Reducing virulence of the human pathogenBurkholderia by altering the substrate specificityof the quorum-quenching acylase PvdQGudrun Kocha,1, Pol Nadal-Jimeneza,2, Carlos R. Reisa,3, Remco Muntendama, Marcel Bokhoveb,4, Elena Melilloa,Bauke W. Dijkstrab, Robbert H. Coola, and Wim J. Quaxa,5

aDepartment of Pharmaceutical Biology, University of Groningen, 9713 AV, Groningen, The Netherlands; and bLaboratory of Biophysical Chemistry, Universityof Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands

Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved December 23, 2013 (received for review June 19, 2013)

The use of enzymes to interfere with quorum sensing representsan attractive strategy to fight bacterial infections. We used PvdQ, aneffective quorum-quenching enzyme from Pseudomonas aeruginosa,as a template to generate an acylase able to effectively hydrolyzeC8-HSL, the major communication molecule produced by the Burkhol-deria species. We discovered that the combination of two singlemutations leading to variant PvdQLα146W,Fβ24Y conferred high activitytoward C8-HSL. Exogenous addition of PvdQLα146W,Fβ24Y dramaticallydecreased the amount of C8-HSL present in Burkholderia cenocepaciacultures and inhibited a quorum sensing-associated phenotype. Theefficacy of this PvdQ variant to combat infections in vivo was furtherconfirmed by its ability to rescue Galleria mellonella larvae upon in-fection, demonstrating its potential as an effective agent towardBurkholderia infections. Kinetic analysis of the enzymatic activitiestoward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrateswitch. This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In ad-dition, we demonstrate that their substrate range can be easilyswitched, thereby paving the way to selectively target only spe-cific bacterial species inside a complex microbial community.

computational design | enzyme engineering | antibiotic | cystic fibrosis

The Burkholderia cepacia complex (Bcc) comprises a group of17 related bacterial species able to colonize different envi-

ronmental niches (1). Over the years the Bcc has gained specialattention, as some of its members have been associated with life-threatening human infections (2, 3). Especially immunocom-promised patients and people suffering from cystic fibrosis aregenerally infected with these pathogens; in particular, infectionwith Burkholderia cenocepacia has been correlated with a poorprognosis (1, 4). B. cenocepacia is often found cocolonizing thelungs of cystic fibrosis patients alongside the opportunisticpathogen Pseudomonas aeruginosa (5–9).Reports on the occurrence of these two pathogens are ap-

pearing more and more frequently, underlining the difficultyin eradicating these pathogens with common antibiotics (10).Hence, novel strategies are needed to target bacterial infectionswithout applying too much selective pressure (11). An importantbacterial Achilles’ heel is quorum sensing (QS), a cell density-reliant regulatory system dependent on the secretion of N-acylhomoserine lactones (AHLs) (12). These molecules have beenlargely associated with virulence traits, as they are pivotal for theexpression of genes involved in toxin production, motility, plas-mid transfer, antibiotic synthesis, and biofilm formation (13, 14).In the last several years, many ways to interfere with QS have

been explored, as interference with the action of AHLs has beendemonstrated to reduce pathogenesis (15–17). The use of enzymesin targeting QS paves a new way in combating pathogens. A majorfinding in the field was the discovery of two families of quorum-quenching enzymes: the AHL lactonases and the AHL acylases(18–21). Lactonases target the lactone ring, whereas acylaseshydrolyze the amide bond of AHLs; both enzymes render the

autoinducer inactive, reducing bacterial virulence and path-ogenesis in vivo (22–25).Recently, we have shown that the quorum-quenching acylase

PvdQ, produced by fluorescent pseudomonads, decreases thelevels of the P. aeruginosa signal molecule 3-oxo-C12-HSL.Consequently, by either overexpression or exogenous addition ofPvdQ, expression of virulence-related genes was reduced (21, 26,27) in a model system measuring the survival of Caenorhabditiselegans upon infection by P. aeruginosa (23). PvdQ is most ef-fective against AHLs with side chains longer than 10 carbonatoms (21), whereas showing little to no activity toward AHLswith shorter acyl chains such as C8-HSL, which induces virulencein members of the Bcc (6, 9). The recently solved structure ofPvdQ with a bound 3-oxo-C12 fatty acid revealed a large hy-drophobic substrate-binding cleft that properly accommodatesthis fatty acid side chain (28). Altering the substrate range ofPvdQ toward shorter AHLs, such as C8-HSL, might therefore

Significance

Resistance toward commonly used antibiotics is becominga serious issue in the fight against bacterial pathogens. Onepromising strategy lies in the interference of bacterial quorumsensing by the hydrolysis of the signaling molecules. In thisstudy, we present a structure-aided computational design ap-proach to alter the substrate specificity of the quorum-quenchingacylase PvdQ. Introduction of two point mutations in residueslining the active site led to a switch in substrate specificity, ren-dering the enzyme highly active toward C8-HSL and thereby re-ducing virulence caused by Burkholderia cenocepacia. Thus, thiswork not only provides a structural insight into the substratespecificity of quorum-quenching acylases but also indicates theirpotential in the fight against specific bacterial pathogens.

Author contributions: G.K., P.N.-J., R.H.C., and W.J.Q. designed research; G.K., P.N.-J., C.R.R.,R.M., M.B., E.M., and R.H.C. performed research; G.K., P.N.-J., C.R.R., R.M., M.B., E.M.,B.W.D., and R.H.C. analyzed data; and G.K., P.N.-J., C.R.R., M.B., B.W.D., R.H.C., andW.J.Q. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The crystallography, atomic coordinates, and structure factors reportedin this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code4BTH).1Present address: Institut Für Molekulare Infektionsbiologie, Würzburg University, 97080Würzburg, Germany.

2Present address: Bacterial Signalling Group, Instituto Gulbenkian de Ciência, 2781-901Oeiras, Portugal.

3Present address: Department of Cell Biology, University of Texas Southwestern MedicalCenter, Dallas, TX 75390-9039.

4Present address: Department of Biosciences and Nutrition and Center for Biosciences,Karolinska Institutet, SE 14183 Huddinge, Sweden.

5To whom correspondence should be addressed. E-mail: w.j.quax@rug.nl.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311263111/-/DCSupplemental.

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shift the antibacterial scope of PvdQ as a therapeutic agent,potentially providing an effective therapy for Bcc infections.In this study, we report a structure-aided design approach to

modify the substrate specificity of a quorum-quenching acylasesuch that it targets explicitly the signaling molecules of a re-stricted range of pathogens. We have identified a PvdQ variantcontaining two amino acid substitutions, Leuα146Trp andPheβ24Tyr, which showed a substantially increased C8-HSL–degrading activity compared with the wild-type enzyme. Kineticanalysis was in-line with a substrate switch from 3-oxo-C12-L-HSLto C8-L-HSL caused by these mutations. Using an in vivo modelfor B. cenocepacia infection (29), we demonstrate that this quorum-quenching PvdQ variant can be successfully used to attenuatepathogen virulence and increase host survival. These resultsvalidate PvdQLα146W,Fβ24Y as a promising and effective potentialagent to combat emerging Bcc infections.

Results and DiscussionDesign of PvdQ Variants for Increased C8-HSL Activity. Using therecently elucidated crystal structure of PvdQ (28), we adopteda rational design approach to scan for PvdQ variants that wouldbe better capable of accommodating C8-HSL than the wild-typeenzyme. PvdQ is a heterodimeric Ntn hydrolase with an un-usually large binding pocket that can accommodate the long acylchain of an HSL substrate (28). The acyl chain of 3-oxo-C12-HSL has been structurally characterized in complex with PvdQ,but not the homoserine lactone part. Therefore, we performedmolecular docking experiments using 3-oxo-C12-HSL and C12-HSL in the active site of PvdQWT. The results show that thecarbonyl oxygen of these substrates has good hydrogen-bondinginteractions with the Nδ2 group of Asnβ269 and the Valβ70backbone amide (Fig. 1 A and B and Fig. S1), which constitutethe oxyanion hole (28). The most favorable substrate posesobtained for 3-oxo-C12-HSL show the acyl chain positioned inthe hydrophobic pocket in a conformation similar to that adopted

by the C12 acyl chain in the C12-PvdQ crystal structure. Incontrast, a similar analysis using C8-HSL resulted in a substantialconformational heterogeneity within the active site of PvdQWT,with the most energetically favorable poses showing that the car-bonyl oxygen of C8-HSL is no longer within H-bonding distancewith Asnβ269 and the Valβ70 backbone amide (more than 3.5 Å),and a significant distance between the carbonyl carbon of C8-HSLand the catalytic Serβ1 of PvdQ is observed (6.1 Å) (Fig. S2).Based on the differences shown for C8-HSL and C12-HSL duringmolecular docking experiments, the following amino acid residueswere selected for in silico mutagenesis: Thrα143, Leuα146,Glyα150, Pheβ24, Leuβ50, Leuβ53, Asnβ57, Valβ158, Trpβ162,Proβ185, Trpβ186, and Valβ187. Each amino acid was substitutedby all other 19 possible amino acids, and the new model structureswere energy-minimized, after which C8-HSL was docked in theactive site of PvdQ. A final minimization was used to refine theligand poses. The most energetically favorable substrate-dockedposes were analyzed with respect to the positioning of the substrateand the new distances obtained between the catalytic Serβ1 Oγ andthe carbonyl carbon atom of C8-HSL. Eighteen amino acid sub-stitutions out of 218 resulting in a reduction of the distance betweenSerβ1 and the carbonyl carbon of C8-HSL of less than 4 Å weretherefore considered for further analysis. Positions Thrα143Met,Thrα143Lys, Leuα146Ile, Leuα146Arg, Leuα146Trp, Pheβ24Tyr,Leuβ53Phe, Leuβ53Lys, Leuβ53Ile, Leuβ53Arg, Asnβ57Arg,Asnβ57His, Valβ158Met, Valβ158Ile, Trpβ162Phe, Trpβ162Tyr,Valβ187Phe, and Valβ187Tyr substitutions were therefore se-lected for site-directed mutagenesis (Table S1). The eight se-lected residues were also mutated to alanine.

Screening for C8-HSL Quenching. Based on this in silico screeningapproach, the 26 proposed site-directed mutants of PvdQ wereconstructed, produced, and purified. The variants containingValβ158Ala and Asnβ57Arg were affected in protein maturation(Fig. S3), a property often observedwhenmutagenizing acylases thatrely on the same residues to perform substrate conversion and pro-tein maturation. These were therefore excluded from further anal-ysis. As seen with fluorescence experiments, mutations of Trpβ162and Valβ187 had no effect on C8-HSL hydrolysis, but substitutionsat the positions Thr-143 and Leu-146 of the α-subunit and Phe-24, Leu-53, Asn-57, and Val-158 of the β-subunit resulted inincreased hydrolytic activity toward C8-HSL. In particular, var-iants Thrα143Lys, Leuα146Trp, Pheβ24Tyr, Leuβ53Ile, Asnβ57His,Valβ158Met, and Valβ158Ile resulted in a significant increase inC8-HSL hydrolysis compared with PvdQWT, as shown by a morethan 50% decrease in mean specific fluorescence activity (Fig. 2A).A second round of computational analysis was performed, to

identify possible combinatorial mutations in PvdQ with a furtherenhanced activity and specificity toward C8-HSL. The best singlemutants were combined in silico and analyzed as previously for thedistance between the catalytic Serβ1 Oγ and the carbonyl carbonatom of C8-HSL. Based on the results obtained by this analysis,the double mutants Leuα146Trp/Pheβ24Tyr and Pheβ24Tyr/Asnβ57His were generated and tested for their activity. Mostimportantly, the Leuα146Trp/Pheβ24Tyr variant displayed thehighest hydrolytic activity on C8-HSL: 5 ng/μL PvdQLα146W,Fβ24Y

was sufficient to quench 5 μM C8-HSL, corresponding to a five-fold increased activity compared with the best single mutanttested (Fig. 2A).Hydrolytic activity of these enzymes toward 3-oxo-C12-HSL was

assessed using a biosensor strain (Fig. 2B and Fig. S4). WhereasPvdQWT displayed high hydrolytic activity, the mutant enzymePvdQLα146W,Fβ24Y was severely impaired in its capacity to hydro-lyze 3-oxo-C12-HSL under the conditions tested, rendering itsfunction of quenching the endogenous signal from P. aeruginosabiologically insignificant. The activities toward 3-oxo-C12-HSL andC8-HSL highlight that a substrate switch had occurred, diminishingactivity toward 3-oxo-C12-HSL but substantially increasing hydro-lytic activity toward C8-HSL, as Fig. 2B clearly indicates. In-terestingly, considering the respective single mutants, deacylaseactivity toward 3-oxo-C12-HSL was hardly affected in PvdQLα146W

and only slightly impaired in PvdQFβ24Y, indicating that the

Fig. 1. Comparative molecular docking simulations. The most favoredconformations of C12-HSL (A) and 3-oxo-C12-HSL (B) in the active site ofPvdQ from CDOCKER, as implemented in Accelrys Discovery Studio 3.0. Hy-drogen atoms were added to the protein molecule and substrates, and theCHARMm force field was used to assign partial charges to the ligands.Substrates were docked into PvdQ using the coordinates of 3-oxo-lauric acidbound in the active site of PvdQWT [PDB ID code 2WYC (28)]. The residuesforming the active site of PvdQ are colored green, and the accessible solventsurface-contoured substrates are represented in yellow and cyan sticks forC12-HSL and 3-oxo-C12-HSL, respectively. Both substrate-docking poses arealigned for nucleophilic attack. The carbonyl oxygen forms hydrogen bondswith the Asnβ269 side-chain Nδ2 and the Valβ70 backbone amide, consistentwith the proposed oxyanion hole residues involved in the stabilization of thetetrahedral transition state (28). [The amino acid numbering follows thesubunit composition of the mature protein; i.e., the α-chain is defined byAspα1–Valα170 (equivalent to D24–V193 of the amino acid sequence of thepreprotein) and the β-chain by Serβ1–Gluβ546 (equivalent to S217–E762).]

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combination of both mutations is responsible for this switch insubstrate specificity (Fig. S4).Previously, Pheβ24 had been shown to allow the entrance of

3-oxo-C12-HSL into the hydrophobic pocket of PvdQWT (28).Position α146 needs to be occupied by a small residue like Leu toallow binding of the C12 acyl chain. We suggest that the con-straints imposed by the Trpα146 side chain on the binding modeof C8-HSL and the stabilization of Trpα146 by the new Tyrβ24contribute to the proper accommodation of C8-HSL in the activesite of PvdQ, whereas impairing its activity toward 3-oxo-C12.

Analysis of Quorum-Quenching Activity. To further characterizePvdQWT and PvdQLα146W,Fβ24Y, the activity of various concen-trations of C8-HSL and 3-oxo-C12-HSL was determined. Theseexperiments confirmed that PvdQWT has a preference for the long-chain substrate 3-oxo-C12-HSL, whereas this preference is shiftedto the shorter-chain substrate C8-HSL in mutant PvdQLα146W,Fβ24Y.Using the biosensor strain Pseudomonas putida (pAS-C8), we de-termined that after incubation of 1 μMC8-HSL with either 5 ng/μLPvdQWT or PvdQLα146W,Fβ24Y, only 0.063 μM in the case ofPvdQWT or 0.0013 μM C8-HSL in the case of PvdQLα146W,Fβ24Y

was detectable. These results indicate that using equal con-centrations of PvdQ proteins, PvdQLα146W,Fβ24Y displaysa 48.5-fold reduction of C8-HSL levels compared with PvdQWT

(Fig. S5).

Structural Effects of PvdQLα146W,Fβ24Y. To investigate the structuraleffects of the Leuα146Trp and Pheβ24Tyr mutations on PvdQ,we elucidated the crystal structure of the double mutantPvdQLα146W,Fβ24Y at a resolution of 1.9 Å to final Rwork and Rfreevalues of 17.9% and 20.7%, respectively. Data collection and refine-ment statistics can be found in Table S2. No major conformational

differences were observed between PvdQLα146W,Fβ24Y and PvdQWT

(rmsd of 0.28 Å for 710 Cα atoms).The crystal structure of the mutant protein shows that the

hydrophobic substrate-binding pocket near the N-terminal nu-cleophile residue Serβ1 is in the closed state (Fig. 3A) (28). Theside chains of the mutated Tyrβ24 and Trpα146 residues line thispocket, with each residue adopting at least two alternate con-formations. The Leuα146Trp mutation introduces a muchbulkier side chain and reduces the volume of the substrate-binding pocket from 260 to 80 Å3 for the closed conformation(Fig. 3 A and B). The cavity with Tyrβ24 in the open confor-mation has a volume of 140 Å3. We propose that the largervolume of the substrate-binding pocket in PvdQWT preferentiallybinds long fatty acid-like acyl chains, whereas the much less vol-uminous pocket of PvdQLα146W,Fβ24Y favors short-chain HSLs.The crystal structures of PvdQWT and PvdQLα146W,Fβ24Y in-

dicate that mutating Pheβ24 to a tyrosine does not cause sub-stantial conformational changes in the active site of PvdQ. Slightlydifferent side-chain orientations for these aromatic residues areobserved, however, with the new tyrosine moving upward relativeto the phenylalanine in the active conformation (Fig. 4). This inturn creates more space at the entrance of the hydrophobic cavity(Fig. 4), and could partially contribute to the better fit of C8-HSL.Additionally, in one of the alternate side-chain conformationsobserved in the crystal structure, the hydroxyl group of Tyrβ24 hasan interaction with the side-chain amine of Trpα146 (Fig. 4). Thisconformation resembles the open conformation that residue β24adopts in the substrate-bound state (28), and thus this interactionmay stabilize the conformation of Trpα146 and provide a better fitof the shorter acyl side chain of C8-HSL.

PvdQLα146W,Fβ24Y Disrupts B. cenocepacia Signaling and Inductionof Virulence. To determine whether PvdQWT and its variant,PvdQLα146W,Fβ24Y, interfere with C8-HSL accumulation and down-stream signaling by B. cenocepacia, cultures of this bacterium wereincubated with either enzyme (Fig. S6). The addition of theenzymes did not influence bacterial growth, as indicated in Fig. S7.Hence, C8-HSL accumulation was assayed after 24 h of incubationat 30 °C. Fig. 5A clearly shows that almost no fluorescence, andtherefore no C8-HSL, could be detected in the presence of themutant acylase PvdQLα146W,Fβ24Y. Significant levels of fluorescencewere measured in the control culture with no enzyme addition; onlya slight decrease was observed in the presence of PvdQWT. Thus,this activity test further substantiates the results obtained in thepreliminary activity screen, namely the limited activity of wild-typeenzyme to hydrolyze C8-HSL compared with the much moreefficient PvdQLα146W,Fβ24Y variant.In addition, and to further substantiate that a substrate switch

had occurred, we performed the same experiment but adding theenzyme to a P. aeruginosa culture. Analysis of 3-oxo-C12-HSLlevels after incubation with the enzymes for 24 h revealed that onlyPvdQWT could significantly decrease the amounts of the signaling

Fig. 2. (A) Relative fluorescence as a measure of the presence of C8-HSLafter incubation of PvdQ variants with the biosensor P. putida F117 (pAS-C8)after 13 h of incubation. The protein concentration was 5 ng/μL. Valuesreported indicate the mean specific fluorescence activity of each PvdQ variantnormalized as a percentage of the fluorescence level in an assay with PvdQWT.(B) Activity of PvdQWT and PvdQLα146W,Fβ24Y toward AHLs. Enzymes were in-cubated with 5 μM 3-oxo-C12-HSL or C8-HSL. 3-Oxo-C12 levels were analyzedusing E. coli (pSB1075) and C8-HSL levels with P. putida F117 (pAS-C8). Valuesreported indicate themean specific fluorescence/luminescence activity normalizedto the units measured by AHLs only. Error bars indicate SD.

Fig. 3. Residues lining the substrate-binding pocket in PvdQWT andPvdQLα146W,Fβ24Y. (A) PvdQWT; the main chain is indicated by a Cα trace, theblue ball and sticks indicate the residues lining the substrate-binding pocket,and the mesh shows the cavity as calculated by VOIDOO (Uppsala SoftwareFactory). (B) PvdQLα146W,Fβ24Y; the main chain is indicated by a Cα trace, thegreen ball and sticks indicate the residues lining the substrate-binding pocket,except for the mutated residues, which are indicated in orange, and the meshshows the cavity as calculated by VOIDOO. The Trpα146 mutation decreasesthe volume of the cavity, making it more suitable for short-chain fatty acids.

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molecule, as shown by a clear decrease in luminescence. Hardly anyeffect, however, on AHL levels was observed upon addition ofPvdQLα146W,Fβ24Y (Fig. S6). Taken together, these AHL quantifi-cations show that PvdQLα146W,Fβ24Y affects shorter acyl chains ina more effective manner than the wild-type enzyme does.To determine the downstream effects of quorum quenching

in Bcc, we monitored the proteolytic activity, as the production ofextracellular protease, which plays an important role in the in-vasion of lung tissue by B. cenocepacia, is positively regulated byC8-HSL (30, 31). Addition of PvdQLα146W,Fβ24Y to B. cenocepaciacultures significantly decreased the number of protease unitsdetected in culture supernatants, almost to the level of that pro-duced by the QS-negative strain H111-I, which is unable to induceprotease activity (Fig. 5A). In contrast, addition of PvdQWT resultedinprotease production similar to the control culture ofB. cenocepaciawild-typeH111 (Fig. 5A). These data show that PvdQLα146W,Fβ24Y

decreases the level of C8-HSL present in B. cenocepacia cul-tures, thereby reducing the expression of virulence traits.

PvdQLα146W,Fβ24Y Attenuates Burkholderia Virulence upon in VivoInfection of Galleria mellonella Larvae. The promising resultsobtained in degrading C8-HSL and protease activity after exogenousaddition of PvdQLα146W,Fβ24Y to B. cenocepacia in vitro prompted usto investigate the quorum-quenching effects of this enzyme in vivo.As depicted in Fig. 5B, injection of larvae of the great wax mothGalleria mellonella with B. cenocepacia H111 culture kills nearly alllarvae, whereas injection with the QS-negative strain B. cenocepaciaH111-I does not affect survival, in accordance with the importance ofQS in B. cenocepacia infection and pathogenesis. Whereas pre-incubation of B. cenocepacia H111 bacteria with PvdQWT hardlyaffected the survival rates of the larvae, preincubation withPvdQLα146W,Fβ24Y led to a nearly complete attenuation of bacterialvirulence and increased the overall survival of the larvae (Fig. 5B).This result demonstrates that PvdQLα146W,Fβ24Y, but not PvdQWT, isable to diminish the virulence of B. cenocepacia H111.

Kinetic Analysis of the Enzymatic Activities of the PvdQ Enzymes.Enzymatic activities were determined by an end-point assay andderivatization with ortho-phthaldialdehyde. The low solubility of3-oxo-C12-L-HSL allowed us to screen for activity in the range of0–0.2mM, whereas the activity toward C8-L-HSLwasmeasured upto 0.6 mM. The activity plots (Fig. S8) clearly show a substrate

switch due to the mutations: Whereas PvdQWT preferentiallyhydrolyzes 3-oxo-C12-L-HSL, PvdQLα146W,Fβ24Y has a preferencefor C8-L-HSL. The concentration window is not sufficiently largeto determine the kinetic parameters separately, but an estimationof the kcat:Km ratio can be obtained. For 3-oxo-C12-L-HSL, theseparameters were 5.8 × 103 and 1.5 × 103 M−1s−1 for PvdQWT andPvdQLα146W,Fβ24Y, respectively, resulting in a 3.8-fold difference.For the substrate C8-L-HSL, these values were 0.8 × 103 and 3.4 ×103 M−1s−1 for PvdQWT and PvdQLα146W,Fβ24Y, respectively,resulting in a 4.3-fold difference. Thus, in total, the mutations resultin a 16-fold difference in catalytic efficiency.

ConclusionsWe identified and characterized PvdQLα146W,Fβ24Y, a PvdQ var-iant with a shifted substrate range that is highly active toward C8-HSL. The in vivo quorum-quenching activity of PvdQLα146W,Fβ24Y

against B. cenocepacia, demonstrated by the decrease in pro-teolytic activity in the culture supernatant and the increase in hostsurvival, confirms its potential as a possible therapeutic, especiallyas this protease activity has been associated with the pathogen’sinvasion of lung tissue (30, 31). Our results obtained withPvdQLα146W,Fβ24Y together with previous results on PvdQWT (23)

Fig. 4. Structural impression of mutations on docking of C8-HSL and 3-oxo-C12-HSL. Close-up view of the two superimposed active sites of P. aeruginosaPvdQWT (shown in green) and PvdQLα146W,Fβ24Y (in white) with the most fa-vored docked conformations for C8-HSL (A) and 3-oxo-C12-HSL (B), using thecoordinates given by the crystal structure of PvdQLα146W,Fβ24Y. As shown, theintroduction of residue Trpα146 clearly reduces the hydrophobic pocket sizeand the protrusion into the interior of the enzyme, and contributes to theproper accommodation of the acyl chain of C8-HSL (A). The hydroxyl groupof the new Tyrβ24 forms a 3.2-Å hydrogen bond with the side-chain amineof Trpα146, stabilizing the conformation of Trpα146 and providing a betterfit of the alternate acyl chain of C8-HSL. Inversely, the mutant PvdQLα146W,Fβ24Y

no longer allows the proper accommodation of the acyl chain of 3-oxo-C12-HSL (B), with a distance between the carbonyl carbon of the substrate and thecatalytic serine of 6.1 Å for the most favorable conformation.

Fig. 5. Effects of PvdQWT and PvdQLα146W,Fβ24Y on B. cenocepacia QS. (A)Cells of B. cenocepacia were incubated for 24 h without PvdQ (first set ofbars) or in the presence of PvdQWT (indicated as WT) or PvdQLα146W,Fβ24Y

(indicated as MUT) and tested for C8-HSL levels with the P. putida F117 (pAS-C8) biosensor strain (gray bars). Fluorescence units were calculated relativeto the activity in the presence of PvdQWT (equal to 1). Cultures were alsoanalyzed for the production of protease by the activity on skim milk (dashedbars). One unit of protease was defined as the activity that produceda change in the OD600 of 0.1 per h (30). Lower protease units were found tobe produced when PvdQLα146W,Fβ24Y was added to the cultures (dashedbars). (B) PvdQLα146W,Fβ24Y protects for B. cenocepacia H111 infection inthe insect model. G. mellonella larvae were injected with B. cenocepacia H111wild-type cells either untreated or treated with PvdQWT or PvdQLα146W,Fβ24Y.After 48 h of incubation, larval survival was assessed. G. mellonella injectedwith buffer only resulted in 100% survival, B. cenocepacia H111 had a lethaloutcome for the larvae, whereas pretreatment of the bacterial cultures withPvdQLα146W,Fβ24Y rescued survival. For all determinations, a quorum-sensingnegative-strain H111-I served as control. Error bars indicate SD.

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provide the first steps toward the development of future antimi-crobial therapies aiming to effectively combat P. aeruginosa and B.cenocepacia infections, the two most important Gram-negativepathogens isolated from the lungs of cystic fibrosis patients. Fur-thermore, we illustrate how the design approach used in this studycan be applied to successfully produce quorum-quenching variantswith specific substrate ranges to target a selected group of bacteria.The application of highly active enzymes as potential treatment canhave a number of beneficial effects not only with respect to lunginfections but also to those occurring in the gastrointestinal tract.Specific targeting of the quorum-sensing systems of pathogenswould leave the beneficial microbiota unharmed. Decreasing thedeleterious side effects on the normal host microbiota is a majorfactor contributing to host protection against invading competitive,opportunistic bacteria and increasing the recovery of the hostafter infection (32, 33). We aim with our findings to substantiate thehigh potential of quorum-quenching enzymes in targeting bacterialpathogens.

Materials and MethodsStructure-Based Design of PvdQ Mutants by Molecular Docking of C8-HSLs.Based on the crystal structure of the P. aeruginosa quorum-quenching acy-lase (PvdQ) in complex with 3-oxo-lauric acid [Protein Data Bank (PDB) IDcode 2WYC (28)], molecular docking experiments were performed using the3-oxo-C12-L-HSL and C8-L-HSL substrates. Molecular docking simulations ofthe 10 lowest-energy poses were done using the grid-based approachCDOCKER, a molecular dynamics simulated annealing-based algorithm inwhich the receptor is held rigid while the ligands are allowed to flex duringthe refinement (34, 35), implemented in Discovery Studio 3.0 (Accelrys). All ofthe structures were further energy-minimized using CHARMm (36), consistingof 150 steps of steepest descent followed by 2,000 iterations of theadopted basis set Newton–Raphson algorithm using an energy toleranceof 0.01 kcal·mol−1·Å−1. To obtain a structural overview of the new dockedsubstrate conformations with regard to the structure of 3-oxo-lauric acidbound to PvdQWT, an overlay was made of the structures containing themost favorable ligand poses for 3-oxo-C12-HSL and C8-HSL. Analysis ofligand-binding pattern tools in Discovery Studio 3.0 allowed us to char-acterize and compare ligand-binding poses in PvdQ and visualize specificinteractions between protein residues and bound substrates, such as res-idues involved in hydrogen bonding, charge or polar interactions, and vander Waals interactions. Analysis of the residues involved in interactionswith the fatty acid chain of C12-HSL and 3-oxo-C12-HSL permitted us tofurther curtail our search and select for determinant residues involved ininteractions with the substrates of interest. Selected amino acid residueswere mutated in silico into all other possible 19 amino acid residues andenergy-minimized as described above. The new energy-minimized struc-tures were then used to perform docking as described above using C8-HSLas substrate. The five most favorable ligand poses were inspected forbinding energy and distance between the carbonyl carbon of the substrateand the catalytic Serβ1 of the β-subunit of PvdQ. Finally, targeted aminoacid substitutions were selected for site-directed mutagenesis.

Mutagenesis of pvdQ. The plasmid pMCT-pvdQ was under the control of iso-propyl β-D-1-thiogalactopyranoside-inducible expression from the lacZ pro-moter/operator (21). Point mutations were generated using the MEGAWHOPmethod as described in ref. 37. Briefly, a megaprimer (200–500 bp) containingthe desired mutation was generated using Phusion polymerase (Finnzymes).After purification, the megaprimer was used to amplify the whole plasmid. ThePCR mixtures were subsequently digested with DpnI (Fermentas) to remove thetemplate. All constructs were verified by DNA sequencing (Macrogen).

Protein Expression and Purification. Escherichia coli strain DH10B was used forexpression of recombinant PvdQ protein as described previously (21). Briefly,E. coli DH10B cells harboring pMCT-pvdQ and variants were grown for 48 hat 30 °C in 2× trypton-yeast medium (38) supplemented with chloramphenicol(50 μg/mL). After harvesting cells by centrifugation at 5000 × g, pellets wereresuspended in Tris·EDTA buffer (50 mM Tris·HCl, pH 8.8, 2 mM EDTA) andlysed by sonication. PvdQ was purified by a two-step procedure as previouslydescribed (28). In short, the flow-through of an anion-exchange chromatog-raphy column (Q-Sepharose; GE Healthcare) was collected and adjusted toa 0.7 M final concentration of ammonium sulfate and loaded onto a phenyl-Sepharose column. PvdQ and variants eluted at a final concentration of 0%ammonium sulfate in T50 (50 mM Tris·HCl, pH 8.8) with a purity of ≥95% asshown by SDS/PAGE (Invitrogen) and Coomassie staining.

Initial Screening of Mutants. An initial screening for C8-HSL degradation wasconducted using the bioreporter strain P. putida F117 carrying the plasmidpAS-C8 (39). This plasmid encodes a cepR-PcepI::gfp fusion that drives gfpexpression in response to C8-HSL, allowing quantification of the amount ofremaining C8-HSL after exposure to the PvdQ mutants. An overnight cultureof this strain was diluted 100 times in LB medium (38) containing gentamicin(20 μg/mL) and a final concentration of 0.5, 1, or 5 μM C8-HSL. The concen-tration of tested proteins used in this assay was either 5 or 10 ng/μL; at theseconcentrations, PvdQWT does not affect the amount of GFP fluorescenceproduced by the biosensor. Reaction conditions without enzyme or AHLs alonewere used as controls. GFP expression was monitored every 30 min duringa 20-h experiment in a multifunctional microplate reader (FLUOstar Omega;BMG Labtech; excitation wavelength, 485 nm; emission wavelength, 520 nm).Screenings were conducted in at least three independent experiments.3-Oxo-C12-HSL degradation was assayed as previously described (40) usingthe biosensor strain E. coli JM109 containing the plasmid pSB1075 (lasR-PlasI::luxCDABE) (41) that produces luminescence in the presence of thisautoinducer. All PvdQ variants that caused a 50% decrease in fluorescencecorrelating to an increase in activity toward C8-HSL were scored positive.

Analysis of Quorum-Quenching Activity of PvdQWT and PvdQLα146W,Fβ24Y. Enzy-matic activitywas investigated using the above-mentioned biosensor systems.Luminescence and fluorescence, respectively, were followed over a period in thepresence of various concentrations of AHL substrates (0–10 μM)with andwithoutthe addition of 5 ng/μL enzyme. Linearity of the reaction was checked.

Data Collection, Crystal Structure Determination, and Refinement. PurifiedPvdQLα146W,Fβ24Y was crystallized as previously described (28). Crystals wereflash-cooled in liquid nitrogen using mother liquor supplemented with 25%(vol/vol) glycerol as cryoprotectant. One hundred and twenty degrees of datawere collected at 100 K with an oscillation range of 0.2° at the PX-1 beamline,Swiss Light Source (Villigen) supplied with a Pilatus detector. Data were in-tegrated and scaled using XDS (42) and SCALA (43) using the CCP4 interface(44). Initial phase information of the double mutant was obtained with Phaser(45) using PDB ID code 2WYE (28) as a search model. Model building andrefinement were done with Coot (46) and REFMAC5 (47) using translation/libration/screw refinement (48). Structure validation was done with Mol-Probity (49).

Virulence Assays of B. cenocepacia H111. Purified proteins were tested for re-duction of C8-HSL levels present in B. cenocepacia H111 cultures. Overnightcultures of wild-type B. cenocepacia H111 (50) and the synthase-negative strainB. cenocepacia H111-I (51) were diluted 100-fold in LB medium. RecombinantPvdQWT or PvdQLα146W,Fβ24Y was added to the cultures at a final concentrationof 0.045 mg/mL, cultures containing buffer only served as controls, and wereincubated at 30 °C for 24 h. Bacterial growth was measured in a Tecan platereader over a period. In addition, 2-mL aliquots were collected by centrifuga-tion at 5000 × g and supernatants were filtered through a 0.2-μm filter andstored at −20 °C until further analysis. To determine AHL concentrations, 900 μLof supernatant was first acidified with 1 M HCl (100 μL) and incubated at 37 °Cfor 18 h to revert spontaneous hydrolysis of the AHLs. Detection was per-formed as mentioned above with the biosensor strain F117 (pAS-C8) byincubating 180 μL bacterial strain with 10 μL of bacterial supernatant. Fluo-rescence was measured every 30 min for 20 h.

Detection of bacterial protease activity was measured upon mixing 300 μLof bacterial supernatant with 700 μL of skim milk (2%) in LB medium. TheOD600 was measured every 30 min for 18 h. In this assay, proteolytic activityresults in a decrease of absorbance due to the breakdown of milk proteins.One unit of protease was defined as the activity that produced a change inthe OD600 of 0.1 per h (30).

P. aeruginosa AHL Analysis. The same procedure was followed as describedpreviously for B. cenocepacia. In brief, an overnight culture of P. aeruginosaΔpvdQ (40) was diluted 100-fold in LB medium. Recombinant PvdQWT orPvdQLα146W,Fβ24Y was added to the cultures at a final concentration of 0.045mg/mL, cultures containing buffer only served as controls, and were in-cubated at 30 °C for 24 h. To determine AHL concentrations, 900 μL of su-pernatant (of a given time point) was first acidified with 1 M HCl (100 μL)and incubated at 37 °C for 18 h to revert spontaneous hydrolysis of the AHLs.Detection was performed as mentioned above with the biosensor strainE. coli JM109 containing the plasmid pSB1075 (lasR-PlasI::luxCDABE) (41).

G. mellonella Infection Assay. Infection assays were performed as previouslydescribed (29). Briefly, bacterial overnight cultures were diluted 1:100 in LBmedium and grown to an OD600 of 0.6–0.8. Cultures were collected and adjusted

1572 | www.pnas.org/cgi/doi/10.1073/pnas.1311263111 Koch et al.

to a final concentration of 4 × 107 cfu/mL (OD600 0.125) using 10 mM MgSO4.Before injection, cultures were incubated at 30 °C for 1 h with or without 0.15mg/mL enzyme, after which the cultures were immediately transferred to ice. Aninsulin pen (HumaPen Luxura; Lilly Nederland) was used to inject 10-μL aliquotsinto the hindmost proleg of G. mellonella. Fifteen healthy larvae were injectedper strain and incubated at 30 °C. Animals only injected with MgSO4 served ascontrols. Larvae were monitored after 24 and 48 h, respectively, and were scoreddead if they did not respond to touch or had turned black. Assays were per-formed in three independent experiments.

Kinetic Analysis. The enzymatic activities of PvdQWT and PvdQLα146W,Fβ24Y

were tested with an end-point assay using a derivatization with ortho-phthaldialdehyde (52). Stocks of the substrates 3-oxo-C12-L-HSL and C8-L-HSL (Bio-Connect) were made in methanol. These substrates were added toreaction vials, after which the methanol was removed by evaporation. Thesubstrate was then carefully solubilized in PBS at 30 °C. Enzyme was addedto 5 μg/mL and samples of 60 μL were taken immediately and at 2- to 5-minintervals. Each sample was immediately heat-inactivated and stored on ice.At the end of the assay, 50 μL of each sample was transferred to a well of a384-well plate (Greiner Bio-One) and mixed with 50 μL of phthaldialdehyde

reagent (Sigma-Aldrich). The absorbance at 340 nm was recorded after15–20 min in a microplate reader (FLUOstar Omega; BMG Labtech). A cali-bration curve was made with 0–0.5 mM homoserine lactone (Sigma-Aldrich)and showed linearity up to an absorbance of 1.4. The absorbance valueswere plotted as a function of time and the initial rates were calculatedusing the slope of the calibration curve. Finally, the initial rates wereplotted as a function of the substrate concentration. For each substrateconcentration at least three, but mostly five, experiments were per-formed. Controls with only enzyme or only substrates were performed,and all steps of the experimental setup were checked for effectiveness andintroduction of artifacts.

ACKNOWLEDGMENTS. We thank Leo Eberl and Kathrin Riedel for providing B.cenocepaciaH111 and H111-I strains as well as the C8 biosensor strain F117 (pAS-C8). We gratefully acknowledge Rien Hoge for helpful discussions regardingGalleria infection assays, Eli LillyNederland for providing empty insulin cartridges,and Rita Setroikromo, Ronald van Merkerk, and Putri Dwi Utari for technicalassistance with the assays. We thank the beamline staff of PX-1 (Swiss LightSource) for their assistance. Jessica A. Thompson is thanked for carefully readingand correcting the manuscript. This research was partly funded by EuropeanUnion Grant Antibiotarget MEST-CT-2005-020278 (to G.K. and P.N.-J.).

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