Attenuation of Vibrio fischeri Quorum Sensing ... - ACS Publications

Mar 15, 2010 - Hospital, NHS Trust, Kempston Road, Bedford, Bedfordshire, MK42 9DJ, United Kingdom, and School of. Biosciences, University of Kent, ...
0 downloads 0 Views 1MB Size
Download PDF
Biomacromolecules 2010, 11, 975–980

975

Attenuation of Vibrio fischeri Quorum Sensing Using Rationally Designed Polymers Elena V. Piletska,*,† Georgios Stavroulakis,† Kal Karim,† Michael J. Whitcombe,† Iva Chianella,† Anant Sharma,‡ Kevin E. Eboigbodin,§ Gary K. Robinson,§ and Sergey A. Piletsky† Cranfield Health, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, United Kingdom, Bedford Hospital, NHS Trust, Kempston Road, Bedford, Bedfordshire, MK42 9DJ, United Kingdom, and School of Biosciences, University of Kent, Canterbury, CT2 7NJ, United Kingdom Received December 21, 2009; Revised Manuscript Received March 2, 2010

A first attempt to attenuate the quorum sensing (QS) of a marine heterotroph microorganism, Vibrio fischeri, using signal molecule-sequestering polymers (SSPs) is presented. A set of rationally designed polymers with affinity toward a signal molecule of V. fischeri, N-(β-ketocaproyl)-L-homoserine lactone (3-oxo-C6-AHL) was produced. It is reported that computationally designed polymers could sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes (in this case, bioluminescence) from being up-regulated. It was proven that the attenuation of bioluminescence of V. fischeri was due to sequestration of the signal molecule by specific polymers and not due to the toxicity of polymer or nonspecific depletion of nutrients. The ability to disrupt the bacterial communication using easy to synthesize and chemically inert polymers could provide a new concept for the development of pharmaceuticals and susceptible device coatings such as catheters.

Introduction Throughout the long history of humanity, people have investigated the causes and treatment of the various bacterial diseases that have claimed millions of human lives. During the middle ages, the poor sanitation and overcrowded living conditions of European cities were responsible for the numerous outbreaks of epidemics such as bubonic plague, cholera, leprosy, and tuberculosis. It is surprising that, even now, in the 21st century, despite the wealth of accumulated knowledge on the nature and treatment of the various bacterial diseases, bacterial infections still pose a danger to people. For instance, cholera, which was eradicated for a century, again appeared in the countries of Africa and Latin America.1 Cases of tuberculosis are also on the rise.2 The discovery of penicillin as an antibacterial marked the start of some of the greatest advances in pharmaceutical development in the last century. A multitude of therapeutics have been developed for the control of pathogenic and spoilage organisms in a range of application domains, for example, infection control, food preservation, and surface fouling/biofilm formation. Unfortunately, the essential cellular processes that have previously been targeted in antibacterial discovery (e.g., replication, transcription, and translation) are required for growth and therefore impose high selective pressure to develop resistance. The latter has resulted in an increased incidence of multidrug resistance and a reduction in the antibacterial arsenal that is available for treatment.3,4 As resistance to antibiotics has developed and it has become increasingly difficult to treat certain bacterial infections, the interest in controlling bacterial infections through alternative approaches is growing. The question is how to fight bacterial infections without inducing mutagenesis and * To whom correspondence should be addressed. Tel.: +44(0)1234 758325. E-mail: [email protected]. † Cranfield University. ‡ Bedford Hospital. § University of Kent.

resistance. One of the alternative antibacterial approaches was advocated by Hentzer.5 Hentzer envisaged that a bacterial communication system that regulates bacterial virulence could be used for the control of infectious bacteria without affecting the growth and inducing the bacterial mutagenesis. It is known that the majority of Gram-negative and Grampositive bacteria communicate via production and sensing of small signal molecules or autoinducers.6 Certain phenotypic traits, particularly virulence factors and production of biofilms, are expressed in a population density-dependent manner. The ability of bacteria to sense the population density is termed as quorum sensing (QS). Due to its relation to virulence factor production, antibiotic sensitivity, and biofilm development QS could be considered as a potential therapeutic target.7 The development of potential therapeutics for the control of QS currently includes the production of vaccines and antagonists of the sensing molecules.8-10 The obvious problem with the use of vaccines lies in their high price and the risk of induction of the immune response after reinfection, which limits the scope of their application. Antagonists such as halogenated furanones are usually toxic compounds and could not be considered for applications in medical practice. Another promising approach involves chemical inactivation of the signal molecules. Unfortunately, its objectives, such as the development of catalytic antibodies or enzymes for controlling QS through selective hydrolysis or lactonolysis of the signaling molecules, are still far from completion and only offer a practical solution if they can be engineered into a host organism or plant.11,12 We are developing a new solution for attenuation of QS, which involves selective sequestering of the signal molecules using computationally designed synthetic polymers. The idea is that, if the signal molecule is adsorbed by the polymer, it will be removed from the system, leading to disruption of the communication between the bacteria and preventing the quorum sensing threshold being reached. Consequently, expression of virulence genes could be either reduced or prevented. In contrast

10.1021/bm901451j  2010 American Chemical Society Published on Web 03/15/2010

976

Biomacromolecules, Vol. 11, No. 4, 2010

to traditional antibiotic therapy, treatment with signal sequestering polymers (SSPs) is less likely to induce resistance in the exposed bacterial population, because suppression of QS is nonlethal. It is also less likely that polymers will produce undesirable side effects compared to drugs. Similar cross-linked polymers are already widely used as dental resins. To get the approval for dental application, polymerized methacrylate resins successfully passed the stringent tests against any neurotoxic, carcinogenic, or allergic activity.13 Polymeric materials capable of controlling/attenuating the expression of virulence determinants and biofilm formation would have tremendous advantages over antibodies in a number of applications, especially surface applications. Unlike conventional antibodies the SSPs can be synthesized in bulk, using easily available substrates and technologies. SSPs can be fabricated in any form, including films and injection moldings, offering the chance to attenuate QSregulated infections by coating surfaces that are susceptible to contamination and biofilm accumulation, for example, catheters, prosthetics, and so on. A further possibility would be to combine SSPs with traditional antibiotics in order to reduce their dosage and minimize possible side effects. This can be particularly attractive for pharmaceutical companies that can expand their range of drug candidates, currently limited to less active agents. The Gram-negative, marine heterotroph bacterium, Vibrio fischeri, was selected as the model system for this study. In the V. fischeri quorum sensing system, the cognate signal molecule is 3-oxo-C6-AHL and it controls the synthesis of luciferase and the development of bioluminescence. The bioluminescence characteristic for V. fischeri is a sensitive marker for quorum sensing bacterial communication.14

Materials and Methods Materials. The wild strain of V. fischeri ATCC 7744 was used throughout the study. N-(β-ketocapryloyl)-DL-homoserine lactone (3oxo-C6-AHL) and phosphate-buffered saline (PBS) was purchased from Sigma (Sigma-Aldrich, Gillingham, U.K.). Acrylamide (AA), N,N′methylene bisacrylamide (MBAA), itaconic acid (IA), methacrylic acid (MAA), N,N-diethylaminoethyl methacrylate (DEAEM), ethylene glycol methacrylate phosphate (EGMP), dimethylformamide (DMF), and sodium chloride (NaCl) were purchased from Aldrich (Sigma-Aldrich, Gillingham, U.K.). Methanol was obtained from Fisher, U.K. Nutrient broth No. 2 (NB) and Agar Bacteriological (Agar No.1) were purchased from Oxoid (Basingstoke, U.K.). Computational Modeling. The molecular modeling was made using a workstation from Research Machines running the CentOS 5 GNU/ Linux operating system. This system was used to execute the software packages SYBYL 7.0 (Tripos Inc., St. Louis, Missouri). The LEAPFROG algorithm was used to screen the library of functional monomers for their possible interactions with the template, resulting in a table, ranking the monomers with the highest binding score (kcal mol-1) as the best candidates for polymer preparation. The library contained 20 functional monomers commonly used in molecular imprinting that possess polymerizable residues and residues able to interact with a template through ionic and hydrogen bonds, van der Waals’, and dipole-dipole interactions.15 The charges for each atom were calculated and the structures of the monomers were refined using molecular mechanical methods. Energy minimization was performed on each of the monomers in the database to a value of 0.01 kcal mol-1. Polymer Preparation. Based on the computational modeling six functional monomers were selected for the preparation of SSPs: MBAA, DEAEM, IA, MAA, EGMP, and AA. Six polymers were prepared with high concentration (20 or 30%) of the functional monomers (P1-P6), two polymers (P7 and P8) were prepared with reduced concentration (5%) of the functional monomers, and one polymer (P9) was prepared without functional monomer. The composition of polymers is shown in the Table

Piletska et al. 1S (Supporting Information). All polymers were prepared by thermal polymerization at 80 °C for 12 h. After synthesis, polymers were ground, washed with methanol, and sieved. The fraction with particles in the size range 38-125 µm was collected. Polymers were thoroughly washed in methanol using Soxhlet extraction, dried, and used for SPE screening. The surface area of the polymers was characterized using Nova 1000e Series High Speed Gas Sorption Analyzer (Quantachrome, U.K.). Screening of the Polymers against of 3-Oxo-C6-AHL. A total of 100 mg of each polymer (P1-P6) was packed in empty 1 mL capacity SPE cartridges between two polyethylene frits. To perform the screening of the polymers under physiological conditions, 3-oxo-C6-AHL was dissolved in PBS, pH 7.4. Cartridges were conditioned with 2 mL of water and loaded with 1 mL of 3-oxo-C6-AHL solution in PBS (1 mg mL-1). The filtrate was collected and evaluated using a spectrophotometer by determining the absorbance at 211 nm. Quantification of 3-Oxo-C6-AHL Using HPLC-MS. To increase the sensitivity of the quantification of 3-oxo-C6-AHL the HPLC-MS method was optimized. HPLC separation was conducted using a Waters 2975 HPLC system equipped with Luna C18 (2) column (150 × 3 mm, 3 µm, Phenomenex). Mobile phase: water (A) and methanol (B) in a binary system with 0.1% of formic acid as an additive. The gradient elution conditions were as follows: 0-10 min, 100% solution A (water/ 0.1% formic acid), then a linear gradient to from 0 to 70% B over 20 min, immediately switching to 100% A for a further 10 min to wash the column before the next injection (total analysis time, 40 min). To minimize the transfer of salt to the mass spectrometer, measurement of the sample was performed between the 25 to 30 min of the run, at all other times liquid was diverted to waste using the “solvent delay” option. The flow rate was 0.2 µL min-1; the injection volume, 10 µL; the column temperature, +20 °C. Fragment of AHL m/z 102 was detected by mass spectrophotometer Micromass Quattro Micro (Waters, U.K.) equipped with an ESI interface in positive ion mode.16 The MS parameters were following: desolvation gas, 850 L h-1; cone gas- 50 L h-1, capillary- 4.5 kV, cone-25 V, CE- 20, source temperature- +120 °C, desolvation temperature, +350 °C; collision energy, 25 V; multiplier, 650 V. Determination of Binding Capacity of Itaconic Acid-Based Polymers. The polymers that contained 30% of IA (P6), 5% of IA (P7), and without IA (P9) correspondingly were tested for adsorption of 3-oxo-C6-AHL from PBS, pH 7.4, and from nutrient broth containing 2% NaCl (NB/NaCl). A total of 100 mg of each polymer was packed into 1 mL SPE cartridge. 3-Oxo-C6-AHL (1.5 mg mL-1) was dissolved in PBS or in NB/NaCl and filtered through the polymer in 1 mL aliquots. To determine the polymer capacity, the concentration of 3-oxoC6-AHL in each fraction was quantified using HPLC-MS. The volume from which more than 50% of 3-oxo-C6-AHL was adsorbed was considered as a “breakthrough” volume and used to evaluate the capacity of the resin. The adsorbed AHL was then eluted with 2 × 1 mL of acetonitrile and quantified using HPLC-MS. Effect of the Polymer on Growth and Bioluminescence of V. fischeri. A single colony of V. fischeri ATCC 7744 was inoculated into NB/NaCl and incubated overnight with orbital shaking (Yellowline OS 2 basic shaker, Fisher) at 200 rpm at 25 °C. A 0.25 mL aliquot of an overnight cell culture was added to 10 mL of NB/NaCl. Polymer (200 mg of the P7 or P8 polymer) was also added where required. All cultures were incubated at 25 °C in 15 mL Falcon tubes with constant inversion. The growth of bacteria and the effect of polymers on growth were determined during 10 h by optical density measurements at 600 nm after 10-fold dilution with NB/NaCl, using a spectrophotometer (UVPC 2100, Shimadzu, Japan). The development of bioluminescence by the bacterial suspension was studied in the presence and absence of the P7 and P8 polymers. The bioluminescence was measured every 2.5 h using a HY-LiTE 2 luminometer (Merck, Darmstadt, Germany). For the measurement, all samples incubated for 2.5 h were diluted 10-fold with NB/NaCl and the bioluminescence was measured. The samples that were incubated for 5, 7.5, and 10 h were diluted 100-fold with NB/NaCl.

Attenuation of Vibrio fischeri Quorum Sensing

Biomacromolecules, Vol. 11, No. 4, 2010

977

Table 1. Computational Screening of Functional Monomers and SPE Screening of the Polymers Prepared with Corresponding Monomers (in the Brackets are the Names of the Corresponding Polymers)

Figure 1. (a) 3D molecular structure of 3-oxo-C6-AHL, which was computationally minimized in vacuum; (b) 2D molecular structure of 3-oxo-C6-AHL.

To prove that it was specifically the 3-oxo-C6-AHL that was adsorbed from the cell culture and, therefore, unavailable to up-regulate bacterial luminescence, 100 mg of the P7 polymer was recovered from the cell culture and was extracted with acetonitrile. The polymer was packed in an SPE tube, washed with 3 mL of distilled water, and dried on a vacuum manifold. The elution was carried out with 1 mL of acetonitrile. The sample was filtered through a 0.22 µm nylon filter (Phenomenex, U.K.) and analyzed using HPLC-MS. Biofilm Study. A single colony of V. fischeri ATCC 7744 was inoculated into NB/NaCl and was incubated for 24 h at 25 °C with reciprocal shaking at 200 rpm. The inoculum was diluted 100 times with NB/NaCl, and, where required, 100 µM of 3-oxo-C6-AHL in PBS was added. Portions of the diluted cell suspension (200 µL; either with or without 3-oxo-C6-AHL) were added to each microtiter plate well that contained, where required, 4 ( 0.1 mg of sterile P7 polymer. The colorimetric assay of biofilms with crystal violet staining was performed as described by Burton et al.17 After 22 h of incubation without shaking, the media was gently removed and the microtiter plate wells were washed three times with 200 µL of PBS buffer (0.1 M, pH 7.4) using a multichannel pipet, and the plate was allowed to dry for 15 min. The biofilm on the microtiter plate wells was stained with 200 µL of a 0.4% solution of crystal violet for 15 min. Then unbound crystal violet stain was removed, and the wells were washed gently three times with PBS buffer. The wells were air-dried for 15 min, and the crystal violet in each well was solubilized by adding 200 µL of 95% ethanol. The plate was read at 600 nm using a microtiter plate reader from Dynex (Dynex Technologies, U.S.A.).

Results and Discussion Analysis of the Molecular Structure of 3-Oxo-C6-AHL. The primary structural features that can be identified in the 3-oxo-C6-AHL molecule include the lactone functionality, the secondary amide group, and the β-dicarbonyl function (Figure 1). β-Keto amide and (β-dicarbonyl) functional elements are rather rare in the literature describing the synthesis of affinity polymers, although isolated amide groups and to some extent keto-groups are common. The consensus is that acidic monomers (TFMAA or MAA)18 are suitable for binding to keto-functional groups, but the interactions tend to be weak. Both acidic monomers (MAA)19 and neutral amides (acrylamide)20 are proposed in the literature for structures interacting with secondary amide groups, but two-center bonds can easily be disrupted by conformational effects in the template. Acrylamide has also been used with lactone templates, as the fixed geometry allows two-centered H-bonding involving the -NH2 function to be easily established.21,22 Protonated acidic monomers, such as MAA and related compounds, are also H-bond donors and can coordinate to either (or both) of the oxygen atoms of lactone. Acrylamide is a common monomer for many of the structural features of 3-oxo-C6-AHL, however, safety issues may preclude its use in this application. Acidic monomers such as MAA may

monomers

binding energy, kcal/mol

adsorption, %

MBAA (P1) AA (P2) DEAEM (P3) EGMP (P4) MAA (P5) IA (P6)

-28.23 -27.32 -26.83 -25.64 -15.33 -25.44

87 ( 4 88 ( 1 73 ( 2 15 ( 2 80 ( 5 79 ( 1

Figure 2. Complex of 3-oxo-C6-AHL with acrylamide (a) and IA (b).

also prove useful, however, conformational features of AHL will determine which monomer is most appropriate. Computational Screening. A molecular model of 3-oxoC6-AHL was designed and minimized to a value of 0.001 kcal mol-1 (Figure 1). Then in silico screening of a library of functional monomers against 3-oxo-C6-AHL was performed as described earlier.15 The monomers that demonstrated the highest binding scores represented the best candidates for polymer preparation, forming the strongest complexes with the target. There is experimental evidence that calculated binding energy correlates with adsorption properties of synthesized polymers toward the modeled compound of interest.15,23 The top six monomers with the highest binding energies were identified as acrylamide, MBAA, DEAEM, IA, EGMP, and MAA (Table 1) and they were selected for polymer preparation. It was necessary to highlight that acrylamide, MBAA, and IA monomers formed two-point interactions with 3-oxo-C6AHL and therefore were expected to demonstrate stronger interactions and higher affinity (Figure 2) than DEAEM and EGMP, which showed only a single-point interaction during molecular modeling. The rational selection of the monomers was also influenced by the site of interaction with 3-oxo-C6AHL. To increase the specificity of the binding for 3-oxo-C6AHL the preference was made for the monomers which could interact with functional groups close to the lactone ring. IA represented a good example of such a monomer (Figure 2) and was selected for further testing. In Vitro Screening of the Polymers for Adsorption of 3-Oxo-C6-AHL. The P1-P6 polymers were polymerized as described in the Materials and Methods and their physical properties were characterized. The average surface area of all tested polymers was 255 ( 25 m2 per g and average pore size was 43 ( 3 Å. Then P1-P6 polymers were packed into the 1 mL SPE cartridges. The 3-oxo-C6-AHL solution in PBS was filtered through the polymer cartridges and the concentration of 3-oxo-C6-AHL in the filtrate was measured. It was found that AA, MBAA, MAA, and IA-based polymers demonstrated the highest retention of 3-oxo-C6-AHL (88, 87, 80, and 79%, correspondingly, Table 1). Both screenings, in silico and by filtration, were in good agreement with each other and with literature references discussed earlier. Due to the perceived high

978

Biomacromolecules, Vol. 11, No. 4, 2010

toxicity of acrylamide and of acrylamide derivatives, such as MBAA, which could preclude polymers containing these monomers from some potential applications, they were not considered for further development, and IA-based polymers were therefore selected for the continuation of the testing. Determination of Binding Capacity of Itaconic Acid-Based Polymers. Three polymers containing different quantity of IA (P6, P7, and P9) were tested for adsorption of 3-oxo-C6-AHL from PBS and NB/NaCl. NB/NaCl was selected for testing because it was used as the growth medium for V. fischeri. It was necessary to evaluate if such rich media as NB would hinder the binding of 3-oxo-C6-AHL to the polymer. The polymer made without functional monomer (P9) was used as a reference polymer to estimate the specificity of binding. The capacity of the polymers was also evaluated accordingly to their surface area. The P7 polymer based on 5% IA demonstrated the highest binding capacity per surface area (∼150 µg m-2) in comparison with P6 polymer, which contained 30% IA (binding capacity, 122 µg m-2), and P9 polymer, which was made without functional monomer (binding capacity, 105 µg m-2; Supporting Information, Table 2S). It was observed that the adsorption of 3-oxo-C6-AHL from NB/ NaCl was indeed reduced in comparison with adsorption from PBS. The decrease in polymer binding capacity toward 3-oxoC6-AHL could be explained by higher nonspecific adsorption of other components of the nutrient broth by the polymer. The P9 polymer which was prepared without functional monomer was affected the most by nonspecific adsorption. Its binding capacity toward 3-oxo-C6-AHL in NB/NaCl was reduced by 24% in comparison with adsorption from PBS. This polymer demonstrated very high nonspecific adsorption of hydrophobic components of the nutrient broth, which led to a reduction of the polymer capacity and prevention of the binding of 3-oxoC6-AHL. At the same time, the P7 polymer (5% IA) adsorbed as much 3-oxo-C6-AHL in NB/NaCl as in PBS and it was selected for the further testing. Effect of the Polymer on Growth and Bioluminescence of V. fischeri. The effect of the P7 (5% IA) and P8 (5% MAA) polymers on the growth of V. fischeri was tested by measuring the optical density of the suspension over a 10 h period. It was found that none of the polymers inhibited the growth of V. fischeri (Figure 3), indicating that the polymers were not toxic nor were they depleting nutrients from the media. In parallel, each sample was analyzed on development of the bioluminescence, a phenotype of V. fischeri, which is controlled by quorum sensing. The bioluminescence of the bacterial suspensions was monitored in the presence and absence of polymers. Figure 4 shows that the significant development of the bioluminescence in the control bacterial suspension, which was growing without addition of the polymers, took place between 5 and 7.5 h. At the same time, the bioluminescence of the samples that contained polymers was substantially reduced (Figure 4). The V. fischeri culture that was incubated with either the P7 or P8 polymer for 10 h showed marked reductions in bioluminescence equal to 85 ( 8 and 84 ( 6%, respectively. The P7 polymer, which was incubated with the bacterial suspension, was then extracted with acetonitrile and analyzed using HPLC-MS to give the spectrum in Figure 5. It was found that the P7 polymer had adsorbed 0.27 ( 0.02 µg of 3-oxoC6-AHL per g of polymer (12.6 nM), explaining the quenching of bioluminescence, which had been demonstrated in Figure 4. It correlates with a publication that reports that even as low as 10 nM of 3-oxo-C6-AHL, which is equivalent to 1 or 2

Piletska et al.

Figure 3. The effect of the IA- and MAA-based polymers on the growth of V. fischeri: P7, 5% IA-based polymer; P8, 5% MAA-based polymer; C, control without polymers. The experiments were made in duplicates with standard deviation under 5%.

Figure 4. The effect of the IA- and MAA-based polymers on the bioluminescence of V. fischeri: P7, 5% IA-based polymer; P8, 5% MAA-based polymer; C, control without polymers. All experiments were made in duplicates with standard deviation under 5%.

molecules per cell, is sufficient for induction of bioluminescence in V. fischeri.24 P7 polymer had a much lower effect on the bioluminescence of V. fischeri when structurally different signal molecule (3hydroxy-C4-AHL) was added (data not shown), which could suggest about the specificity of the polymer toward the 3-oxoC6-AHL. Biofilm Study. The effect of the polymers on biofilm formation by V. fischeri was studied. Because V. fischeri is a symbiotic bacterium that populates the light organs of squids, it is able to form biofilms, as has already been reported in the literature.25,26 Although the role of QS in biofilm formation by V. fischeri has not yet been thoroughly studied, it was interesting to find out whether this process could be influenced by the addition of 3-oxo-C6-AHL. It was shown that the external addition of the cognate signal molecule increased the amount of biofilm by 40% (Figure 6), which suggests that 3-oxo-C6AHL might play some part in helping to trigger biofilm formation in this microorganism. The addition of P7 polymer to the suspension reduced biofilm production by V. fischeri by

Attenuation of Vibrio fischeri Quorum Sensing

Biomacromolecules, Vol. 11, No. 4, 2010

979

Figure 5. HPLC-MS spectrum of pure 3-oxo-C6-AHL (a) and a spectrum of the compounds extracted from P7 polymer after its incubation with a bacterial suspension for 10 h (* 3-oxo-C6-AHL-related fragments) (b).

The successful example of the attenuation of QS-controlled phenotype of V. fischeri by selective sequestering of the signal molecules suggests that it would also be possible to develop SSPs for more problematic microorganisms that also possess QS-regulated virulence determinants, such as Pseudomonas aeruginosa or Staphylococcus aureus. Custom-made SSPs are a new tool for the control of life-threatening infections in a multitude of applications (e.g., catheter-associated infections), while minimizing the risk of inducing bacterial resistance.

Figure 6. Effect of the P7 polymer (“polymer”) on V. fischeri (V.f.) biofilm formation with and without (+ and -) exogenous 3-oxo-C6AHL (AHL). All standard deviations were based on six replicates.

56%, removing all effect of exogenous AHL (Figure 6). These results suggest that 5% IA polymer was effective in the sequestration of the signal molecules, which resulted in reduced biofilm formation by V. fischeri.

Conclusions The concept of QS attenuation was proven through in vitro tests, which showed that it is possible to produce polymers with affinity to the signal molecule of the bacterium V. fischeri, N-(βketocapryloyl)-L-homoserine lactone. It confirmed that removal of the signal molecule disrupted the communication between the bacterial cells leading to inhibition of the expression of a postquorate phenotype, that is, to reduction of bioluminescence in V. fischeri. At the same time, the polymer did not inhibit bacterial growth and in this aspect was superior to other nonspecific commercial adsorbents (such as activated charcoal), which inhibited bacterial growth through the nonspecific absorption of nutrients. Although an influence of 3-oxo-C6-AHL on V. fischeri biofilm formation was observed, a more detailed study of this phenomenon will be required.

Acknowledgment. Authors acknowledge with gratitude Dr. Sandy Primrose for useful discussion. G.S. would like to thank Bedford Hospital and British Council in Germany for financial support of his studentship. Supporting Information Available. Table with polymer compositions (Table 1S) and the information on the binding capacity of the polymers toward 3-oxo-C6-AHL in PBS and NB/NaCl (Table 2S). This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Gaffga, N. H.; Tauxe, R. V.; Mintz, E. D. Am. J. Trop. Med. Hyg. 2007, 77, 705–713. (2) Global tuberculosis control: surVeillance, planning, financing: WHO report, 2008; Available from http://www.who.int/tb/publications/ global_report/2008/pdf/fullreport.pdf. (3) Alanis, A. J. Arch. Med. Res. 2005, 36, 697–705. (4) Rossolini, G. M.; Mantengoli, E. Clin. Microbiol. Infect. 2005, 11, 17–32. (5) Hentzer, M.; Wu, H.; Andersen, J. B.; Riedel, K.; Rasmussen, T. B.; Bagge, N.; Kumar, N.; Schembri, M. A.; Song, Z.; Kristoffersen, P.; Manefield, M.; Costerton, J. W.; Molin, S.; Eberl, L.; Steinberg, P.; Kjelleberg, S.; Høiby, N.; Givskov, M. EMBO J. 2003, 22, 3803– 3815. (6) Camara, M.; Williams, P.; Hardman, A. Lancet Infect. Dis. 2002, 2, 667–676. (7) Smith, R. S.; Iglewski, B. H. J. Clin. InVest. 2003, 112, 1460–1465. (8) Park, J.; Jagasia, R.; Kaufmann, G. F.; Mathison, J. C.; Ruiz, D. I.; Moss, J. A.; Meijler, M. M.; Ulevitch, R. J.; Janda, K. D. Chem. Biol. 2007, 14, 1119–1127. (9) Suga, H.; Smith, K. M. Curr. Opin. Chem. Biol. 2003, 7, 586–591. (10) Rasmussen, T. B.; Givskov, M. Microbiology 2006, 152, 895–904.

980

Biomacromolecules, Vol. 11, No. 4, 2010

(11) Borchardt, S. A.; Allain, E. J.; Michels, J. J.; Stearns, G. W.; Kelly, R. F.; McCoy, W. F. Appl. EnViron. Microbiol. 2001, 67, 3174–3179. (12) Rasmussen, T. B.; Givskov, M. Int. J. Med. Microbiol. 2006, 296, 149–161. (13) Nie, J.; Lovell, L. G.; Bowman, C. N. Biomaterials 2001, 22, 535– 540. (14) Kuttler, C.; Hense, B. A. J. Theor. Biol. 2008, 251, 167–180. (15) Piletsky, S. A.; Karim, K.; Piletska, E. V.; Day, C. J.; Freebairn, K. W.; Legge, C.; Turner, A. P. F. Analyst 2001, 126, 1826–1830. (16) Morin, D.; Grasland, B.; Velle´e-Re´hel, K.; Dufau, C.; Haras, D. J. Chromatogr. A 2003, 1002, 79–92. (17) Burton, E.; Yakandawala, N.; LoVetri, K.; Madhyastha, M. S. J. Ind. Microb. Biotechnol. 2007, 34, 1–4. (18) Weiss, R.; Freudenschuss, M.; Krska, R.; Mizaikoff, B. Food Addit. Contam. 2003, 20, 386–395.

Piletska et al. (19) Lu, C. Y.; He, H. C.; He, X. W.; Zeng, X. S. Chin. J. Chem. 2004, 22, 275–278. (20) Lepisto¨, M.; Sellergren, B. J. Org. Chem. 1989, 54, 6010–6012. (21) Zhu, X. F.; Cao, Q.; Hou, N. B.; Wang, G. S.; Ding, Z. T. Anal. Chim. Acta 2006, 561, 171–177. (22) Hu, S. G.; Li, L.; He, X. W. J. Chromatogr. A 2005, 1062, 31–37. (23) Breton, F.; Rouillon, R.; Piletska, E. V.; Karim, K.; Guerreiro, A.; Chianella, I.; Piletsky, S. A. Biosens. Bioelectron. 2007, 22, 1948– 1954. (24) Kaplan, H. B.; Greenberg, E. P. J. Bacteriol. 1985, 163, 1210–1214. (25) Darnell, C. L.; Hussa, E. A.; Visick, K. L. J. Bacteriol. 2008, 190, 4941–4950. (26) Yildiz, F. H.; Visick, K. L. Trends Microbiol. 2009, 17, 109–118.

BM901451J