Reducing virulence of the human pathogen Burkholderia by altering the substrate specificity of the quorum-quenching acylase PvdQ

Significance

Resistance toward commonly used antibiotics is becoming a serious issue in the fight against bacterial pathogens. One promising strategy lies in the interference of bacterial quorum sensing by the hydrolysis of the signaling molecules. In this study, we present a structure-aided computational design approach to alter the substrate specificity of the quorum-quenching acylase PvdQ. Introduction of two point mutations in residues lining the active site led to a switch in substrate specificity, rendering the enzyme highly active toward C8-HSL and thereby reducing virulence caused by Burkholderia cenocepacia. Thus, this work not only provides a structural insight into the substrate specificity of quorum-quenching acylases but also indicates their potential in the fight against specific bacterial pathogens.

Abstract

The use of enzymes to interfere with quorum sensing represents an attractive strategy to fight bacterial infections. We used PvdQ, an effective quorum-quenching enzyme from Pseudomonas aeruginosa, as a template to generate an acylase able to effectively hydrolyze C8-HSL, the major communication molecule produced by the Burkholderia species. We discovered that the combination of two single mutations leading to variant PvdQ^{Lα146W,Fβ24Y} conferred high activity toward C8-HSL. Exogenous addition of PvdQ^{Lα146W,Fβ24Y} dramatically decreased the amount of C8-HSL present in Burkholderia cenocepacia cultures and inhibited a quorum sensing-associated phenotype. The efficacy of this PvdQ variant to combat infections in vivo was further confirmed by its ability to rescue Galleria mellonella larvae upon infection, demonstrating its potential as an effective agent toward Burkholderia infections. Kinetic analysis of the enzymatic activities toward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrate switch. This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In addition, we demonstrate that their substrate range can be easily switched, thereby paving the way to selectively target only specific bacterial species inside a complex microbial community.

The Burkholderia cepacia complex (Bcc) comprises a group of 17 related bacterial species able to colonize different environmental niches (1). Over the years the Bcc has gained special attention, as some of its members have been associated with life-threatening human infections (2, 3). Especially immunocompromised patients and people suffering from cystic fibrosis are generally infected with these pathogens; in particular, infection with Burkholderia cenocepacia has been correlated with a poor prognosis (1, 4). B. cenocepacia is often found cocolonizing the lungs of cystic fibrosis patients alongside the opportunistic pathogen Pseudomonas aeruginosa (5–9).

Reports on the occurrence of these two pathogens are appearing more and more frequently, underlining the difficulty in eradicating these pathogens with common antibiotics (10). Hence, novel strategies are needed to target bacterial infections without applying too much selective pressure (11). An important bacterial Achilles’ heel is quorum sensing (QS), a cell density-reliant regulatory system dependent on the secretion of N-acyl homoserine lactones (AHLs) (12). These molecules have been largely associated with virulence traits, as they are pivotal for the expression of genes involved in toxin production, motility, plasmid 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 been demonstrated to reduce pathogenesis (15–17). The use of enzymes in targeting QS paves a new way in combating pathogens. A major finding 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 acylases hydrolyze the amide bond of AHLs; both enzymes render the autoinducer inactive, reducing bacterial virulence and pathogenesis in vivo (22–25).

Recently, we have shown that the quorum-quenching acylase PvdQ, produced by fluorescent pseudomonads, decreases the levels of the P. aeruginosa signal molecule 3-oxo-C12-HSL. Consequently, by either overexpression or exogenous addition of PvdQ, expression of virulence-related genes was reduced (21, 26, 27) in a model system measuring the survival of Caenorhabditis elegans upon infection by P. aeruginosa (23). PvdQ is most effective against AHLs with side chains longer than 10 carbon atoms (21), whereas showing little to no activity toward AHLs with shorter acyl chains such as C8-HSL, which induces virulence in members of the Bcc (6, 9). The recently solved structure of PvdQ with a bound 3-oxo-C12 fatty acid revealed a large hydrophobic substrate-binding cleft that properly accommodates this fatty acid side chain (28). Altering the substrate range of PvdQ toward shorter AHLs, such as C8-HSL, might therefore 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 acylase such that it targets explicitly the signaling molecules of a restricted range of pathogens. We have identified a PvdQ variant containing two amino acid substitutions, Leuα146Trp and Pheβ24Tyr, which showed a substantially increased C8-HSL–degrading activity compared with the wild-type enzyme. Kinetic analysis was in-line with a substrate switch from 3-oxo-C12-L-HSL to C8-L-HSL caused by these mutations. Using an in vivo model for B. cenocepacia infection (29), we demonstrate that this quorum-quenching PvdQ variant can be successfully used to attenuate pathogen virulence and increase host survival. These results validate PvdQ^{Lα146W,Fβ24Y} as a promising and effective potential agent to combat emerging Bcc infections.

Results and Discussion

Design of PvdQ Variants for Increased C8-HSL Activity.

Using the recently elucidated crystal structure of PvdQ (28), we adopted a rational design approach to scan for PvdQ variants that would be better capable of accommodating C8-HSL than the wild-type enzyme. PvdQ is a heterodimeric Ntn hydrolase with an unusually large binding pocket that can accommodate the long acyl chain 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 performed molecular docking experiments using 3-oxo-C12-HSL and C12-HSL in the active site of PvdQ^WT. The results show that the carbonyl oxygen of these substrates has good hydrogen-bonding interactions with the Nδ2 group of Asnβ269 and the Valβ70 backbone amide (Fig. 1 A and B and Fig. S1), which constitute the oxyanion hole (28). The most favorable substrate poses obtained for 3-oxo-C12-HSL show the acyl chain positioned in the hydrophobic pocket in a conformation similar to that adopted by the C12 acyl chain in the C12-PvdQ crystal structure. In contrast, a similar analysis using C8-HSL resulted in a substantial conformational heterogeneity within the active site of PvdQ^WT, with the most energetically favorable poses showing that the carbonyl oxygen of C8-HSL is no longer within H-bonding distance with Asnβ269 and the Valβ70 backbone amide (more than 3.5 Å), and a significant distance between the carbonyl carbon of C8-HSL and the catalytic Serβ1 of PvdQ is observed (6.1 Å) (Fig. S2). Based on the differences shown for C8-HSL and C12-HSL during molecular docking experiments, the following amino acid residues were selected for in silico mutagenesis: Thrα143, Leuα146, Glyα150, Pheβ24, Leuβ50, Leuβ53, Asnβ57, Valβ158, Trpβ162, Proβ185, Trpβ186, and Valβ187. Each amino acid was substituted by all other 19 possible amino acids, and the new model structures were energy-minimized, after which C8-HSL was docked in the active site of PvdQ. A final minimization was used to refine the ligand poses. The most energetically favorable substrate-docked poses were analyzed with respect to the positioning of the substrate and the new distances obtained between the catalytic Serβ1 Oγ and the carbonyl carbon atom of C8-HSL. Eighteen amino acid substitutions out of 218 resulting in a reduction of the distance between Serβ1 and the carbonyl carbon of C8-HSL of less than 4 Å were therefore 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 selected for site-directed mutagenesis (Table S1). The eight selected residues were also mutated to alanine.

Fig. 1.

Comparative molecular docking simulations. The most favored conformations of C12-HSL (A) and 3-oxo-C12-HSL (B) in the active site of PvdQ from CDOCKER, as implemented in Accelrys Discovery Studio 3.0. Hydrogen atoms were added to the protein molecule and substrates, and the CHARMm force field was used to assign partial charges to the ligands. Substrates were docked into PvdQ using the coordinates of 3-oxo-lauric acid bound in the active site of PvdQ^WT [PDB ID code 2WYC (28)]. The residues forming the active site of PvdQ are colored green, and the accessible solvent surface-contoured substrates are represented in yellow and cyan sticks for C12-HSL and 3-oxo-C12-HSL, respectively. Both substrate-docking poses are aligned for nucleophilic attack. The carbonyl oxygen forms hydrogen bonds with the Asnβ269 side-chain Nδ2 and the Valβ70 backbone amide, consistent with the proposed oxyanion hole residues involved in the stabilization of the tetrahedral transition state (28). [The amino acid numbering follows the subunit composition of the mature protein; i.e., the α-chain is defined by Aspα1–Valα170 (equivalent to D24–V193 of the amino acid sequence of the preprotein) and the β-chain by Serβ1–Gluβ546 (equivalent to S217–E762).]

Screening for C8-HSL Quenching.

Based on this in silico screening approach, the 26 proposed site-directed mutants of PvdQ were constructed, produced, and purified. The variants containing Valβ158Ala and Asnβ57Arg were affected in protein maturation (Fig. S3), a property often observed when mutagenizing acylases that rely on the same residues to perform substrate conversion and protein maturation. These were therefore excluded from further analysis. As seen with fluorescence experiments, mutations of Trpβ162 and Valβ187 had no effect on C8-HSL hydrolysis, but substitutions at 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 in increased hydrolytic activity toward C8-HSL. In particular, variants Thrα143Lys, Leuα146Trp, Pheβ24Tyr, Leuβ53Ile, Asnβ57His, Valβ158Met, and Valβ158Ile resulted in a significant increase in C8-HSL hydrolysis compared with PvdQ^WT, as shown by a more than 50% decrease in mean specific fluorescence activity (Fig. 2A).

Fig. 2.

(A) Relative fluorescence as a measure of the presence of C8-HSL after incubation of PvdQ variants with the biosensor P. putida F117 (pAS-C8) after 13 h of incubation. The protein concentration was 5 ng/µL. Values reported indicate the mean specific fluorescence activity of each PvdQ variant normalized as a percentage of the fluorescence level in an assay with PvdQ^WT. (B) Activity of PvdQ^WT and PvdQ^{Lα146W,Fβ24Y} toward AHLs. Enzymes were incubated with 5 µM 3-oxo-C12-HSL or C8-HSL. 3-Oxo-C12 levels were analyzed using E. coli (pSB1075) and C8-HSL levels with P. putida F117 (pAS-C8). Values reported indicate the mean specific fluorescence/luminescence activity normalized to the units measured by AHLs only. Error bars indicate SD.

A second round of computational analysis was performed, to identify possible combinatorial mutations in PvdQ with a further enhanced activity and specificity toward C8-HSL. The best single mutants were combined in silico and analyzed as previously for the distance between the catalytic Serβ1 Oγ and the carbonyl carbon atom 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. Most importantly, the Leuα146Trp/Pheβ24Tyr variant displayed the highest hydrolytic activity on C8-HSL: 5 ng/µL PvdQ^{Lα146W,Fβ24Y} was sufficient to quench 5 µM C8-HSL, corresponding to a fivefold increased activity compared with the best single mutant tested (Fig. 2A).

Hydrolytic activity of these enzymes toward 3-oxo-C12-HSL was assessed using a biosensor strain (Fig. 2B and Fig. S4). Whereas PvdQ^WT displayed high hydrolytic activity, the mutant enzyme PvdQ^{Lα146W,Fβ24Y} was severely impaired in its capacity to hydrolyze 3-oxo-C12-HSL under the conditions tested, rendering its function of quenching the endogenous signal from P. aeruginosa biologically insignificant. The activities toward 3-oxo-C12-HSL and C8-HSL highlight that a substrate switch had occurred, diminishing activity toward 3-oxo-C12-HSL but substantially increasing hydrolytic activity toward C8-HSL, as Fig. 2B clearly indicates. Interestingly, considering the respective single mutants, deacylase activity toward 3-oxo-C12-HSL was hardly affected in PvdQ^Lα146W and only slightly impaired in PvdQ^Fβ24Y, indicating that the combination of both mutations is responsible for this switch in substrate specificity (Fig. S4).

Previously, Pheβ24 had been shown to allow the entrance of 3-oxo-C12-HSL into the hydrophobic pocket of PvdQ^WT (28). Position α146 needs to be occupied by a small residue like Leu to allow binding of the C12 acyl chain. We suggest that the constraints imposed by the Trpα146 side chain on the binding mode of C8-HSL and the stabilization of Trpα146 by the new Tyrβ24 contribute to the proper accommodation of C8-HSL in the active site of PvdQ, whereas impairing its activity toward 3-oxo-C12.

Analysis of Quorum-Quenching Activity.

To further characterize PvdQ^WT and PvdQ^{Lα146W,Fβ24Y}, the activity of various concentrations of C8-HSL and 3-oxo-C12-HSL was determined. These experiments confirmed that PvdQ^WT has a preference for the long-chain substrate 3-oxo-C12-HSL, whereas this preference is shifted to the shorter-chain substrate C8-HSL in mutant PvdQ^{Lα146W,Fβ24Y}. Using the biosensor strain Pseudomonas putida (pAS-C8), we determined that after incubation of 1 µM C8-HSL with either 5 ng/μL PvdQ^WT or PvdQ^{Lα146W,Fβ24Y}, only 0.063 µM in the case of PvdQ^WT or 0.0013 µM C8-HSL in the case of PvdQ^{Lα146W,Fβ24Y} was detectable. These results indicate that using equal concentrations of PvdQ proteins, PvdQ^{Lα146W,Fβ24Y} displays a 48.5-fold reduction of C8-HSL levels compared with PvdQ^WT (Fig. S5).

Structural Effects of PvdQ^{Lα146W,Fβ24Y}.

To investigate the structural effects of the Leuα146Trp and Pheβ24Tyr mutations on PvdQ, we elucidated the crystal structure of the double mutant PvdQ^{Lα146W,Fβ24Y} at a resolution of 1.9 Å to final R_work and R_free values of 17.9% and 20.7%, respectively. Data collection and refinement statistics can be found in Table S2. No major conformational differences were observed between PvdQ^{Lα146W,Fβ24Y} and PvdQ^WT (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 nucleophile residue Serβ1 is in the closed state (Fig. 3A) (28). The side chains of the mutated Tyrβ24 and Trpα146 residues line this pocket, with each residue adopting at least two alternate conformations. The Leuα146Trp mutation introduces a much bulkier side chain and reduces the volume of the substrate-binding pocket from 260 to 80 ų for the closed conformation (Fig. 3 A and B). The cavity with Tyrβ24 in the open conformation has a volume of 140 ų. We propose that the larger volume of the substrate-binding pocket in PvdQ^WT preferentially binds long fatty acid-like acyl chains, whereas the much less voluminous pocket of PvdQ^{Lα146W,Fβ24Y} favors short-chain HSLs.

Fig. 3.

Residues lining the substrate-binding pocket in PvdQ^WT and PvdQ^{Lα146W,Fβ24Y}. (A) PvdQ^WT; the main chain is indicated by a Cα trace, the blue ball and sticks indicate the residues lining the substrate-binding pocket, and the mesh shows the cavity as calculated by VOIDOO (Uppsala Software Factory). (B) PvdQ^{Lα146W,Fβ24Y}; the main chain is indicated by a Cα trace, the green ball and sticks indicate the residues lining the substrate-binding pocket, except for the mutated residues, which are indicated in orange, and the mesh shows the cavity as calculated by VOIDOO. The Trpα146 mutation decreases the volume of the cavity, making it more suitable for short-chain fatty acids.

The crystal structures of PvdQ^WT and PvdQ^{Lα146W,Fβ24Y} indicate that mutating Pheβ24 to a tyrosine does not cause substantial conformational changes in the active site of PvdQ. Slightly different side-chain orientations for these aromatic residues are observed, however, with the new tyrosine moving upward relative to the phenylalanine in the active conformation (Fig. 4). This in turn 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 conformations observed in the crystal structure, the hydroxyl group of Tyrβ24 has an interaction with the side-chain amine of Trpα146 (Fig. 4). This conformation resembles the open conformation that residue β24 adopts in the substrate-bound state (28), and thus this interaction may stabilize the conformation of Trpα146 and provide a better fit of the shorter acyl side chain of C8-HSL.

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. aeruginosa PvdQ^WT (shown in green) and PvdQ^{Lα146W,Fβ24Y} (in white) with the most favored docked conformations for C8-HSL (A) and 3-oxo-C12-HSL (B), using the coordinates given by the crystal structure of PvdQ^{Lα146W,Fβ24Y}. As shown, the introduction of residue Trpα146 clearly reduces the hydrophobic pocket size and the protrusion into the interior of the enzyme, and contributes to the proper accommodation of the acyl chain of C8-HSL (A). The hydroxyl group of the new Tyrβ24 forms a 3.2-Å hydrogen bond with the side-chain amine of Trpα146, stabilizing the conformation of Trpα146 and providing a better fit of the alternate acyl chain of C8-HSL. Inversely, the mutant PvdQ^{Lα146W,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 the catalytic serine of 6.1 Å for the most favorable conformation.

PvdQ^{Lα146W,Fβ24Y} Disrupts B. cenocepacia Signaling and Induction of Virulence.

To determine whether PvdQ^WT and its variant, PvdQ^{Lα146W,Fβ24Y}, interfere with C8-HSL accumulation and downstream signaling by B. cenocepacia, cultures of this bacterium were incubated with either enzyme (Fig. S6). The addition of the enzymes did not influence bacterial growth, as indicated in Fig. S7. Hence, C8-HSL accumulation was assayed after 24 h of incubation at 30 °C. Fig. 5A clearly shows that almost no fluorescence, and therefore no C8-HSL, could be detected in the presence of the mutant acylase PvdQ^{Lα146W,Fβ24Y}. Significant levels of fluorescence were measured in the control culture with no enzyme addition; only a slight decrease was observed in the presence of PvdQ^WT. Thus, this activity test further substantiates the results obtained in the preliminary activity screen, namely the limited activity of wild-type enzyme to hydrolyze C8-HSL compared with the much more efficient PvdQ^{Lα146W,Fβ24Y} variant.

Fig. 5.

Effects of PvdQ^WT and PvdQ^{Lα146W,Fβ24Y} on B. cenocepacia QS. (A) Cells of B. cenocepacia were incubated for 24 h without PvdQ (first set of bars) or in the presence of PvdQ^WT (indicated as WT) or PvdQ^{Lα146W,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 relative to the activity in the presence of PvdQ^WT (equal to 1). Cultures were also analyzed for the production of protease by the activity on skim milk (dashed bars). One unit of protease was defined as the activity that produced a change in the OD₆₀₀ of 0.1 per h (30). Lower protease units were found to be produced when PvdQ^{Lα146W,Fβ24Y} was added to the cultures (dashed bars). (B) PvdQ^{Lα146W,Fβ24Y} protects for B. cenocepacia H111 infection in the insect model. G. mellonella larvae were injected with B. cenocepacia H111 wild-type cells either untreated or treated with PvdQ^WT or PvdQ^{Lα146W,Fβ24Y}. After 48 h of incubation, larval survival was assessed. G. mellonella injected with buffer only resulted in 100% survival, B. cenocepacia H111 had a lethal outcome for the larvae, whereas pretreatment of the bacterial cultures with PvdQ^{Lα146W,Fβ24Y} rescued survival. For all determinations, a quorum-sensing negative-strain H111-I served as control. Error bars indicate SD.

In addition, and to further substantiate that a substrate switch had occurred, we performed the same experiment but adding the enzyme to a P. aeruginosa culture. Analysis of 3-oxo-C12-HSL levels after incubation with the enzymes for 24 h revealed that only PvdQ^WT could significantly decrease the amounts of the signaling molecule, as shown by a clear decrease in luminescence. Hardly any effect, however, on AHL levels was observed upon addition of PvdQ^{Lα146W,Fβ24Y} (Fig. S6). Taken together, these AHL quantifications show that PvdQ^{Lα146W,Fβ24Y} affects shorter acyl chains in a 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 of extracellular protease, which plays an important role in the invasion of lung tissue by B. cenocepacia, is positively regulated by C8-HSL (30, 31). Addition of PvdQ^{Lα146W,Fβ24Y} to B. cenocepacia cultures significantly decreased the number of protease units detected in culture supernatants, almost to the level of that produced by the QS-negative strain H111-I, which is unable to induce protease activity (Fig. 5A). In contrast, addition of PvdQ^WT resulted in protease production similar to the control culture of B. cenocepacia wild-type H111 (Fig. 5A). These data show that PvdQ^{Lα146W,Fβ24Y} decreases the level of C8-HSL present in B. cenocepacia cultures, thereby reducing the expression of virulence traits.

PvdQ^{Lα146W,Fβ24Y} Attenuates Burkholderia Virulence upon in Vivo Infection of Galleria mellonella Larvae.

The promising results obtained in degrading C8-HSL and protease activity after exogenous addition of PvdQ^{Lα146W,Fβ24Y} to B. cenocepacia in vitro prompted us to investigate the quorum-quenching effects of this enzyme in vivo. As depicted in Fig. 5B, injection of larvae of the great wax moth Galleria mellonella with B. cenocepacia H111 culture kills nearly all larvae, whereas injection with the QS-negative strain B. cenocepacia H111-I does not affect survival, in accordance with the importance of QS in B. cenocepacia infection and pathogenesis. Whereas preincubation of B. cenocepacia H111 bacteria with PvdQ^WT hardly affected the survival rates of the larvae, preincubation with PvdQ^{Lα146W,Fβ24Y} led to a nearly complete attenuation of bacterial virulence and increased the overall survival of the larvae (Fig. 5B). This result demonstrates that PvdQ^{Lα146W,Fβ24Y}, but not PvdQ^WT, is able 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 and derivatization with ortho-phthaldialdehyde. The low solubility of 3-oxo-C12-L-HSL allowed us to screen for activity in the range of 0–0.2 mM, whereas the activity toward C8-L-HSL was measured up to 0.6 mM. The activity plots (Fig. S8) clearly show a substrate switch due to the mutations: Whereas PvdQ^WT preferentially hydrolyzes 3-oxo-C12-L-HSL, PvdQ^{Lα146W,Fβ24Y} has a preference for C8-L-HSL. The concentration window is not sufficiently large to determine the kinetic parameters separately, but an estimation of the k_cat:K_m ratio can be obtained. For 3-oxo-C12-L-HSL, these parameters were 5.8 × 10³ and 1.5 × 10³ M⁻¹s⁻¹ for PvdQ^WT and PvdQ^{Lα146W,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 PvdQ^WT and PvdQ^{Lα146W,Fβ24Y}, respectively, resulting in a 4.3-fold difference. Thus, in total, the mutations result in a 16-fold difference in catalytic efficiency.

Conclusions

We identified and characterized PvdQ^{Lα146W,Fβ24Y}, a PvdQ variant with a shifted substrate range that is highly active toward C8-HSL. The in vivo quorum-quenching activity of PvdQ^{Lα146W,Fβ24Y} against B. cenocepacia, demonstrated by the decrease in proteolytic activity in the culture supernatant and the increase in host survival, confirms its potential as a possible therapeutic, especially as this protease activity has been associated with the pathogen’s invasion of lung tissue (30, 31). Our results obtained with PvdQ^{Lα146W,Fβ24Y} together with previous results on PvdQ^WT (23) provide the first steps toward the development of future antimicrobial therapies aiming to effectively combat P. aeruginosa and B. cenocepacia infections, the two most important Gram-negative pathogens isolated from the lungs of cystic fibrosis patients. Furthermore, we illustrate how the design approach used in this study can be applied to successfully produce quorum-quenching variants with specific substrate ranges to target a selected group of bacteria. The application of highly active enzymes as potential treatment can have a number of beneficial effects not only with respect to lung infections but also to those occurring in the gastrointestinal tract. Specific targeting of the quorum-sensing systems of pathogens would leave the beneficial microbiota unharmed. Decreasing the deleterious side effects on the normal host microbiota is a major factor contributing to host protection against invading competitive, opportunistic bacteria and increasing the recovery of the host after infection (32, 33). We aim with our findings to substantiate the high potential of quorum-quenching enzymes in targeting bacterial pathogens.

Materials and Methods

Structure-Based Design of PvdQ Mutants by Molecular Docking of C8-HSLs.

Based on the crystal structure of the P. aeruginosa quorum-quenching acylase (PvdQ) in complex with 3-oxo-lauric acid [Protein Data Bank (PDB) ID code 2WYC (28)], molecular docking experiments were performed using the 3-oxo-C12-L-HSL and C8-L-HSL substrates. Molecular docking simulations of the 10 lowest-energy poses were done using the grid-based approach CDOCKER, a molecular dynamics simulated annealing-based algorithm in which the receptor is held rigid while the ligands are allowed to flex during the refinement (34, 35), implemented in Discovery Studio 3.0 (Accelrys). All of the structures were further energy-minimized using CHARMm (36), consisting of 150 steps of steepest descent followed by 2,000 iterations of the adopted basis set Newton–Raphson algorithm using an energy tolerance of 0.01 kcal⋅mol⁻¹⋅Å⁻¹. To obtain a structural overview of the new docked substrate conformations with regard to the structure of 3-oxo-lauric acid bound to PvdQ^WT, an overlay was made of the structures containing the most favorable ligand poses for 3-oxo-C12-HSL and C8-HSL. Analysis of ligand-binding pattern tools in Discovery Studio 3.0 allowed us to characterize and compare ligand-binding poses in PvdQ and visualize specific interactions between protein residues and bound substrates, such as residues involved in hydrogen bonding, charge or polar interactions, and van der Waals interactions. Analysis of the residues involved in interactions with the fatty acid chain of C12-HSL and 3-oxo-C12-HSL permitted us to further curtail our search and select for determinant residues involved in interactions with the substrates of interest. Selected amino acid residues were mutated in silico into all other possible 19 amino acid residues and energy-minimized as described above. The new energy-minimized structures were then used to perform docking as described above using C8-HSL as substrate. The five most favorable ligand poses were inspected for binding energy and distance between the carbonyl carbon of the substrate and the catalytic Serβ1 of the β-subunit of PvdQ. Finally, targeted amino acid substitutions were selected for site-directed mutagenesis.

Mutagenesis of pvdQ.

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

Protein Expression and Purification.

Escherichia coli strain DH10B was used for expression of recombinant PvdQ protein as described previously (21). Briefly, E. coli DH10B cells harboring pMCT-pvdQ and variants were grown for 48 h at 30 °C in 2× trypton-yeast medium (38) supplemented with chloramphenicol (50 µg/mL). After harvesting cells by centrifugation at 5000 × g, pellets were resuspended in Tris⋅EDTA buffer (50 mM Tris⋅HCl, pH 8.8, 2 mM EDTA) and lysed by sonication. PvdQ was purified by a two-step procedure as previously described (28). In short, the flow-through of an anion-exchange chromatography column (Q-Sepharose; GE Healthcare) was collected and adjusted to a 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% as shown by SDS/PAGE (Invitrogen) and Coomassie staining.

Initial Screening of Mutants.

An initial screening for C8-HSL degradation was conducted using the bioreporter strain P. putida F117 carrying the plasmid pAS-C8 (39). This plasmid encodes a cepR-PcepI::gfp fusion that drives gfp expression in response to C8-HSL, allowing quantification of the amount of remaining C8-HSL after exposure to the PvdQ mutants. An overnight culture of 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 concentration of tested proteins used in this assay was either 5 or 10 ng/µL; at these concentrations, PvdQ^WT does not affect the amount of GFP fluorescence produced by the biosensor. Reaction conditions without enzyme or AHLs alone were used as controls. GFP expression was monitored every 30 min during a 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) using the biosensor strain E. coli JM109 containing the plasmid pSB1075 (lasR-PlasI::luxCDABE) (41) that produces luminescence in the presence of this autoinducer. All PvdQ variants that caused a 50% decrease in fluorescence correlating to an increase in activity toward C8-HSL were scored positive.

Analysis of Quorum-Quenching Activity of PvdQ^WT and PvdQ^{Lα146W,Fβ24Y}.

Enzymatic activity was investigated using the above-mentioned biosensor systems. Luminescence and fluorescence, respectively, were followed over a period in the presence of various concentrations of AHL substrates (0–10 µM) with and without the addition of 5 ng/µL enzyme. Linearity of the reaction was checked.

Data Collection, Crystal Structure Determination, and Refinement.

Purified PvdQ^{Lα146W,Fβ24Y} was crystallized as previously described (28). Crystals were flash-cooled in liquid nitrogen using mother liquor supplemented with 25% (vol/vol) glycerol as cryoprotectant. One hundred and twenty degrees of data were 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 integrated 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 and refinement were done with Coot (46) and REFMAC5 (47) using translation/libration/screw refinement (48). Structure validation was done with MolProbity (49).

Virulence Assays of B. cenocepacia H111.

Purified proteins were tested for reduction of C8-HSL levels present in B. cenocepacia H111 cultures. Overnight cultures of wild-type B. cenocepacia H111 (50) and the synthase-negative strain B. cenocepacia H111-I (51) were diluted 100-fold in LB medium. Recombinant PvdQ^WT or PvdQ^{Lα146W,Fβ24Y} was added to the cultures at a final concentration of 0.045 mg/mL, cultures containing buffer only served as controls, and were incubated at 30 °C for 24 h. Bacterial growth was measured in a Tecan plate reader over a period. In addition, 2-mL aliquots were collected by centrifugation at 5000 × g and supernatants were filtered through a 0.2-µm filter and stored at −20 °C until further analysis. To determine AHL concentrations, 900 µL of supernatant 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 strain F117 (pAS-C8) by incubating 180 µL bacterial strain with 10 µL of bacterial supernatant. Fluorescence was measured every 30 min for 20 h.

Detection of bacterial protease activity was measured upon mixing 300 µL of bacterial supernatant with 700 µL of skim milk (2%) in LB medium. The OD₆₀₀ was measured every 30 min for 18 h. In this assay, proteolytic activity results 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 in the OD₆₀₀ of 0.1 per h (30).

P. aeruginosa AHL Analysis.

The same procedure was followed as described previously for B. cenocepacia. In brief, an overnight culture of P. aeruginosa ΔpvdQ (40) was diluted 100-fold in LB medium. Recombinant PvdQ^WT or PvdQ^{Lα146W,Fβ24Y} was added to the cultures at a final concentration of 0.045 mg/mL, cultures containing buffer only served as controls, and were incubated at 30 °C for 24 h. To determine AHL concentrations, 900 µL of supernatant (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 strain E. coli JM109 containing the plasmid pSB1075 (lasR-PlasI::luxCDABE) (41).

G. mellonella Infection Assay.

Infection assays were performed as previously described (29). Briefly, bacterial overnight cultures were diluted 1:100 in LB medium and grown to an OD₆₀₀ of 0.6–0.8. Cultures were collected and adjusted 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 0.15 mg/mL enzyme, after which the cultures were immediately transferred to ice. An insulin pen (HumaPen Luxura; Lilly Nederland) was used to inject 10-µL aliquots into the hindmost proleg of G. mellonella. Fifteen healthy larvae were injected per strain and incubated at 30 °C. Animals only injected with MgSO₄ served as controls. Larvae were monitored after 24 and 48 h, respectively, and were scored dead if they did not respond to touch or had turned black. Assays were performed in three independent experiments.

Kinetic Analysis.

The enzymatic activities of PvdQ^WT and PvdQ^{Lα146W,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 to reaction vials, after which the methanol was removed by evaporation. The substrate was then carefully solubilized in PBS at 30 °C. Enzyme was added to 5 μg/mL and samples of 60 μL were taken immediately and at 2- to 5-min intervals. 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 a 384-well plate (Greiner Bio-One) and mixed with 50 μL of phthaldialdehyde reagent (Sigma-Aldrich). The absorbance at 340 nm was recorded after 15–20 min in a microplate reader (FLUOstar Omega; BMG Labtech). A calibration curve was made with 0–0.5 mM homoserine lactone (Sigma-Aldrich) and showed linearity up to an absorbance of 1.4. The absorbance values were plotted as a function of time and the initial rates were calculated using the slope of the calibration curve. Finally, the initial rates were plotted as a function of the substrate concentration. For each substrate concentration at least three, but mostly five, experiments were performed. Controls with only enzyme or only substrates were performed, and all steps of the experimental setup were checked for effectiveness and introduction of artifacts.