2013). Biostimulation of AHL-degrading rhodococci by treatment with ␥-caprolactone, a non-toxic and cheap chemical that is also used as a food additive, is a particularly attractive approach, because of the dual role of the substrate as an inducer of QsdA synthesis and as substrate for growth. Due to its rapid degradation, biostimulation by ␥-caprolactone is transient (Latour et al., 2013). 7.3. Wild-type quorum quenching bacteria to interfere with animal pathogens in aquaculture Diseases caused by pathogenic or opportunistic pathogenic bacteria such as Aeromonas spp. and Vibrio spp. are a major problem in aquaculture. One critical aspect in fish and shrimp farming is the highly variable survival during larval rearing. Among different alternative or additional strategies to the use of disinfectants and antibiotics, quorum quenching approaches have been tested (Defoirdt et al., 2004, 2011a). Enrichment cultures of AHLdegrading bacteria, originating from shrimp gut, were observed to increase the survival of turbot larvae (Scophthalmus maximus L.) that were treated with AHLs (Tinh et al., 2008). In a related study on the giant freshwater prawn Macrobrachium rosenbergii, adding AHL-degrading enrichment cultures obtained from the gastrointestinal tract of sea bass to the rearing water of the larvae, or adding them through inoculation of the larval food, was found to increase survival of larvae challenged with V. harveyi (Nhan et al., 2010). Additional mechanisms besides AHL degradation may contribute to the protective effect. AHL-degrading Bacillus spp. strains were isolated from these AHL-degrading enrichment cultures for possible use as probiotics in aquaculture (Defoirdt et al., 2011b). 7.4. Quorum quenching enzymes to interfere with human and animal pathogens Purified AHL hydrolyzing enzymes such as the acylases AhlM (Park et al., 2005) and PvdQ (Sio et al., 2006) were shown to reduce the production of virulence factors when added to P. aeruginosa cultures. The potential of isolated AHL degrading enzymes to combat P. aeruginosa or B. cenocepacia infections in vivo has also been assessed in infection models such as C. elegans (Papaioannou et al., 2009) and G. mellonella (Koch et al., 2014). The ring-cleaving dioxygenase Hod, which up to now is the only enzyme reported to attack PQS, despite its comparatively low activity toward PQS is able to reduce the expression of key virulence factors when added to P. aeruginosa cultures. It also quenched P. aeruginosa virulence in a plant leaf infection model (Pustelny et al., 2009). However, in P. aeruginosa cultures, the enzyme lost 50% of its activity within 6 h due to proteolytic degradation (Pustelny et al., 2009). This illustrates that for the application of isolated enzymes as QQ tools, the proteins need to be protected, e.g., by immobilization or encapsulation. This is especially important when targeting pathogens like P. aeruginosa that are potent producers of proteolytic extracellular enzymes. Ng et al. (2011) immobilized recombinant SsoPox lactonase, which can hydrolyze C4-HSL as well as 3OC12-HSL, on nanoalumina membranes, retaining 25% of its activity. In P. aeruginosa PAO1 cultures grown in the presence of SsoPox-immobilized membranes, the production of the virulence factors elastase, protease, and pyocyanin was significantly reduced. The enzyme retained 90% of its activity after 23 days of storage in water or buffer, however, the effect of immobilization on protease resistance was not investigated in this study. Quorum

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Interestingly, Bacillus lactonase AiiA when heterologously produced in Pichia pastoris exhibited remarkable stability and protease resistance, which was ascribed to post-translational modifications of the recombinant protein. Co-injection of AiiA with the pathogen Aeromonas hydrophila in the common carp attenuated the infection (Chen et al., 2010). The enzyme was also tested as a food additive in a zebrafish infection model (Cao et al., 2014). 7.5. Quorum quenching enzymes and bacteria to control biofouling Membrane bioreactors (MBR) – combinations of bioreactors and membrane filtration units for biomass retention – have become increasingly popular in wastewater treatment. However, membrane fouling, i.e., coverage of the membrane surface by deposits, limits the efficiency and lifespan of the membrane reactors and causes high operation costs (Drews, 2010). Since biofouling in MBRs can be correlated with QS activity, approaches for biofouling control by QS signal degradation have been developed. The feasibility of this strategy was first tested by incubating cultures of biofilmforming bacteria on different surfaces with porcine kidney acylase I (Paul et al., 2009), and by adding the enzyme to a lab-scale MBR (Yeon et al., 2009a). In a subsequent study the acylase was immobilized by a cross-linking reaction onto magnetic particles which can be recovered by magnetic capture (Yeon et al., 2009b). Using various enzyme immobilization techniques, the activity of the AHL acylase could be stabilized, and biofouling could be reduced in MBRs as well as in nanofiltration systems (Jiang et al., 2013; Kim et al., 2011; Lee et al., 2014; Yeon et al., 2009b). In recent studies on biofouling control in MBRs, QQ bacteria rather than QQ enzymes were applied in order to reduce the costs of the process. AHL degrading strains, such as recombinant E. coli producing an AHL lactonase, or the wild-type QQ strains Rhodococcus sp. BH4 and Pseudomonas sp. 1A1, which both were isolated from MBRs, were encapsulated in polymeric or ceramic microporous vessels that are inserted in the MBR, or entrapped in beads that circulate freely in the MBR (Cheong et al., 2013, 2014; Kim et al., 2013; Oh et al., 2012, 2013). Pseudomonas sp. 1A1 has an extracellular QQ activity, whereas the QQ activity of Rhodococcus sp. BH4 is cell-associated (Cheong et al., 2013; Oh et al., 2013). Both “microbial vessels” and “QQ beads” were successful in mitigating membrane biofouling in MBRs used for wastewater treatment. 8. Concluding remarks QQ enzymes are potential antivirulence tools, because they can inactivate signaling molecules involved in control of pathogenicity. However, for applications in aquaculture and agriculture, wild-type signal-degrading bacteria (rather than isolated enzymes) appear to be most promising. In aquaculture, probiotic treatment with AHL-degrading bacteria may be an effective way to protect larvae from Vibrio and Aeromonas infections. In agriculture, release of AHL-degrading R. erythropolis into the soil, or spraying crops with formulations containing a biostimulating compound such as ␥caprolactone, which induces the AHL lactonase QsdA and promotes growth of AHL-degrading rhodococci, may be effective biocontrol strategies against soft-rot bacteria. QQ bacteria also have been used with some success in membrane bioreactors for wastewater treatment to control biofouling. These fields of potential applications, however, present new challenges to elucidate the impact of QQ on complex (natural) communities. Compared with QQ compounds that enter the target cell, QQ enzymes that act extracellularly are thought to prevent or at least reduce selective pressure toward resistance development. A major problem in the application of isolated enzymes is their Please cite this article in press as: http://dx.doi.org/10.1016/j.jbiotec.2014.09.001

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susceptibility to denaturation and proteolytic degradation. Nevertheless, reduction of the virulence of plant or human pathogens by QQ enzymes has been demonstrated in various model systems. While QS interference holds great promise as antivirulence strategy, issues like substrate specificity, catalytic efficiency, stability, enzyme delivery, and potential side effects need to be addressed in more detail for the development of QQ enzymes as protective or therapeutic proteins. Thus, most approaches are still in the research phase, and practical tools to be applied in agricultural, industrial or even clinical settings are still scarce.

Acknowledgement The Deutsche Forschungsgemeinschaft (DFG) is gratefully acknowledged for financial support through grant no. FE 383/15-1 and GRK1409.

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