Characterization of a Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus and Its Immobilization for Disruption of Quorum Sensing

Abstract

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K_m values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k_cat/K_m = 5.5 × 10³ M⁻¹·s⁻¹), but SsoPox can also degrade N-butyryl homoserine lactone (C₄-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C₁₂-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C₄-HSL- and oxo-C₁₂-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.

Quorum sensing is a cell-cell communication strategy that enables single-celled organisms to synchronize their gene expression in a population-dependent manner. The best-characterized signaling molecules involved in quorum sensing are N-acyl homoserine lactones (AHLs). Different bacteria produce and respond to AHLs containing a constant homoserine lactone moiety with acyl chains of various lengths and with various substitutions. Genes regulated by quorum sensing include those involved in bioluminescence, biofilm formation, and bacterial virulence. For instance, the opportunistic pathogen Pseudomonas aeruginosa produces N-butyryl homoserine lactone (C₄-HSL) and N-3-oxo-dodecanoyl homoserine lactone (oxo-C₁₂-HSL), which regulate the production of exoproteases, such as elastase and the redox active exotoxin pyocyanin. Both of these molecules ultimately damage tissues and are correlated with bacterial virulence (13, 20). It was postulated that biocontrol by disrupting quorum sensing is less likely than the use of antibiotics to give rise to resistant bacteria, as there is less selective pressure placed on bacteria (26).

Given the roles of quorum sensing in bacterial virulence and biofilm formation, enzymes that can degrade AHLs, known as quorum quenchers, are potentially useful for a variety of environmental and medical applications. Two general types of quorum-quenching enzymes have been discovered: lactonases, which hydrolyze the lactone ring of AHL, and acylases, which remove the acyl chains attached to the lactone ring. Some promising results have been obtained using transgenic plants expressing the aiiA gene, encoding the lactonase from Bacillus thuringiensis (10). The plants were demonstrated to possess increased resistance to Erwinia carotovora, a bacterial organism that uses N-(3-oxohexanoyl)-l-homoserine lactone (oxo-C₆-HSL) for quorum sensing. In addition, the acylase AhlM, from Streptomyces spp., has been added to P. aeruginosa cultures and found to attenuate production of virulence factors regulated by AHLs (24).

Recently, AHL-degrading enzymes have been found in other prokaryotes, including SsoPox, which is produced by the hyperthermophilic archaeon Sulfolobus solfataricus (aryldialkylphosphatase, EC 3.1.8.1) (22). This enzyme was first reported to hydrolyze organophosphates. Organophosphates are commonly used as pesticides, as they can form a covalent bond with the active-site serine residue in acetylcholinesterase, thereby inhibiting nerve functions irreversibly (7). Subsequent studies showed that SsoPox is also a lactonase that can hydrolyze AHLs (1, 22). However, only semiquantitative bioassays of SsoPox activity toward the AHLs oxo-C₆-HSL and C₄-HSL have been reported, and therefore, the specificity of this enzyme toward homoserine lactones of various acyl chains is not known. The three-dimensional structure of the enzyme has been solved, and it adopts a (β/α)₈ barrel fold containing a binuclear divalent metal center composed of Co²⁺ and Fe³⁺ that assists with substrate binding and an activated water molecule which is involved in the hydrolysis reaction (11). SsoPox is evolutionarily distinct and shares no sequence similarities with better-characterized AHL lactonases, such as AiiA from B. thuringiensis, which belong to the β-lactamase family of enzymes (23). Instead, SsoPox is a member of a subgroup of the amidohydrolase family of metalloenzymes known as phosphotriesterase-like lactonases (1).

Here we overexpressed SsoPox in Pseudomonas putida with good yield. Kinetic parameters of the purified enzyme toward different AHL substrates are reported for the first time. Finally, we immobilized SsoPox on membranes functionalized with nanoalumina (AlOOH) fibers. These positively charged membranes have been used to concentrate a variety of viruses in water samples through electrostatic interactions (14, 16). We showed here that this membrane can also be used to immobilize SsoPox and can then be applied to bacterial cultures to disrupt quorum sensing. This is the first report of the successful use of an immobilized lactonase for quorum quenching.

MATERIALS AND METHODS

Chemicals.

Chelex-100, paraoxon, methyl-paraoxon, m-cresol purple, and all N-acyl homoserine lactones were purchased from Sigma-Aldrich (Oakville, ON, Canada). Restriction enzymes, T4 DNA ligase, and Taq polymerase were from Invitrogen (Burlington, ON, Canada) or New England BioLabs (Pickering, ON, Canada). All other chemicals were analytical grade and were obtained from Sigma-Aldrich and Fisher Scientific (Nepean, ON, Canada). Disruptor grade 4601 (cellulose, unlaminated) nanoalumina-functionalized membranes were kindly provided by Ahlstrom (PA).

Bacterial strains and plasmids.

Escherichia coli DH5α and P. putida KT2442 were routinely grown in Luria-Bertani broth. pTZ57R/T (Fermentas, Burlington, ON, Canada) and pT7-7 (27) were used as cloning vectors, while pVLT-31 (9) was used for expression in P. putida. Strains containing pTZ57R/T or pT7-7 were cultivated in medium containing 100 μg/ml ampicillin, while strains containing pVLT-31 were cultivated in medium containing 15 μg/ml tetracycline.

DNA manipulation and transformation.

The native gene which encodes SsoPox (SSO_2522) was amplified from Sulfolobus solfataricus P2 genomic DNA by PCR. Forward primer 5′-GCGCCATATGAGAATACCATTAGTTG-3′ and reverse primer 5′-GACGGTCGACTTAGCTGAAGAACTTTTTCGG-3′ were used to amplify the 945-bp gene sequence from genomic DNA of Sulfolobus solfataricus P2. Introduced NdeI and SalI restriction sites are underlined. The following amplification profile was used: 94°C for 2 min; 30 cycles of 94°C for 30 s, 48°C for 30 s, and 68°C for 1 min; and finally, 68°C for 5 min. The 945-bp amplified DNA was digested with the NdeI and SalI, purified using a QIAex II gel extraction kit (Qiagen, Mississauga, ON, Canada), according to the manufacturer's instructions, and then ligated into cloning vector pTZ57R/T, which had been digested with the same enzymes. The gene from a positive clone was sequenced at the Guelph Molecular Supercenter (University of Guelph, Guelph, ON, Canada) and subcloned into pT7-7 via NdeI/SalI restriction sites. To express the encoded protein in P. putida, site-directed mutagenesis was performed to generate a silent mutation (CTT to CTC) which abolishes the HindIII restriction site (AGGCTT) within the coding sequence at 817 bp, and then the ribosomal binding site located in pT7-7 was excised along with the gene using restriction enzymes XbaI and HindIII and ligated into pVLT-31, which had been digested with the same restriction enzymes. Codon-optimized SsoPox (see Fig. SA1 in the supplemental material) was synthesized by BioBasic (Markham, Canada). The gene was inserted into pT7-7 using NdeI and HindIII restriction sites. From the pT7-7 construct, XbaI and HindIII restriction sites were used to insert the gene into pVLT-31.

Protein expression.

Large-scale expression was carried out in 1-liter cultures. At early logarithmic phase (optical density [OD], between 0.4 and 0.6), isopropyl-β-d-thiogalactopyranoside and CoCl₂ were added to the culture at final concentrations of 0.5 mM and 0.2 mM, respectively. The presence of CoCl₂ is necessary for optimal protein expression (22). After overnight incubation at 37°C with shaking (210 rpm), cells were harvested by centrifugation at 3,000 × g. The cells were washed with 250 ml of 20 mM Tris-HCl (pH 8.0) to remove salts prior to storage at −20°C.

Protein purification.

Cell pellets were resuspended in 20 mM sodium HEPES-0.2 mM CoCl₂ (pH 8.0) (3 ml lysis buffer per gram cells). The resuspended cells were treated with lysozyme and DNase (final concentrations, 0.2 mg/ml and 40 U/ml, respectively) for 20 min at room temperature and then frozen at −20°C overnight. Cells were then disrupted by sonication, and the lysate was centrifuged at 58,545 × g for 20 min to sediment cellular debris. Subsequently, a method similar to that described by Merone et al. (22) was followed. The clarified lysate was heated at 50°C for 15 min and centrifuged at 75,600 × g for 20 min to precipitate host proteins. The same procedure was repeated at 60°C and then 70°C. The remaining proteins were loaded onto a Source 15Q anion-exchange column (2 by 15 cm; GE Healthcare, Baie d'Urfe, QC, Canada) that had been equilibrated with 20 mM sodium HEPES (pH 8.0). The column was washed with 3 column volumes of the equilibration buffer and then with a linear gradient of NaCl from 0 to 1 M over 10 column volumes. Fractions containing paraoxonase activity were eluted at 0.2 M NaCl. Active fractions were combined, concentrated, and stored in 20 mM sodium HEPES-0.2 mM CoCl₂ (pH 8.0). As protein of high purity was already obtained at this step (see Fig. SA2 in the supplemental material), we modified the method published by Merone et al. (22) and separation by hydrophobic interactions was not performed.

Determination of protein concentration, purity, molecular mass, identity, and metal ion content.

Protein concentrations were determined by the Bradford assay (3), using bovine serum albumin as the standard. To determine purity and verify molecular mass, SDS-PAGE was performed, and the gels were stained with Coomassie blue according to established procedures (19). The BenchMark protein ladder (Invitrogen) containing proteins ranging from 10 to 220 kDa in mass was used for molecular mass markers. Gel slices for in-gel trypsin digestion were prepared according to the method described by Shevchenko et al. (25). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis on trypsin-digested protein was performed by the Mass Spectrometry Facility at the University of Guelph to confirm protein identity. Purified protein was treated with Chelex-100 and washed with distilled water which had been pretreated with Chelex-100 to remove traces of metal ions. Inductively coupled plasma mass spectroscopy (ICP-MS) analysis was performed by ALS Laboratory Group (Waterloo, ON, Canada).

SsoPox immobilization.

Twenty micrograms of SsoPox was immobilized onto circular discs (diameter, 6 mm) of a Disruptor grade 4601 (cellulose, unlaminated) nanoalumina functionalized membrane by taking advantage of inherent negative charges on the enzyme and the positively charged alumina nanofibers in the membrane. As a control, enzyme storage buffer (20 mM sodium HEPES, pH 8.0, 0.2 mM CoCl₂) was spotted. These membranes were then incubated at 4°C for 1 h to allow binding. Unbound protein was removed by washing membranes with 10 to 20 ml of distilled water three times with shaking for 5-min intervals. For quorum-quenching assays, membranes were sterilized by UV light for 5 min on each side. Enzyme solutions were sterilized by filtration through 0.45-μm-pore-size filters. All washes were performed under sterile conditions.

To quantify the amount of protein immobilized upon the membrane, two discs containing immobilized enzyme were placed in a 1.5-ml microcentrifuge tube and washed with 500 μl of distilled water, 2 M NaCl, or 0.2 M HCl for 15 min, and the protein concentrations of washes were determined by the Bradford assay (3). The amount of protein immobilized was calculated from the difference between the total amount of protein spotted and the amount of protein in the wash.

Enzyme assays.

Paraoxonase and methyl-paraoxonase activities were measured spectrophotometrically by determination of p-nitrophenolate formation at 405 nm (ɛ = 16.5 OD mM⁻¹ cm⁻¹) or 450 nm (ɛ = 3.3 OD mM⁻¹ cm⁻¹) using a Varian Cary 100 Bio UV-visible spectrophotometer equipped with a thermojacketed cuvette holder at 25°C, unless otherwise stated. All assays were performed at least in duplicate in a total volume of 1 ml containing 20 mM Tris-HCl, 0.02 mM CoCl₂, and 10% acetonitrile (pH 8.0). Assays were performed at pH 8.0, which is the pH optimum for this reaction, as shown by Merone et al. (22). Paraoxon and methyl-paraoxon concentrations used in the kinetic assays were between 0.1× K_m and 3× K_m. Lactonase activity was measured using a pH indicator assay similar to the procedure described by Afriat and coworkers (1). The product of AHL cleavage is a carboxylic acid which reacts with the pH indicator m-cresol purple to induce a change in absorbance. Extinction coefficients of m-cresol purple were determined by titration with acetic acid. For determination of kinetic parameters, the lactonase reaction was measured at 560 nm (ɛ = 2.41 cm⁻¹ mM⁻¹) using a BMG FLUOstar Optima microplate reader at 25°C in 96-well plates. Reaction mixtures contained 2 mM Tris-HCl, 160 mM NaCl, 0.3 mM m-cresol purple, 0.02 mM CoCl₂, 2% (vol/vol) dimethyl sulfoxide (pH 8.0), and N-acyl homoserine lactone substrates at concentrations between 0.4× K_m and 3× K_m. Linear regression was used to determine the reaction velocity for each substrate concentration. Data were fitted by nonlinear regression to the Michaelis-Menten equation using the program LEONORA (6).

Since the degradation of paraoxon produces a yellow product, p-nitrophenolate, the paraoxonase reaction can be used to directly evaluate the efficiency of SsoPox immobilization on membranes. Comparison of the specific activities of immobilized and free SsoPox was performed by adding 10 to 20 μg of enzyme onto Disruptor membranes (immobilized enzyme) and Whatman paper (free enzyme). Twenty microliters of 1 mM paraoxon solution was spotted onto the membrane, and then the membrane was incubated at 45°C for 15 min. An image was obtained every minute by scanning. The amount of yellow p-nitrophenolate produced was determined by analyzing the image using ImageJ software (5). Color intensity was calibrated against a standard curve with known concentrations of pure p-nitrophenolate. The stability of SsoPox after immobilization on membranes when they were stored dry or in buffer was determined by a method similar to that described above over a period of 23 days. Images were obtained at the assay endpoint using a scanner.

Evaluation of SsoPox-immobilized membranes on growth and quorum sensing in P. aeruginosa.

P. aeruginosa PAO1 cultures were grown in ABt medium [15 mM (NH₄)₂SO₄, 15 mM Na₂HPO₄·2H₂O, 10 mM KH₂PO₄, 25 mM NaCl, 0.9 mM MgCl₂, 0.09 mM CaCl₂, 0.009 mM FeCl₂, 2.25 mg/liter thiamine, 0.5% (wt/vol) glucose, 0.5% (wt/vol) Casamino Acids] containing sterile SsoPox-immobilized membrane. At 6, 18, 26, and 48 h, bacterial growth was monitored by measuring the optical density at 600 nm. The rest of the culture was collected in microcentrifuge tubes and centrifuged at 17,000 × g to sediment the bacterial cells. The supernatant was used for protease assays and determination of pyocyanin levels.

One milliliter of the elastase reaction mixtures was composed of 20 mg of elastin-Congo red, 0.1 M Tris-HCl (pH 7.5), 1 mM CaCl₂, and 100 μl of culture supernatant (24). The mixtures were incubated at 37°C for 3 h, rotating continuously. After the incubation, the samples were centrifuged at 17,000 × g for 10 min to sediment the insoluble substrate. The absorbance of the supernatant was measured at 495 nm. Azocasein degradation of culture supernatants was measured in reaction mixtures with a final volume of 1 ml composed of 0.33% (wt/vol) azocasein, 0.1 M Tris-HCl (pH 7.5), 1 mM CaCl₂, and 100 μl of culture supernatant (24). The reaction mixture was incubated at 37°C for 30 min, and then the reaction was terminated by adding 500 μl of 10% (wt/vol) trichloroacetic acid. The absorbance of the supernatant was measured at 400 nm spectrophotometrically.

Pyocyanin was extracted from 1 ml of culture supernatant by chloroform extraction, followed by acidification. In brief, 1 ml of culture supernatant was added to 500 μl of chloroform and inverted 10 times to mix. The sample was centrifuged at 17,000 × g for 5 min to separate the organic phase from the aqueous phase. The aqueous phase was discarded, and 1 ml of 0.2 M HCl was added to acidify the pyocyanin within the organic phase (12). This sample was inverted 10 times to mix and was then centrifuged at 17,000 × g for 5 min before the absorbance was read at 520 nm (ɛ = 2.46 × 10⁶ cm⁻¹ M⁻¹) (2).

RESULTS AND DISCUSSION

Expression and purification of SsoPox.

The native and codon-optimized genes encoding SsoPox were subcloned into the broad-host-range expression plasmid pVLT-31 and transformed into P. putida KT2442 for protein production. Expression of metalloenzymes in P. putida, including 2,3-dihydroxybiphenyl dioxygenase and phosphotriesterase from Burkholderia xenovorans LB400 and Pseudomonas diminuta, respectively, has been documented previously (9, 15). Since improved expression levels had been observed for the former enzymes by using P. putida, we hypothesized that SsoPox expression may also work well in this host. We found that both the native and codon-optimized constructs expressed well and yielded soluble SsoPox. More soluble protein was obtained in this host than in E. coli (see Fig. SA3 in the supplemental material), which enabled the purification of the protein using heat treatment and only one chromatographic step. The yield of purified SsoPox was 9.4 mg per liter of culture (3.6 mg of protein per gram of cells), with a typical purification and its results being illustrated in Table SA1 and Fig. SA2 in the supplemental material. The molecular mass of the purified protein estimated by SDS-PAGE is 34 kDa, which is in agreement with the predicted molecular mass calculated from the amino acid sequence. The identity of the purified protein was further verified by analysis of trypsin-digested protein fragments identified by MALDI-TOF mass spectrometry.

Substrate specificity.

The substrate specificity of SsoPox was determined by steady-state kinetics. k_cat and K_m values for paraoxon at 70°C for our enzyme preparations were 2.29 s⁻¹ and 2.36 mM, respectively, whereas k_cat and K_m values of 0.24 s⁻¹ and 0.060 mM, respectively, were previously reported for the enzyme purified from E. coli (22). This discrepancy may be attributed to differences in metal ion cofactors in the active site of the enzyme. Metal ion analysis was done by ICP-MS, which showed 2.2 Co²⁺ ions per active site in the enzyme that we purified from recombinant P. putida. In contrast, one Co²⁺ ion and one Fe³⁺ ion were present in the active site of the SsoPox purified from E. coli, despite that fact that only Co²⁺ was added to the culture medium (11). This difference may have arisen from the host organism used to express SsoPox, as it was previously suggested that P. putida is more efficient at metal incorporation than E. coli (9).

Assays for AHL hydrolysis were performed at 25°C instead of 70°C for several reasons. First, AHLs are heat labile; in particular, AHLs with short acyl chains or ketone substituents are most susceptible to lactonolysis at elevated temperatures (28). Second, our aim is to evaluate the effectiveness of the enzyme at a temperature conducive for growth of mesophilic bacteria that produce AHLs, and lastly, kinetic parameters obtained can be directly compared with those for other homologous enzymes that have been determined at 25°C (4). SsoPox can hydrolyze a number of AHLs with different acyl chain lengths and substitutions, with the highest specificity constant obtained for 3-oxo-C₁₀-l-HSL (Table 1). This is mainly due to a low K_m value for this substrate. Specificity constants for AHLs with short acyl chains (C₄-HSL and 3-oxo-C₆-HSL) are generally lower than those for AHLs with longer acyl chains, mainly due to higher K_m values for these substrates.

TABLE 1.

Steady-state kinetic parameters of SsoPox with AHL substrates containing different acyl substituents at 25°C

Substrate	Km (mM)	kcat (s−1)	kcat/Km (M−1·s−1)
C4-dl-HSLa	NDb	ND	18 ± 7
3-oxo-C6-l-HSL	5.6 ± 0.9	0.52 ± 0.05	92 ± 5
3-oxo-C8-l-HSL	0.16 ± 0.03	1.0 ± 0.4	(6.6 ± 1) × 103
C8-dl-HSLb	0.093 ± 0.027	2.0 ± 0.3	(2.1 ± 0.3) × 105
3-oxo-C10-l-HSL	0.050 ± 0.0001	1.5 ± 0.2	(2.9 ± 0.5) × 105
3-oxo-C12-l-HSL	0.17 ± 0.002	0.95 ± 0.03	(5.5 ± 0.1) × 103

^aDue to low substrate solubility and a high apparent K_m for this substrate (>50 mM), the specificity constant can only be estimated from the gradient of the specific activity-versus-substrate concentration graph.

^bND, not determined.

This observation is in accordance with the characteristics of the substrate binding site in the crystal structure of SsoPox (11). The substrate binding site consists of a long pocket lined with hydrophobic residues which accommodates the long acyl chain in the inhibitor decanoyl homoserine thiolactone (11). The favorable interactions between the hydrophobic residues in the binding site and the long acyl chain in AHLs likely contribute to the higher level of activity observed in the substrates tested. Interestingly, other characterized members of the phosphotriesterase-like lactonase family have substrate specificities that are distinct from those of SsoPox. For instance, the homologous enzyme in Mycobacterium avium K-10 (38% sequence identity and 61% sequence similarity to SsoPox) exhibits a preference for AHL substrates with longer acyl chains. As the acyl chain length of the AHL substrate increases from 7 to 12, the specificity constants for the M. avium enzyme increases due to a decrease in K_m (4). However, unlike SsoPox, the M. avium enzyme has no detectable activity with C₄-HSL and 3-oxo-C₆-HSL (4). On the other hand, the homologous enzymes from Rhodococcus erythropolis and Mycobacterium tuberculosis (39% and 40% sequence identities to SsoPox, respectively) are more active toward C₄-HSL and 3-oxo-C₈-HSL. These two enzymes display specificity constants on the same order of magnitude of those for C₄-HSL and oxo-C₈-HSL, at 10⁵ and 10⁶ M⁻¹ s⁻¹, respectively, but the specificity constant for C₁₀-HSL is at least 3-fold lower than the constants for C₄-HSL and N-3-oxo-C₈-HSL (1).

Immobilization of SsoPox on nanoalumina membranes.

Positively charged nanoalumina membranes have been used to bind viruses from water samples due to their electrostatic interactions with negatively charged virus particles and in water purification systems (14, 16, 21). Since SsoPox has an estimated pI of 6.1, giving the protein an overall negative charge at pH 8.0, we predicted that it would also be able to interact with the positively charged nanoalumina membranes. Five microliters of 4 μg/μl SsoPox was manually spotted on membranes containing positively charged alumina nanofibers, and then the secondary activity of the enzyme toward the organophosphate paraoxon was used to determine immobilization efficiency and enzyme stability by colorimetric measurement of the yellow p-nitrophenolate product. When the enzyme was exposed to 12.5 nmol of paraoxon, formation of p-nitrophenolate was observed after incubation for 15 min at 45°C, indicating that enzyme was successfully retained on the membrane. To determine immobilization efficiency, the membrane was washed with water after enzyme was spotted, but no protein could be detected in the washes by the Bradford protein concentration assay (3). From the sensitivity of this assay, immobilization efficiency was estimated to be at least 95%. Moreover, no paraoxonase activity could be detected from the washes. These data show that the enzyme is tightly bound to the membrane and cannot be eluted by repeated wash with water or high-ionic-strength solutions (2 M NaCl). Membranes were also incubated in ABt medium with shaking at 37°C over 24 h. There was only a 3% decrease in color intensity on the membranes upon assaying for paraoxonase activity, which indicates that little or no enzyme leached into solution.

Specific activities of immobilized and free SsoPox were 1.69 U/mg and 6.80 U/mg, respectively, which indicates that 25% of the activity is retained upon immobilization. When tested with 1 mM paraoxon, the membranes produced at least 90% of their original color intensity after 23 days of storage in water or 20 mM sodium HEPES buffer (pH 8.0). Storage under dry conditions was less favorable for the stability of the enzyme, as the amount of p-nitrophenolate produced from 1 mM paraoxon was reduced to 60 to 75% of the initial amount after 23 days.

Evaluation of immobilized SsoPox for quorum quenching.

As SsoPox can degrade a broad range of AHLs, we hypothesized that it would be effective for disrupting quorum sensing in P. aeruginosa, which uses both C₄-HSL and 3-oxo-C₁₂-HSL for quorum sensing. Exogenous pyocyanin production is regulated by the C₄-HSL-controlled quorum-sensing system, whereas elastase and casein-hydrolyzing activities are regulated by the oxo-C₁₂-HSL-controlled quorum-sensing system. Therefore, to determine if SsoPox-immobilized papers can act as quorum quenchers, we examined the effect of SsoPox on the production of exogenous protease and pyocyanin synthesis in P. aeruginosa PAO1 cultures. For the negative control, nanoalumina membranes were spotted with enzyme storage buffer instead of SsoPox. These membranes were stored and treated in the same way as the membranes that contain immobilized SsoPox. As seen in Fig. 1A, immobilized SsoPox had little to no effect on bacterial growth in late stationary phase. From 0 to 6 h, no elastase or casein-hydrolyzing activity was detected in any of the treatments. From 18 to 48 h, the amounts of elastin and casein hydrolyzed by culture supernatant were lower in the cultures treated with immobilized SsoPox (P < 0.04 and P < 0.02, respectively). At 18 h, no elastase activity was detected in the cultures treated with immobilized enzyme (Fig. 1B). At 26 and 48 h, elastase activity was reduced by at least 5-fold for both the 40-μg and 80-μg enzyme treatments. For casein-hydrolyzing activity, cultures treated with immobilized enzyme displayed a decrease in activity of at least 10-fold from 18 to 48 h (Fig. 1C). For the same time points, levels of pyocyanin were at least 8-fold and 11-fold lower than the level for the control when 40 μg and 80 μg of protein were spotted, respectively (P < 0.04) (Fig. 1D). These results indicated that SsoPox immobilized on nanoalumina membranes can indeed attenuate the production of P. aeruginosa quorum-sensing-associated virulence factors. To our knowledge, this is the first report of quorum sensing disruption using an immobilized lactonase.

FIG. 1.

Effect of immobilized SsoPox on growth and quorum sensing in P. aeruginosa PAO1. Cultures were incubated with Disruptor discs containing 20 mM sodium HEPES (pH 8.0)-0.2 mM CoCl₂, 40 μg of SsoPox, or 80 μg of SsoPox. Error bars indicate standard deviations of at least three replicates for panels A to C and at least two replicates for panel D. (A) SsoPox-immobilized membranes had little effect on bacterial growth. Culture supernatants were tested for elastase activity (B), casein-hydrolyzing activity (C), and pyocyanin levels (D). No elastase or casein-hydrolyzing activity was detected in the culture supernatant at 0 and 6 h. Significant differences in protease activities and pyocyanin levels were found for cultures incubated with SsoPox-immobilized membranes in late stationary phase.

Since AHL signals are host specific, enzymes which are active toward a broad spectrum of AHLs, like SsoPox, would be useful for disrupting quorum sensing in more bacterial species. They would also be more effective on bacteria that harbor quorum-sensing systems which are activated by different AHLs, such as P. aeruginosa. The ability to hydrolyze multiple signals is especially important in species where there is cross talk between quorum-sensing systems, as one system can compensate for the absence of the other (8).

Previously, Kato et al. have successfully attenuated quorum sensing in P. aeruginosa by using cellulose ether gel matrices immobilized with 2-hydroxypropyl-β-cyclodextrin to sequester AHLs through hydrophobic interactions (17, 18). In addition, quorum-quenching enzymes have been shown to reduce biofouling by bacteria in water treatment reactors (29). However, there have not been any reports of enzyme-mediated quorum quenching by solid supports to date. We have shown here that a lactonase can be immobilized onto nanoalumina membranes as solid supports simply by adsorption and then used for quorum quenching. Since the functionalized membranes can indeed disrupt quorum sensing successfully and positively charged nanoalumina membranes have been used in water purification systems, the use of filtration membranes containing immobilized lactonases may provide a possible means to control undesirable bacterial activities.