WO2011055987A2

WO2011055987A2 - Method for screening of quorum sensing inhibitors

Info

Publication number: WO2011055987A2
Application number: PCT/KR2010/007733
Authority: WO
Inventors: Hak-Sung Kim; Jin-Hyun Kim; Sang-Chul Lee; Young-Su Jeong
Original assignee: Korea Advanced Institute Of Science And Technology
Priority date: 2009-11-04
Filing date: 2010-11-04
Publication date: 2011-05-12
Also published as: WO2011055987A3; KR101141787B1; KR20110049193A

Abstract

The present invention relates to a vector comprising an expression cassette for screening quorum sensing inhibitors, which comprises a gene encoding a transcriptional regulator that recognizes and binds to acyl homoserine lactone, a gene encoding an inhibitor of antibiotic-degrading enzyme that is operably linked to a promoter regulated by the transcriptional regulator, and a gene encoding an antibiotic-degrading enzyme that is inhibited by the inhibitor of antibiotic-degrading enzyme, a method for screening a quorum sensing inhibitor and a quorum sensing inhibitor screened by the method.

Description

METHOD FOR SCREENING OF QUORUM SENSING INHIBITORS

The present invention relates to a vector comprising an expression cassette for screening quorum sensing inhibitors, and a method for screening quorum sensing inhibitors by use of a strain introduced with the vector. In particular, the present invention relates to a vector comprising an expression cassette for screening quorum sensing inhibitors, which comprises a gene encoding a transcriptional regulator that recognizes and binds to acyl homoserine lactone, a gene encoding an inhibitor of antibiotic-degrading enzyme that is operably linked to a promoter regulated by the transcriptional regulator, and a gene encoding an antibiotic-degrading enzyme that is inhibited by the inhibitor of antibiotic-degrading enzyme, a strain transformed with the vector, a method for screening a quorum sensing inhibitor by contacting the strain with quorum sensing inhibitor candidates, and a quorum sensing inhibitor screened by the method.

Quorum sensing is a population density-dependent mechanism for gene regulation, which allows bacterial cells to communicate with each other using their chemical language, thereby expressing specific genes only when they reach a certain cell density. A quorum sensing mechanism, a biological phenomenon whereby bacteria actively proliferate by accumulation of a low-molecular weight signal molecule such as an autoinducer or pheromone in the extracellular environment and reach a certain quorum to induce gene expression, refers to a bacterial cell-to-cell communication process using specific signal molecules. A quorum sensing mechanism is required to regulate numerous physiological activities needed for the bacteria to survive, such as protein expression, biofilm formation, migration, symbiosis, and cell population control. Quorum sensing is mediated by signal molecules and signal-binding transcriptional regulators for regulation of gene expression.

Quorum-sensing regulation in many Gram-negative bacteria involves acyl homoserine lactone (N-acyl homoserine lactone, AHL). Acyl homoserine lactone is synthesized by LuxI protein and perceived by LuxR protein, and functions as a transcription factor required for specific gene expression. Acyl homoserine lactones have various structures depending on hydrocarbon chain length that forms the acyl chain, and each species has differences in the length and form, and the carbon number of the acyl chain. In addition to acyl homoserine lactone, Gram-negative bacteria have been known to utilize 3-hydroxy-palmitic acid methyl ester and heptyl-hydroxy-quinolone, also called PQS (Pseudomonas quinolone signal), as an autoinducer (Int. J. Infect. Dis. 2004. 8, 81-95).

Infectious diseases, when they are caused by pathogens in mammalian individuals, are also the result of molecular mechanisms of quorum sensing, and representative pathogens that cause pathogenesis by regulating production of various factors via quorum sensing are exemplified by a respiratory pathogen in patients with cystic fibrosis, Pseudomonas aeruginosa or Burkholderia cepacia, an opportunistic mammalian pathogen, Serratia liquefaciens, a fish pathogen, Aeromonas hydrophila or Vibrio anguillarum, and a plant pathogen, Erwinia carotovora or Agrobacterium tumefaciens. Meanwhile, microorganisms present in foods, in particular, chilled or frozen foods, do not cause pathogenesis under normal conditions, but produce signal molecules in response to environmental changes, leading to pathogenesis. Bacteria present in a sample can be detected and quantified by analysis of the signal molecules in the sample. It has been reported that quorum sensing is also involved in the pathogenesis of various animals, plants and human including crops and fish, as well as foods. Therefore, in order to prevent or treat infectious diseases caused by such pathogens, many studies have been conducted to develop quorum-sensing inhibitors. Disease treatment by use of quorum-sensing inhibitors has been mostly achieved by development of derivatives being chemically similar to signal molecules as a quorum-sensing antagonist or by quenching through enzymatic degradation of quorum-sensing signal molecules. These approaches aim to selectively block a direct mechanism involved in pathogenesis, unlike the conventional therapeutic strategies of using antibiotics for inhibition of cell survival. Thus, emergence of resistant bacteria can be prevented, thereby attracting much attention to these new therapies. In order to accomplish this, many researchers have designed and synthesized AHL derivatives, but not yet reached the point of being able to clarify a structure-activity relationship or species selectivity.

Further, despite the high industrial applicability of the quorum-sensing inhibitors in medical fields, studies of a high throughput screening method, which is essential for the development of effective inhibitors, are still at a very early stage. Recent studies have reported a method of developing a quorum sensing-quenching enzyme using a thiolactone derivative of acyl homoserine lactone autoinducer as a substrate, a fluorescent dye recognizing degradation of the thiolactone derivative and binding thereto, and fluorescence-activated cell sorting (FACS) based on fluorescence intensity (Nat. Methods. 2006. 3, 561-570). However, application of this method is rather limited in that it is to screen an enzyme capable of degrading specific thiolactone derivative only. Further, a method of screening quorum sensing inhibitors using the lux operon of Vibrio fischeri that is involved in quorum sensing has been suggested (Biochemistry. 2009. 48, 4344-4353), but this method is problematic in that as the inhibition activity increases, the luminescence intensity decreases, thereby reducing the accuracy.

Accordingly, there is a need to develop more effective methods for screening quorum sensing inhibitors from natural or synthetic candidates, in order to discover, develop, and improve the quorum sensing inhibitors useful in medical fields.

Therefore, the present inventors have made an effort to develop a method for screening quorum sensing inhibitors. As a result, they developed a novel genetic circuit system by association of quorum sensing inhibition with antibiotic resistance, in which antibiotic resistance of host cells used as a screening system increases, as quorum quenching activity increases.

It is an object of the present invention to provide a vector comprising an expression cassette for screening quorum sensing inhibitors, which comprises (i) a gene encoding a transcriptional regulator that recognizes and binds to acyl homoserine lactone; (ii) a promoter regulated by the transcriptional regulator and a gene encoding an inhibitor of antibiotic-degrading enzyme that is operably linked to the promoter; and (iii) a gene encoding an antibiotic-degrading enzyme that is inhibited by the inhibitor of the antibiotic-degrading enzyme.

It is another object of the present invention to provide a strain introduced with the vector.

It is still another object of the present invention to provide a method for screening quorum sensing inhibitors, comprising the steps of (i) determining the concentrations of acyl homoserine lactone and antibiotic, which inhibit growth of the strain for screening quorum sensing inhibitors; (ii) contacting the growth-inhibited strain with quorum sensing inhibitor candidates; and (iii) determining the quorum sensing inhibitor candidate as a quorum sensing inhibitor when the strain contacted with the quorum sensing inhibitor candidate grows under the higher concentrations of acyl homoserine lactone or antibiotic than those determined in step (i).

It is still another object of the present invention to provide a quorum sensing inhibitor screened by the screening method.

The method for screening quorum sensing inhibitors of the present invention is used to easily select quorum sensing inhibitors by growth analysis of host cells against antibiotics, to readily screen compounds, enzymes or proteins having quorum-quenching effect from a large-scale compound library, metagenome library, mutant library, or artificial gene library, and to screen improved inhibitors.

Further, the method for screening quorum sensing inhibitors of the present invention can be widely utilized for the development of therapeutic agent for infectious diseases and industrial antimicrobial agents.

FIG. 1 shows a schematic representation of the quorum sensing circuit that is designed for screening quorum sensing inhibitors according to the present invention;

FIG. 2 shows a schematic representation of the quorum sensing inhibitor selection plasmid pQS that is constructed for the circuit of FIG. 1;

FIG. 3 shows a C6-L-HSL concentration-dependent growth pattern of E.coli host cells transformed with the plasmid pQS of FIG. 2;

FIG. 4 shows a schematic representation of E.coli host cell that is transformed with both of the quorum sensing inhibitor selection plasmid and the plasmid expressing lactonase as a quorum sensing inhibitor;

FIG. 5 shows a C6-L-HSL concentration-dependent growth pattern of E.coli host cells in the presence or absence of lactonase as a quorum sensing inhibitor;

FIG. 6 shows a C6-L-HSL concentration-dependent growth pattern of E.coli host cells that are transformed with the weak expression vector pZS*24DN possessing lactonase;

FIG. 7 shows a C6-L-HSL concentration-dependent growth pattern of E.coli host cells that express the wild type lactonase and the mutant lactonase V69L with improved activity; and

FIGs. 8 and 9 show C6-L-HSL concentration-dependent growth patterns of E.coli host cells that express the wild type lactonase and the mutant lactonase V69L/I190F with improved activity.

In one aspect to achieve the above objects, the present invention relates to a vector comprising an expression cassette for screening quorum sensing inhibitors, which comprises (i) a gene encoding a transcriptional regulator that recognizes and binds to acyl homoserine lactone; (ii) a promoter regulated by the transcriptional regulator and a gene encoding an inhibitor of antibiotic-degrading enzyme that is operably linked to the promoter; and (iii) a gene encoding an antibiotic-degrading enzyme that is inhibited by the inhibitor of antibiotic-degrading enzyme.

As used herein, the term "quorum sensing" means cell to cell communication through synthesis, extracellular release and detection of specific materials in response to environmental changes, in which communication is mediated by low-molecular weight signal molecules called autoinducers. Typically, Gram-negative bacteria use acyl homoserine lactones and Gram-positive bacteria use peptide fragments as autoinducers. The term "quorum" means "the fixed minimum number of bacteria", in which bacteria do not respond to signal molecules at low cell population density, but accumulation of acyl homoserine lactones with increasing cell population enables bacteria to sense their cell population density. When the signal molecules reach a critical threshold concentration, they bind with regulators and this complex activates or represses specific gene expression, leading to virulence, bioluminescence, biofilm formation, antibiotic resistance or the like.

As used herein, the term "acyl homoserine lactone" refers to an autoinducer, which is a quorum sensing signal molecule of Gram-negative bacteria. Many pathogenic bacteria employ acyl homoserine lactone-mediated quorum sensing, and the mechanisms are very similar to each other, whereby it may represent a novel target for anti-infective therapy. In the present invention, the acyl homoserine lactone means a group of compounds that share a common homoserine lactone ring structure, which is a widespread quorum sensing signal molecule in Gram-negative bacteria. Examples thereof include N-β-oxo-hexanoyl-L-homoserine lactone, N-β-oxo-octanoyl-L-homoserine lactone, N-β-oxo-decanoyl-L-homoserine lactone, N-hexanoyl-L-homoserine lactone, N-butanoyl-homoserine lactone, N-β-oxo-dodecanoyl-L-homoserine lactone, but the type of acyl homoserine lactone is not limited thereto. Preferably, the acyl homoserine lactone may be hexanoyl homoserine lactone.

The transcriptional regulator that recognizes and binds with the acyl homoserine lactone refers to a protein which binds with the autoinducer, acyl homoserine lactone to regulate transcription of a target gene. Any protein may be employed without limitation, as long as it binds with acyl homoserine lactone to regulate transcription of a target gene. Examples thereof may include LuxR-type transcriptional regulators such as Pseudomonas aeruginosa-derived LasR and RhlR, Burkholderia cepacia-derived CepR, Serratia liquefacience-derived SwrR, Aeromonas hydrophila-derived AhyR, Vibrio anguillarum-derived VanR, Erwinia carotovora-derived ExpR and CarR, Agrobacterium tumefaciens-derived TraR, and Vibrio fischeri-derived LuxR, and preferably Vibrio fischeri-derived LuxR.

The promoter of which transcription is regulated by the transcriptional regulator means a promoter capable of regulating target gene expression by various transcriptional regulators in the strain, and a promoter capable of on-off switching the expression of a gene operably linked to the downstream by a transcriptional regulator bound with acyl homoserine lactone. As the promoter, various quorum sensing-inducing promoters may be used without limitation, and are exemplified by LuxI-type promoters such as Pseudomonas aeruginosa-derived LasI promoter and RhlI promoter, Burkholderia cepacia-derived CepI promoter, Serratia liquefacience-derived SwrI promoter, Aeromonas hydrophila-derived AhyI promoter, Vibrio anguillarum-derived VanI promoter, Erwinia carotovora-derived ExpI promoter and CarI promoter, Agrobacterium tumefaciens-derived TraI promoter, and Vibrio fischeri-derived LuxI promoter, in which the promoter can be paired with a transcriptional regulator derived from each species that is contained in the expression cassette, and preferably a Vibrio fischeri-derived LuxI promoter paired with LuxR.

As used herein, the term "operably linked" means the association of one nucleic acid fragment on the other nucleic acid fragment so that the function or expression thereof is affected by the other, but available combinations of the nucleic acid fragments cause no detectable effect when each fragment performs its function. For instance, the term refers to a functional linkage between a nucleic acid sequence coding for a target protein and a nucleic acid expression control sequence in such a manner as to allow general functions. In addition, the expression cassette of the present invention may further comprise any transcription initiation regulating sequence and transcription termination regulating sequence for transcription regulation. The operable linkage may be prepared using a genetic recombinant technique that is well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes that are generally known in the art.

As used herein, the term "antibiotic-degrading enzyme" is a component to determine the change in antibiotic resistance of host cell depending on quorum-quenching activity, in which the antibiotic resistance is associated with quorum-quenching activity of host cells introduced with the vector. Genes encoding the inhibitor of an antibiotic-degrading enzyme and the antibiotic-degrading enzyme of which catalytic activity is reduced by the inhibitor may be included in one expression cassette. As the antibiotic-degrading enzyme, any antibiotic-degrading enzyme known in the art may be used without limitation, depending on the type of antibiotic used for screening a strain. Examples thereof include extended spectrum beta lactamase (ESBL) such as TEM, SHV and AmpC-type enzymes, but are not limited thereto. Preferably, the antibiotic-degrading enzyme of the present invention is TEM-1 β-lactamase.

In one preferred embodiment, any strain capable of normally operating each component of the expression cassette may be used as the strain without limitation, and exemplified by Gram-negative bacteria such as Actinobacillus actinomycetemcomitans, Acinetobacter baumannii, Bordetella pertussis, Brucella sp., Campylobacter sp., Capnocytophaga sp., Cardiobacterium hominis, Eikenella corrodens, Francisella tularensis, Haemophilus ducreyi, Haemophilus influenzue, Helicobacter pylori, Kingella kingae, Legionella pneumophila, Pasteurella multocida, Citrobacter sp., Enterobacter sp., Escherichia coli, Klebsiella pneumoniae, Proteus sp., Salmonella enteriditis, Salmonella typhi, Serratia marcescens, Shigella sp., Yersinia enterocolitica, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Veillonella sp., Bacteroides fragilis, Bacteroides sp., Prevotella sp., Fusobacterium sp., Spirillum minus, Aeromonas sp., Plesiomonas shigelloides, Vibrio sp., Vibrio fischeri, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Acinetobacter sp., Flavobacterium sp., Pseudomonas aeruginosa, Burkholderia cepacia, Burkholderia pseudomallei, Xanthomonas maltophilia, or Stenotrophomonas maltophila, and preferably Escherichia coli.

As used herein, the term "inhibitor of antibiotic-degrading enzyme" means a protein that targets the antibiotic-degrading enzyme such as various types of ESBL so as to inhibit its function. Depending on the type of antibiotic-degrading enzyme selected, any protein known in the art may be employed without limitation, as long as it is capable of inhibiting function of the antibiotic-degrading enzyme, and preferably β-lactamase inhibitor (BLIP).

As used herein, the term "quorum sensing inhibitor" may include all materials capable of inhibiting quorum sensing without limitation, and may include analogs of acyl homoserine lactone, enzymes or proteins degrading or binding to acyl homoserine lactone, antagonists or binding proteins that bind to quorum-sensing transcriptional regulators so as to inhibit their activity, proteins that specifically bind to quorum-sensing promoter so as to suppress its expression, and also include nucleic acids such as RNAi or compounds capable of inhibiting quorum sensing. The preferred quorum sensing inhibitor is lactonase capable of degrading acyl homoserine lactone.

As used herein, the term "expression cassette" refers to a cassette which is composed of a unit capable of expressing the transcriptional regulator that recognizes and binds to acyl homoserine lactone, a promoter regulated by the transcriptional regulator binding to acyl homoserine lactone and a unit capable of expressing an inhibitor of an antibiotic-degrading enzyme that is operably linked to the promoter, and a unit capable of expressing an antibiotic-degrading enzyme that is targeted by the inhibitor of the antibiotic-degrading enzyme.

Further, as used herein, the term "vector" refers to an expression vector capable of expressing a target protein in suitable host cells, and to a gene construct that comprises essential regulatory elements to which a gene insert is operably linked in such a manner as to be expressed.

The operable linkage to a recombinant vector may be prepared using a genetic recombinant technique that is well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes that are generally known in the art.

The vector of the present invention may include a signal sequence or a leader sequence for targeting membranes or secretion as well as expression regulatory elements, such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal and an enhancer, and can be constructed in various forms depending on the purpose thereof. The promoter of the vector may be constitutive or inducible. In addition, expression vectors include a selectable marker that allows the selection of host cells containing the vector, and replicable expression vectors include a replication origin. The vector may be self-replicable, or may be integrated into the DNA of a host cell.

The vector includes a plasmid vector, a cosmid vector, a viral vector or the like. Preferably, the vector may be the pQS plasmid of SEQ ID NO. 1, pPROTrc2 of SEQ ID NO. 4, or pZS*24DNluc of SEQ ID NO. 10, but is not limited thereto.

In one preferred embodiment, the vector comprising the expression cassette for screening quorum sensing inhibitors is the plasmid pQS of SEQ ID NO. 1, consisting of a gene encoding LuxR protein of Vibrio fischeri, LuxI promoter, a gene encoding Streptomyces clavuligerus-derived β-lactamase inhibitor, and a gene encoding TEM-1 β-lactamase.

In still another embodiment, the present invention provides a strain that is introduced with the expression vector for screening quorum sensing inhibitors.

As used herein, the term "introduced" means insertion of foreign DNA into a cell by transformation or transduction. The transformation may be performed by various methods known in the art, such as a CaCl₂ precipitation, a Hanahan method that is an improved CaCl₂ method by using DMSO (dimethyl sulfoxide) as a reducing material, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicone carbide fiber, Agrobacterium-mediated transformation, PEG-, dextran sulfate-, lipofectamine-, and desiccation/inhibition-mediated transformation. The transduction means the delivery of a gene to a cell using virus or viral vector particle by means of infection.

In one preferred embodiment, Gram-negative bacteria may be used as the strain without limitation, preferably, E.coli, and more preferably, E.coli transformed with pQS of SEQ ID NO. 1, deposited at the Biological Resource Center in the Korea Research Institute of Bioscience and Biotechnology (111 Gwahangno, Yuseong-gu, Daejeon) on October. 27, 2009 under the Accession No. KCTC 11579BP.

In still another embodiment, the present invention provides a method for screening quorum sensing inhibitors, comprising the steps of (i) determining the concentrations of acyl homoserine lactone and antibiotic, which inhibit growth of the strain for screening quorum sensing inhibitors; (ii) contacting the growth-inhibited strain with quorum sensing inhibitor candidates; and (iii) determining the quorum sensing inhibitor candidate as a quorum sensing inhibitor when the strain contacted with the quorum sensing inhibitor candidate grows under higher concentrations of acyl homoserine lactone or antibiotic than those determined in step (i).

As used herein, the term "quorum sensing inhibitor candidates" refer to materials that inhibit quorum sensing mechanism in various steps so as to prevent quorum sensing, and compounds, enzymes, proteins, or nucleic acids may be used without limitation.

As used herein, the term "antibiotic" refers to an antibiotic used to select the strain for screening quorum sensing inhibitors, and any antibiotic may be used without limitation, as long as it is targeted by the antibiotic-degrading enzyme that is introduced into the strain for screening quorum sensing inhibitors. Examples thereof include β-lactam or non-β-lactam antibiotics, and include all antibiotics of penicillin-, cephalosporin-, quinoline-, and aminoglycoside-based antibiotics according to classification. Further, if a suitable antibiotic is selected by those skilled in the art depending on the purpose, a gene encoding the antibiotic-degrading enzyme and a gene encoding the inhibitor of antibiotic-degrading enzyme included in the vector of the present invention are suitably selected according to the selected antibiotic, thereby preparing the expression cassette. The antibiotic is preferably β-lactam antibiotic, and more preferably carbenicillin.

The method for screening quorum sensing inhibitors of the present invention is designed in the type of genetic circuit system by association of quorum sensing inhibition with antibiotic resistance, in which antibiotic resistance of host cells used as a screening system increases, as quorum quenching activity increases.

Schematic representation of the method for screening quorum sensing inhibitors of the present invention is shown in FIG. 1. When the transcriptional regulator binds with acyl homoserine lactone to activate the quorum sensing promoter, the expression level of β-lactamase inhibitor connected to the downstream of the promoter is regulated. If quorum sensing is active so as to increase the expression level of β-lactamase inhibitor, the antibiotic-degrading activity of β-lactamase is suppressed, leading to zero growth of the host cells under antibiotic selection pressure. On the basis of this, the concentration of acyl homoserine lactone, at which quorum sensing becomes active so as to inhibit growth of the host cells, can be determined. In contrast, if quorum sensing is inhibited so as to decrease the expression level of β-lactamase inhibitor, β-lactamase retains the antibiotic-degrading activity to endow the host cells with antibiotic resistance, resulting in the growth of the host cells even at the concentration of acyl homoserine lactone, at which quorum sensing becomes active so as to inhibit growth of the host cells.

Therefore, when the strain is treated with a candidate material capable of inhibiting any route of the quorum sensing circuit, the growth is influenced by the treatment of the corresponding candidate material. Thus, properties of candidate material can be easily determined by the analysis of the growth pattern against the antibiotic. For instance, if the candidate material is enzymes or proteins degrading acyl homoserine lactone, antagonists or binding proteins that bind to quorum-sensing transcriptional regulators so as to inhibit their activity, or proteins that specifically bind to quorum-sensing promoter so as to suppress its expression, the growth of the strain of the present invention increases, thereby screening all quorum sensing inhibitors through the corresponding signals.

In one preferred embodiment, the strain for screening quorum sensing inhibitors may be a strain transformed with a vector that is composed of the expression cassette for screening quorum sensing inhibitors, preferably E.coli transformed with pQS of SEQ ID NO. 1, and more preferably, a strain identified by the Accession No. KCTC 11579BP.

In one preferred embodiment, the present invention may further introduce a vector comprising a gene encoding the wild-type lactonase into the strain of step (i). The wild-type lactonase is, the known quorum sensing inhibitor, an enzyme degrading acyl homoserine lactone. Thus, when the concentration capable of inhibiting the growth of the strain introduced with the wild-type lactonase is determined to perform the screening, it is possible to screen inhibitors with higher activity than the conventional inhibitors.

In one preferred embodiment, the method of contacting the strain with quorum sensing inhibitor candidates of step (ii) may be performed by any method of treating candidates to affect genetic circuit system of the present invention, for example, by introduction of a vector comprising a gene encoding inhibitor candidate into the strain or by treatment of strain media with the inhibitor candidate.

In one preferred embodiment, the present invention provides a method for screening quorum sensing inhibitors, comprising the step of determining a lactonase mutant as an improved quorum sensing inhibitor, when the strain of step (iii) grows under the higher concentrations than those determined in step (i), in which the method of contacting the strain with the quorum sensing inhibitor candidate of step (ii) is to introduce a vector including a gene encoding the lactonase mutant into the strain.

In the method for screening quorum sensing inhibitors of the present invention, when quorum sensing-dependent antibiotic sensitivity of the strain is regulated by varying the concentration of acyl homoserine lactone or β-lactam antibiotic, only the strain expressing quorum sensing inhibitors with higher activity can grow at higher concentrations of acyl homoserine lactone or β-lactam antibiotic, thereby screening more excellent inhibitors.

In one preferred Example, the present inventors constructed a strain identified by the Accession No. KCTC 11579BP, which is prepared by transformation of E.coli DH5α with the pQS vector of SEQ ID NO. 1 (FIG. 2) which is composed of a gene encoding the LuxR quorum sensing transcriptional regulator of Vibrio fischeri, the LuxI promoter regulated thereby, a gene encoding Streptomyces clavuligerus-derived TEM-1 β-lactamase, and a β-lactamase inhibitor targeting TEM-1 β-lactamase. Thereafter, the strain was treated with various hexanoyl homoserine lactones. As the lactone concentration increases, quorum sensing becomes active to inhibit β-lactamase, and growth of the strain was found to be inhibited at the lactone concentration of 10 nM or higher (FIG. 3). Accordingly, the present inventors assumed that the strain is able to grow at the above lactone concentration in the presence of a quorum sensing inhibitor, and thus they constructed an expression vector pPROTrc2 of SEQ ID NO. 4. Specifically, Bacillus thuringiensis-derived lactonase, which is known as the acyl homoserine lactone hydrolase and quorum sensing inhibitor, was inserted into the NcoI and HindIII sites of MCS (multi cloning site), and the strain was transformed with pPROTrc2 including lactonase, constructed by the above method (FIG. 4), followed by treatment of the transformed strain with various concentrations of hexanoyl homoserine lactone. Consequently, it was found that the strain grew at the concentration of 10 nM or higher, at which no growth was observed in the absence of lactonase (FIG. 5). The above experiment demonstrated that the strain and screening method of the present invention can be used to effectively screen quorum sensing inhibitors. This fact indicates that quorum sensing inhibitors can be screened by a simple procedure, including the steps of determining the suitable concentration of acyl homoserine lactone, at which the growth of the strain is affected depending on the presence or absence of an inhibitor, and selecting a strain that is able to grow at the corresponding concentration.

In one preferred Example, the present inventors conducted further experiments to improve catalytic activity of the known lactonase of Bacillus thuringiensis, because the lactonase has lower catalytic activity against hexanoyl homoserine lactone in a level of kcat/K_M = 1.0×10⁴ M^-1s^-1, and also has a similar activity against other lactones that vary in the length and shape of acyl chain. To determine the acyl homoserine lactone concentration, at which host cells possessing the wild-type lactonase do not grow but at which host cells possessing mutant lactonase with improved activity are able to grow, they constructed a pZS*24DNluc vector plasmid of SEQ ID NO. 10, which has a low-copy number and a weak promoter to minimize the expression level of lactonase in the strain. The vector was treated with NcoI and XbaI to prepare the plasmid lactonase/pZS*24DN possessing the wild-type lactonase, which was transformed into the strain identified by the Accession No. KCTC 11579BP. Consequently, it was confirmed that 100 nM of hexanoyl homoserine lactone is a growth-limiting concentration (FIG. 6). Thereafter, various lactonase mutants were inserted into pZS*24DNluc of SEQ ID NO. 10, and transformed into the strain identified by the Accession No. KCTC 11579BP to construct a library of lactonase mutants having various amino acid sequences. Strains growing at the hexanoyl homoserine lactone concentration of 1 μM, which is higher than the growth-limiting concentration of 100 nM, were screened to confirm two mutants, and base sequence analysis was revealed that one of them has a single amino acid substitution of Val (Valine, V) with Leu (Leucine, L) at position 69, distant from the enzyme active site directing the acyl chain of the substrate acyl homoserine lactone, and the mutant was designated mutant V69L (SEQ ID NO. 13). It was also confirmed that the other has an amino acid substitution of Val (Valine, V) with Leu (Leucine, L) at position 69, distant from the enzyme active site directing the acyl chain of the substrate acyl homoserine lactone, and amino acid substitution of Ile (Isoleucine, I) with Phe (Phenylalanine, F) at position 190, distant from the enzyme active site directing the acyl chain of the substrate acyl homoserine lactone, and the mutant was designated mutant V69L/I190F (SEQ ID NO. 14). It was confirmed that cells expressing each mutant are able to grow at higher concentration of lactone, compared to the wild-type strain (FIGs. 7, 8, and 9), the mutant V69L activity for hexanoyl homoserine lactone was kcat/K_M=3.7×10⁴ M^-1s^-1, approximately four fold higher than that of the wild-type enzyme, and the mutant V69L/I190F activity for hexanoyl homoserine lactone was kcat/K_M=7.0×10⁴ M^-1s^-1, approximately one order higher than that of the wild-type enzyme, indicating that the screening method of the present invention can be effectively used for screening inhibitors with more improved activity than the conventional inhibitors.

In still another embodiment, the present invention provides a quorum sensing inhibitor screened by the screening method of the present invention.

In one preferred embodiment, the quorum sensing inhibitor may be a compound, enzyme, protein or nucleic acid, and preferably, the mutant lactonase V69L of SEQ ID NO. 13 having amino acid substitution of Val with Leu at position 69 of the known wild-type Bacillus thuringiensis lactonase, or the mutant lactonase V69L/I190F of SEQ ID NO. 14 having amino acid substitution of Val with Leu at position 69 and amino acid substitution of Ile with Phe at position 190 of the known wild-type Bacillus thuringiensis lactonase.

Hereinafter, the present invention will be described in more detail with reference to Examples. Although theses Examples have been disclosed for illustrative purposes, various modifications and variations are possible, and the present invention should not be construed as being limited to these Examples set forth herein.

Example 1: Design and Construction of quorum sensing inhibitor selection system

As the elements constituting the present invention, the LuxR transcriptional regulator and LuxI promoter of Vibrio fischeri were used as a quorum sensing transcriptional regulator and a quorum sensing promoter, respectively. In addition, Streptomyces clavuligerus-derived β-lactamase inhibitor (BLIP) and TEM-1 β-lactamase as a target thereof were used to construct a plasmid of FIG. 2, which was designated pQS. The expression levels of β-lactamase and LuxR remained constant, and the expression level of β-lactamase inhibitor was regulated by the LuxI promoter. The sequence of the constructed pQS plasmid is represented by SEQ ID NO. 1.

E.coli DH5α was used as a host cell, and a quorum sensing circuit was constructed using the plasmid, and E.coli transformed with pQS of SEQ ID NO. 1 was designated Escherichia coli DH5α/pQS, which was deposited at the Biological Resource Center in the Korea Research Institute of Bioscience and Biotechnology (111 Gwahangno, Yuseong-gu, Daejeon) on October. 27, 2009 under the Accession No. KCTC 11579BP. One of acyl homoserine lactones, hexanoyl homoserine lactone (Hexanoyl-L-homo serine lactone, C6-L-HSL) was used as a signal molecule, and carbenicillin was used as a β-lactam antibiotic. When the cells were cultured on LB solid media containing various concentrations of C6-L-HSL and 100 ㎍/㎖ of carbenicillin at 30℃ for 2 days, they showed the growth pattern of FIG. 3. That is, as the lactone concentration increases, quorum sensing becomes more active so that β-lactamase is more inhibited, and thus no growth of E.coli host cells was observed at the lactone concentration of 10 nM or higher. In the presence of a quorum sensing inhibitor, E.coli host cells are able to grow even at a higher lactone concentration, depending on the activity of the inhibitors.

Example 2: Test on quorum sensing selection system using quorum sensing inhibitor, Bacillus thuringiensis-derived lactonase

Bacillus thuringiensis-derived lactonase is, as the acyl homoserine lactone hydrolase, a quorum sensing inhibitor valuable in the medical industry (SEQ ID NO. 5, PNAS. 2005, 102, 17609-17611). This enzyme was introduced into the selection system of quorum sensing inhibitors, which was designed in Example 1, and its quorum-quenching effect was confirmed.

1) Construction of lactonase - expressing vector

A vector plasmid, used for the introduction of lactonase into E.coli host cells and the expression of lactonase, is characterized in that it has a Trc promoter and a multi cloning site (MCS), a kanamycin resistance gene, and a Col E1 replication origin. The vector plasmid was constructed in the following manner.

Trc promoter and MCS:

Trc promoter and MCS, and transcription termination site of the expression vector pTrc99A were used as templates, and a primer having AatII restriction site (SEQ ID NO. 2) and a primer having AvrII restriction site (SEQ ID NO. 3) were used to amplify the corresponding sequences by PCR (95℃ 4 min/ 95℃ 1 min; 55℃ 30 sec; 72℃ 50 sec; 20 cycles/ 72℃ 1 min). Thereafter, the restriction enzymes, AatII and AvrII were used to cleave both ends of the amplified sequences.

Kanamycin resistance gene:

The sequence including a kanamycin resistance gene of pPROLar.A122 (Clontech) expression vector was cleaved using SacI and AatII restriction enzymes, and separated.

Col E1 replication origin:

The sequence including Col E1 replication origin of pPROTet.E133 (Clontech) expression vector was cleaved using AvrII and SacI restriction enzymes, and separated.

Three sequences obtained by the above procedure were ligated to each other by ligase reaction to construct an expression vector plasmid, designated pPROTrc2 (SEQ ID NO. 4).

2) Insertion of lactonase gene for analysis of quorum sensing inhibition

Bacillus thuringiensis lactonase was subjected to PCR (95℃ 3 min/ 95℃ 1 min; 55℃ 30 sec; 72℃ 50 sec; 25 cycles/ 72℃ 1 min) using an N-terminal primer having NcoI restriction site (SEQ ID NO. 6) and a C-terminal primer having HindIII restriction site (SEQ ID NO. 7), and treated with NcoI and HindIII restriction enzymes, followed by insertion into multi cloning site of plasmid pPROTrc2.

3) Test of inhibitory effect of lactonase on quorum sensing

The pPROTrc2 plasmid containing lactonase constructed by the above procedure was inserted into E.coli host cell DH5α together with the quorum sensing inhibitor selection plasmid pQS (FIG. 4). Then, when host cells were cultured on LB solid media containing various concentrations of C6-L-HSL, 100 ㎍/㎖ carbenicillin, 50 ㎍/㎖ kanamycin, and 0.2 mM ZnSO₄ at 30℃ for 2 days, they showed the growth pattern of FIG. 5. That is, when the inhibitor lactonase is expressed, host cells are able to grow even at the C6-L-HSL concentration of 10 nM or higher. At this quorum sensing level, no growth was observed in the absence of inhibitor.

This result indicates that quorum sensing inhibitors can be screened by a simple procedure, including the steps of determining the suitable concentration of acyl homoserine lactone, at which the growth of the host cells is affected depending on the presence or absence of inhibitor, and selecting a strain that is able to grow at the corresponding concentration. For instance, E.coli host cells growing at C6-L-HSL concentration of 10 nM or higher, 100 nM or 1 μM can be said to have a quorum sensing inhibitor. In addition, this result is not limited to lactonase, but applicable to all types of quorum sensing inhibitors.

Example 3: Selection and Improvement of lactonase by directed evolution

In the selection system of quorum sensing inhibitors of the present invention, quorum sensing-dependent antibiotic sensitivity of host cells can be regulated by varying the concentration of acyl homoserine lactone or β-lactam antibiotic. As the concentration of acyl homoserine lactone or β-lactam antibiotic increases, host cells expressing an inhibitor with higher activity are able to grow. This fact provides a tool for selecting and improving excellent inhibitors.

The known Bacillus thuringiensis lactonase has lower catalytic activity against hexanoyl homoserine lactone (C6-L-HSL) in a level of kcat/K_M = 1.0×10⁴ M^-1s^-1, and also has a similar activity against other lactones that vary in the length and shape of acyl chain. A quorum-quenching enzyme having high catalytic activity and substrate specificity is an effective therapeutic agent for infectious diseases. Therefore, the selection system of quorum sensing inhibitors of the present invention was used to improve the activity of lactonase.

1) Vector plasmid for lactonase library

To determine the acyl homoserine lactone concentration, at which host cells possessing the wild-type lactonase do not grow but host cells possessing mutant lactonase with improved activity are able to grow, they constructed a pZS*24DNluc vector plasmid of SEQ ID NO. 10, the expression level of lactonase in host cells should be first minimized. Therefore, the pZS*24luc plasmid (Expressys) having a low-copy number and a weak promoter was used as a library vector.

PCR amplification (95℃ 5 min/ 95℃ 1 min; 55℃ 1 min; 72℃ 6 min; 20 cycles/ 72℃ 6 min) was performed using the entire pZS*24luc plasmid as a template and primers of SEQ ID NOs. 8 and 9 and PrimeStar^TM HS polymerase (TaKaRa) and the plasmid pZS*24DNluc (SEQ ID NO. 10) was constructed from pZS*24luc by substitution of 5'-CCATGG-3' with 5'-CCGTGG-3' in the NcoI endonucleases site of the kanamycin resistance gene.

Lactonase was subjected to PCR (95℃ 3 min/ 95℃ 1 min; 55℃ 30 sec; 72℃ 30 sec; 25 cycles/ 72℃ 1 min) using an N-terminal primer having NcoI restriction site (SEQ ID NO. 11) and a C-terminal primer having XbaI restriction site (SEQ ID NO. 12), and enzyme treatment using NcoI and XbaI restriction enzymes, and the plasmid lactonase/pZS*24DN was constructed by substitution of luciferase gene in the plasmid pZS*24DNluc.

E.coli DH5α transformed with both of the wild-type lactonase/pZS*24DN and the quorum sensing inhibitor selection plasmid pQS was found to have a growth-limiting concentration at 100 nM of C6-L-HSL concentration (FIG. 6). In Example 2 using pPROTrc2 as an expression vector, growth of host cells was observed at higher lactone concentration than the above concentration, indicating that the expression level of lactonase in pZS*24DN is lower than that in pPROTrc2.

In the selection system of quorum sensing inhibitors of the present invention, host cells expressing the mutant lactonase with enhanced activity would be expected to grow at higher lactone concentrations than those expressing the wild-type lactonase.

2) Construction of mutant lactonase library

Lactonase mutants were generated by error-prone PCR due to random mutagenesis, in which Taq polymerase with low polymerization accuracy was used and MgCl₂, MnCl₂, and dNTP of PCR components were controlled to reduce the accuracy of the amplification. The reaction was performed under the following conditions: wild-type lactonase gene template (~1 pg), 1×Taq polymerase buffer (75 mM Tris-HCl, pH8.8, 20 mM (NH₄)₂SO₄, 0.01 % (v/v) Tween 20, 1.25 mM MgCl₂), dNTP (dATP and dGTP, 1.0 mM; dCTP and dTTP, 0.2 mM), 5.5 mM MgCl₂, 0.1 mM MnCl₂, 2.5 U Taq polymerase, 0.5 μM N-terminal primer (SEQ ID NO. 11) and C-terminal primer (SEQ ID NO. 12).

The mutant lactonase genes obtained by the above procedure were treated with NcoI and XbaI restriction enzymes at both ends thereof, and cloned into the expression vector pZS*24DN, and transformed into E.coli DH5α harboring pQS, so as to construct a mutant lactonase library having various amino acid substitutions.

3) Selection and Improvement of mutant lactonase with enhanced activity

The above constructed mutant library was cultured at 30℃ for 3 days under the conditions (LB solid media, C6-L-HSL 1 μM, 100 ㎍/㎖ carbenicillin, 30 ㎍/㎖ kanamycin, 0.2 mM ZnSO₄), where E.coli expressing the wild-type lactonase is not able to grow, followed by selection of E.coli growing under the conditions. To remove false-positive mutants, the lactonase mutant/pZS*24DN plasmid was extracted from the selected E.coli, and re-transformation into E.coli harboring pQS. This procedure was repeated to screen two mutants from the library size of 5×10⁵.

Base sequence analysis was revealed that one of them has a single amino acid substitution of Val with Leu at position 69, distant from the enzyme active site directing the acyl chain of the substrate acyl homoserine lactone, and the mutant was designated mutant V69L (SEQ ID NO. 13). It was also confirmed that the other has amino acid substitution of Val with Leu at position 69 and amino acid substitution of Ile with Phe at position 190, and the mutant was designated mutant V69L/I190F (SEQ ID NO. 14). It was confirmed that E.coli host cells expressing V69L or V69L/I190F are able to grow at higher concentration of lactone, compared to the wild-type strain (FIGs. 7, 8, and 9), the mutant V69L activity for C6-L-HSL was kcat/K_M=3.7×10⁴ M^-1s^-1, approximately four fold higher than that of the wild-type enzyme, and the mutant V69L/I190F activity for C6-L-HSL was kcat/K_M=7.0×10⁴ M^-1s^-1, approximately one order higher than that of the wild-type enzyme.

Claims

A vector comprising an expression cassette for screening quorum sensing inhibitors, which comprises

(i) a gene encoding a transcriptional regulator that recognizes and binds to acyl homoserine lactone;

(ii) a promoter regulated by the transcriptional regulator and a gene encoding an inhibitor of antibiotic-degrading enzyme that is operably linked to the promoter; and

(iii) a gene encoding an antibiotic-degrading enzyme that is inhibited by the inhibitor of antibiotic-degrading enzyme.
The vector according to claim 1, wherein the inhibitor of antibiotic-degrading enzyme is β-lactamase inhibitor, and the antibiotic-degrading enzyme is TEM-1 β-lactamase.
The vector according to claim 1, wherein the transcriptional regulator is LuxR, and the promoter is LuxI promoter.
The vector according to claim 3, wherein the vector is a plasmid pQS of SEQ ID NO. 1.
A strain introduced with the vector of any one of claims 1 to 4.
The strain according to claim 5, wherein the strain is Gram-negative bacteria.
The strain according to claim 6, wherein the strain is identified by the Accession No. KCTC 11579BP.
A method for screening quorum sensing inhibitors, comprising the steps of:

(i) determining the concentrations of acyl homoserine lactone and antibiotic, which inhibit growth of the strain introduced with the vector of claim 1;

(ii) contacting the growth-inhibited strain with quorum sensing inhibitor candidates; and

(iii) determining the inhibitor candidate as a quorum sensing inhibitor when the strain contacted with the inhibitor candidate grows under the higher concentrations of acyl homoserine lactone or antibiotic than those determined in step (i).
The method according to claim 8, wherein the strain is identified by the Accession No. KCTC 11579BP.
The method according to claim 8, wherein the method of contacting with quorum sensing inhibitor candidates of step (ii) is to introduce a vector containing a gene encoding the inhibitor candidate into the strain or to treat culture media with the inhibitor candidate.
The method according to claim 8, wherein a vector containing a gene encoding the wild-type lactonase is further introduced in step (i).
The method according to claim 11, wherein the method of contacting with quorum sensing inhibitor candidates of step (ii) is to introduce a vector containing a gene encoding mutant lactonase as a quorum sensing inhibitor.
A quorum sensing inhibitor screened by the method of claim 8.
The inhibitor according to claim 13, wherein the quorum sensing inhibitor is selected from the group consisting of compounds, enzymes, proteins, and nucleic acids.
The inhibitor according to claim 14, wherein the quorum sensing inhibitor is the mutant lactonase V69L of SEQ ID NO. 13.
The inhibitor according to claim 14, wherein the quorum sensing inhibitor is the mutant lactonase V69L/I190F of SEQ ID NO. 14.