A method for screening quorum sensing inhibitors and simultaneously determining their target

Abstract

The present application belongs to the field of biomedical technology, and particularly relates to a method for screening of quorum sensing system inhibitors of Pseudomonas aeruginosa and simultaneously determining the target of the inhibitors. The method comprises the following steps: constructing a QS inhibitor screening system, and the QS inhibitor screening system takes protease as a screening marker; constructing a mutant with a deleted protease gene, which provides QS signal molecules in the process of co-culture; and screening a compound with inhibitory effect on the QS system and determining the target of the compound by means of co-culture or exogenous addition of QS signal molecules. The present application establishes a simple, fast, effective and high-throughput QSI screening method, and the target of the compound can be determined simultaneously.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for screening quorum sensing inhibitors and simultaneously determining their targets, and more specifically to a method for screening inhibitors of the Pseudomonas aeruginosa quorum sensing system and simultaneously determining the inhibitor targets. Background Technology

[0002] Quorum sensing (QS) is a communication system within bacterial populations, functioning to coordinate gene expression and regulate population behavior. Among these systems, virulence factors regulated by the QS system play a crucial role in the pathogenicity of pathogens. *Pseudomonas aeruginosa* (PA) is an opportunistic pathogen. With the widespread use of antibiotics, PA has developed drug resistance or multidrug resistance through various mechanisms, making it difficult to treat and eradicate. PA possesses at least three QS signaling systems, which interact and play important roles in the development of pathogenicity and drug resistance. Inhibiting the self-induction or biosynthesis of signaling molecules to block the QS system's regulatory processes is considered a novel strategy for controlling pathogenic resistance and pathogenicity; chemical substances capable of achieving this are called quorum sensing inhibitors (QSIs). Studies have shown that reported QSI candidate compounds come from a wide range of sources, and similarly, there are various methods for screening QSIs, such as reporter strain detection methods (including luminescent reporter strains, fluorescent reporter strains, and purple pigment reporter strains), phenotypic detection methods, bioarrays, or qPCR methods. Among these, biomarker strain detection methods and luminescent reporter strain detection methods are widely used. Commonly, they rely on changes in the production of purple pigment in *Violetella violaceum*, using proteins such as GFP or luminescent reporter strains as detection indicators. For example, Toshiko et al. found that actinomycete metabolites reduced the purple pigment production of *Violetella violaceum* CV026, suggesting that its metabolites may contain QSIs. The fluorescent reporter strain method involves fusing green fluorescent protein and QS-related genes, and screening for effective QSIs by detecting GFP expression levels.

[0003] Purple pigment reporter strains, luminescent or fluorescent reporter systems, are not only regulated by QS signaling but also susceptible to other environmental factors. For example, luminescent reporters require a large amount of energy and are therefore significantly affected by cellular metabolism. Furthermore, existing screening systems cannot precisely determine the target of candidate compounds. Further identification of the target of QSIs requires the combined use of multiple methods to ultimately confirm that a candidate compound is a QSI and its target. Therefore, an effective QSI screening method is needed that can simultaneously determine the target during screening. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for screening quorum sensing inhibitors and simultaneously determining their target sites, and more specifically, a method for screening inhibitors of the Pseudomonas aeruginosa quorum sensing system and simultaneously determining the inhibitor's target sites.

[0005] The purpose of this invention is to provide a simple and rapid method for screening inhibitors of the Pseudomonas aeruginosa quorum sensing system and simultaneously determining the mechanism of action of the compounds, comprising:

[0006] A QS inhibitor screening system was constructed, and the QS inhibitor screening system used protease or luminescent reporter as screening markers;

[0007] A mutant lacking the protease gene was constructed, which provided the QS signaling molecule during co-culture.

[0008] Compounds that inhibit the QS system were screened and their target sites were determined by using co-culture or exogenous addition of QS signaling molecules.

[0009] The co-culture refers to the mixed culture of the QS inhibitor screening system, the protease gene deletion mutant, and the compound.

[0010] The aforementioned addition of exogenous QS signaling molecules refers to the co-culturing of QS inhibitor screening systems, compounds, and exogenous QS signaling molecules.

[0011] Preferably, the QS inhibitor screening system includes a Las screening system, an Rhl screening system, and a Pqs screening system containing the luminescent reporter pKD-pqsA.

[0012] Preferably, the Las screening system is constructed as follows: The rhlR, pqsA, and pqsR genes of JP2 are knocked out sequentially using homologous recombination, resulting in a strain JP2ΔrhlRΔpqsAΔpqsR that can be used to screen for Las system inhibitors. The Rhl screening system is constructed as follows: The pqsA, lasR, and pqsR genes of JP2 are knocked out sequentially using homologous recombination, resulting in a strain JP2ΔpqsAΔlasRΔpqsR that can be used to screen for Rhl system inhibitors. The Pqs screening system is constructed as follows: The rhlR, pqsA, and lasR genes of JP2 are knocked out sequentially using homologous recombination, resulting in a strain JP2ΔrhlRΔpqsAΔlasR that can be used to screen for Pqs system inhibitors. The luminescent reporter plasmid pKD-pqsA is then transferred into JP2ΔlasRΔrhlRΔpqsA, which retains only PqsR, to obtain the Pqs screening system.

[0013] Preferably, based on the protease production of the QS inhibitor screening system after co-culturing or exogenously adding QS signaling molecules, it is determined whether the compound is a quorum sensing inhibitor and whether it (quorum sensing inhibitor) acts on the Las system or the Rhl system.

[0014] In addition, the effect of the inhibitory compound on the luminescence of the luminescent reporter in the Pqs screening system can be used to determine whether the compound has an effect on the Pqs system.

[0015] It should be noted that the method of this invention can also determine whether the quorum sensing inhibitor acts on the synthesis process of the signal molecule or the regulatory process after the signal molecule binds to the regulatory protein. For example, after culturing the screening system strains, an exogenous signal molecule is added as a control, and the signal molecule and inhibitor are added as the experimental group. By comparing the yield or luminescence of the two groups of proteases, it can be determined whether the signal molecule regulatory process is inhibited. Without adding exogenous signal molecules, the amount of signal molecules after co-culturing with and without the screening agent is extracted and compared to determine whether the synthesis process of the signal molecule is inhibited.

[0016] Preferably, the co-culture method is as follows: the protease deletion mutants ΔlasA, ΔlasB, and ΔaprA are mixed with the QS inhibitor screening system strains at a 1:1 ratio by cell mass, then the compound is added, and the mixture is co-cultured at 30-37°C.

[0017] Preferably, the exogenous addition of QS signaling molecules is carried out by directly adding different concentrations of signaling molecules to the culture medium during the cultivation of the QS inhibitor screening system; specifically, commercially available N-(3-oxododecanoyl)-L-homoserinelactone (3OC12-HSL) is added to the Las screening system (strain) culture process; N-butanoyl-L-homoserinelactone (C4-HSL) is added to the Rhl screening system (strain) culture process; 2-heptyl-4-quinolone (HHQ) or / and 2-heptyl-3-hydroxy-4-quinolone (PQS) is added to the Pqs screening system (strain) culture process.

[0018] Preferably, the screened quorum sensing inhibitors are pyridine amide compounds.

[0019] Preferably, the pyridine amide compound is N-14.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention aims to establish a simple, rapid, reliable, high-throughput QSI screening system in PA (Polymerase Injection) capable of determining the mechanism of action of candidate compounds, and to utilize this system to screen compounds with clearly defined targets. Specifically, using proteases directly regulated by QS as detection markers, mutants retaining a single QS system (Las system and Rhl system) and lacking the protease gene are constructed. Compounds inhibiting both the Las and Rhl systems are screened using co-culture or exogenous addition of QS signaling molecules, and their mechanisms of action are determined. Based on the production of proteases in the individual systems after activation (exogenous addition or activation of the screening system using signaling molecules provided by co-culture), the compound's QSI status is determined, and its target is identified. For the Pqs screening system, the detection method of luminescent reporter molecules is still used to determine the compound's site of action.

[0022] This invention constructs a mutant retaining a single QS system and a protease deletion through homologous recombination. This mutant is used to target JP2 (i.e., JP2ΔlasIΔrhlI strain), which involves knocking out the lasI and rhlI genes from PAO1. The JP2 strain is described in the literature "Pearson JP, Pesci EC, Iglewski BH: Roles of Pseudomonas aeruginosa las and rhlquorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes." J Bacteriol The strains reported in "1997, 179(18):5756-5767" (which are preserved in our laboratory) and PAO1 (wild-type Pseudomonas aeruginosa) were used for gene knockout, resulting in single-system mutants and protease gene knockout mutants, namely JP2ΔrhlRΔpqsAΔpqsR (single-lasR), JP2ΔlasRΔpqsAΔpqsR (single-rhlR), JP2ΔrhlRΔpqsAΔlasR (single-pqsR), and ΔlasAΔlasBΔaprA (co-culture to provide signal molecules). Under the condition that the drug concentration did not affect the bacterial metabolism, single-lasR or single-rhlR were co-cultured with ΔlasAΔlasBΔaprA, respectively. After adding 2% liquid skim milk to the culture supernatant containing or without the test compound, the degree of protein hydrolysis was measured to determine whether the drug inhibited the corresponding QS system. For the PQS screening system, the pKD-pqsA luminescent reporter is used as the screening marker, and whether the test compound inhibits luminescence is the standard for determining whether it is a PQS system inhibitor.

[0023] Five previously reported QSIs—baicalin, scutellarin, phlorizin, allicin, and tea polyphenols—were selected for validation within this QSI screening system. This invention also used this QSI screening system to screen 20 artificially synthesized compounds, discovering a new chemically synthesized QSI, N-14, which acts on the Las, Rhl, and Pqs systems. In other words, N-14 targets three sites: Las, Rhl, and Pqs. The results of this invention ultimately demonstrate the feasibility of this novel QSI screening system, providing new practical support for the rapid, simple, and high-throughput screening of effective QSIs. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the QSI screening system of the present invention;

[0025] A. Screening diagram for single lasR / rhlR, where single lasR / rhlR represents JP2ΔrhlRΔpqsAΔpqsR (single lasR) and JP2ΔlasRΔpqsAΔpqsR (single rhlR), respectively. lasI / rhlI generate signaling molecules OdDHL / BHL, respectively. lasR / rhlR are both receptors. lasA regulates protease production, lasB regulates elastase production, and aprA regulates alkaline protease production. × indicates gene deletion. B. Screening diagram for single pqsR, i.e., JP2ΔpqsAΔlasRΔpqsR, where pqsA generates the signaling molecule PQS, pqsR is a receptor, ┴ indicates pathway blockade, pKD: plasmid for constructing luminescent reporter, × indicates gene deletion. Figure 1 In the diagram, ① and ② represent the pathways of QSI action.

[0026] Figure 2 Verification diagrams for mutants ΔlasA(A) and ΔlasAΔlasB(B);

[0027] In A, M represents the DNA Marker (all experiments in this invention were conducted by Shanghai Jierui NormalRun). TM 1Kb-ⅠDNAladder), lanes 1-8 use PAO1 as a template, lane 9 uses ΔlasA as a template; in B, M is DNA Marker, lanes 1-5 and 7 use ΔlasA as a template, lanes 6 and 8 use ΔlasAΔlasB as a template.

[0028] Figure 3 The above are verification diagrams for the mutants ΔlasAΔlasBΔaprA(A) and JP2ΔrhlR(B) of this invention.

[0029] In A, M is the DNA Marker, lanes 1-10 use ΔlasAΔlasB as the template, and lane 11 uses ΔlasAΔlasBΔaprA as the template; in B, M is the DNA Marker, lanes 1-3 use JP2 as the template, and lane 4 uses JP2ΔrhlR as the template.

[0030] Figure 4 The images show the verification results of the mutants JP2ΔrhlRΔpqsA(A), JP2ΔrhlRΔpqsAΔpqsR(B), and JP2ΔpqsA(C) of this invention.

[0031] In A, M is the DNA Marker; lane 1 uses JP2ΔrhlR as the template, and lane 2 uses JP2ΔrhlRΔpqsA as the template; in B, M is the DNA Marker; lane 1 uses JP2ΔrhlRΔpqsA as the template, and lane 2 uses JP2ΔrhlRΔpqsAΔpqsR as the template; in C, M is the DNA Marker; lane 1 uses JP2 as the template, and lane 2 uses JP2ΔpqsA as the template.

[0032] Figure 5 Verification diagrams for mutants JP2ΔpqsAΔlasR(A), JP2ΔpqsAΔlasRΔpqsR(B), and JP2ΔrhlRΔpqsAΔlasR(C);

[0033] In A, M is the DNA Marker; lane 1 uses JP2ΔpqsA as the template, and lane 2 uses JP2ΔpqsAΔlasR as the template. In B, M is the DNA Marker; lane 1 uses JP2ΔpqsAΔlasR as the template, and lane 2 uses JP2ΔpqsAΔlasRΔpqsR as the template. In C, M is the DNA Marker; lane 1 uses JP2ΔrhlRΔpqsA as the template, and lane 2 uses JP2ΔrhlRΔpqsAΔlasR as the template.

[0034] Figure 6 This is a diagram illustrating the principle verification of the QSI screening method.

[0035] Figure 7 Verification diagram of furanone C-30 regulated by the two systems of Las(A) and Rhl(B).

[0036] Figure 8 This is a verification diagram of baicalin regulated by the Las(A) and Rhl(B) systems.

[0037] Figure 9 Verification diagram of phloretin regulated by the Las(A) and Rhl(B) systems.

[0038] Figure 10Verification diagram of salicylic acid regulated by the two systems of Las(A) and Rhl(B).

[0039] Figure 11 This is a verification diagram of allicin regulated by the Las(A) and Rhl(B) systems.

[0040] Figure 12 This is a verification diagram of tea polyphenols regulated by the Las(A) and Rhl(B) systems.

[0041] Figure 13 The structure of N-14 and its effect on the growth of Pseudomonas aeruginosa;

[0042] A. Chemical structure of N-14, B. Effect on PAO1, C. Effect on single-residual lasR, D. Effect on single-residual rhlR, E. Effect on single-residual pqsR.

[0043] Figure 14 The impact of N-14 on the QS system;

[0044] A, Las screening system; B, Rhl screening system; C, single pqsR mutant.

[0045] Figure 15 The effect of N-14 on biofilms.

[0046] Figure 16 This is a diagram of the molecular docking of N-14 with LasR.

[0047] In this diagram, A and C are both 3D diagrams, while B and D are both 2D diagrams. A and B are the docking results of C12 and LasR, while C and D are the docking results of N-14 and LasR.

[0048] Figure 17 This is a molecular docking diagram of N-14 with PqsR;

[0049] In this diagram, A and C are both 3D diagrams, B and D are both 2D diagrams, A and B are the docking results of PQS and PqsR, and C and D are the docking results of N-14 and PqsR. Detailed Implementation

[0050] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0051] The strains, plasmids, and primers used in this experiment are shown in Tables 1 and 2, respectively. pEX-18Tc was used to construct the vector for the mutant, and pRK2013 was used to construct the helper plasmid for the mutant; Trelief SoSoo Cloning Kit, catalog number TSV-S1, was manufactured by Beijing Qingke Biotechnology Co., Ltd.

[0052] Table 1. Strains and Plasmids

[0053]

[0054] Note:[1]Stover CK,Pham

[0055] [2] Pearson JP, Pesci EC, Iglewski BH: Roles of Pseudomonas aeruginosalas and rhl quorum-sensing systems in control of elastase and rhamnolipidbiosynthesis genes. J Bacteriol 1997,179(18):5756-5767.

[0056] [3]Schweizer HP: Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriTand the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 1992,6(9):1195-1204.

[0057] [4]Figurski DH, Helinski DR: Replication of an origin-containingderivative of plasmid RK2dependent on a plasmid function provided intrans. Proc Natl Acad Sci USA 1979,76(4):1648-1652.

[0058] Table 2 Primers

[0059]

[0060]

[0061] 1.1 Milk Hydrolysis Experiment

[0062] (1) Activation stage of the strain: Pick colonies and inoculate them into liquid LB medium. Incubate at 37°C in a shaker at 200 r / min for 12 h, and adjust OD. 600 (1) The inoculation amount is 0.5; (2) QS donor bacteria and different system detection bacteria solutions are co-inoculated into 5 mL liquid LB according to the inoculation amount of 1%, and cultured for 20 h with or without the addition of the test substance; (3) After culture, centrifuge at 12,000 r / min for 10 min and take the supernatant. Mix the supernatant and 2% liquid skim milk (2g skim milk powder + 100mL distilled water) in an EP tube at a ratio of 3:7, and place it in a 37℃ water bath for 1-2 h. (4) Detect the treated solution directly on a spectrophotometer at a detection wavelength of OD. 440 .

[0063] 1.2 Computer-aided molecular docking method

[0064] In this study, N-14 was used to dock with the QS transcriptional regulators of LasR and PqsR in *Pseudomonas aeruginosa*. Molecular docking analysis was not performed on RhlR due to the lack of a crystal structure. The 3D structures of LasR (PDB ID: 6D6A) and PqsR (PDB ID: 4JVI) were downloaded from the protein database (http: / / www.pdb.org). Maestro 10.1 docks the N-14 structure with the binding sites of LasR and PqsR. The optimal conformation is determined based on the number of hydrogen bonds, hydrophobic interactions, and binding energy.

[0065] 1.3 Other conventional methods: Refer to "Chen Lin. Screening and study of genes related to intrinsic drug resistance in Pseudomonas aeruginosa [D]. Northwest University, 2010". The primer sequences used in the experiments according to this literature are shown in Table 2.

[0066] 2. Construction of the QSI screening system

[0067] 2.1 Introduction to Principles

[0068] This invention constructs knockout mutants, namely JP2ΔrhlRΔpqsAΔpqsR (LasR only) and JP2ΔlasRΔpqsAΔpqsR (RhlR only), by using a homologous recombination gene knockout method (details in section 2.2). Because proteases are directly regulated by the QS signaling system, the QS system in these two strains cannot initiate protease synthesis without a signal molecule supply. However, with the addition of an exogenous signal molecule, the QS system regulates protease synthesis and secretion extracellularly. The culture supernatant (containing the produced protease) can completely hydrolyze a 2% skim milk solution within a certain time, indicating that the QS system performs its normal regulatory process. This invention utilizes this characteristic to screen for QSIs. Without affecting bacterial growth, compounds with QSI activity can inhibit the production of QS-regulated proteases, ultimately resulting in a significant difference in milk hydrolysis experiments compared to the control. To eliminate potential false positives (e.g., the compound directly inhibiting protease activity), milk hydrolysis experiments were conducted using wild-type strain culture supernatant (containing protease) with and without the compound. The results determined whether the compound directly affected protease activity. To reduce screening costs (compared to purchasing commercially available signaling molecule compounds), a co-culture method was used, leveraging the wild-type strain's own signaling molecule production, to provide exogenous signaling molecules. However, the wild-type strain also produces protease during this process. To minimize interference from the wild-type strain's own protease production during culture, this invention knocked out the relevant protease gene in the wild-type strain, resulting in a protease-deficient mutant—ΔlasAΔlasBΔaprA. This mutant significantly reduced protease production; although a small amount of protease was still produced, it had little impact on the screening process of this system.

[0069] Meanwhile, we also constructed a screening system for Pqs system inhibitors, namely JP2ΔlasRΔrhlRΔpqsA (only PqsR is retained). This system cannot be screened by hydrolysis with proteases. Instead, the luminescent reporter plasmid pKD-pqsA is transferred into JP2ΔlasRΔrhlRΔpqsA (only PqsR is retained) to obtain JP2ΔrhlRΔpqsAΔlasR containing the luminescent reporter plasmid pKD-pqsA. The mechanism of action of the inhibitor is determined by the luminescent reporter. The construction of the pKD-pqsA luminescent reporter is the same as in the literature "Liang H, Li L, Dong Z, Surette MG, Duan K: The YebC family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production. J Bacteriol 2008, 190(18): 6217-6227".

[0070] like Figure 1 As shown in Figure A, the left diagram represents ΔlasAΔlasBΔaprA, which can produce the signaling molecules AHL (OdDHL and BHL) and serve as a signaling molecule donor bacterium. The right diagram represents JP2ΔrhlRΔpqsAΔpqsR (single lasR) and / or JP2ΔlasRΔpqsAΔpqsR (single rhlR), which do not produce signaling molecules or proteases themselves. When both are co-cultured, the signaling molecules provided by the left side bind to the receptors on the right side, activating the Las system and / or the Rhl system, thereby producing proteases.

[0071] like Figure 1 As shown in Figure B, when ΔlasAΔlasBΔaprA is used as the PQS signaling molecule donor bacterium and co-cultured with the JP2ΔrhlRΔpqsAΔlasR strain (containing the luminescent reporter plasmid pKD-pqsA), the PQS signaling molecule produced by the donor bacterium binds to the pqsR receptor, thereby activating the Pqs system and subsequently activating the expression of the luminescent reporter pKD-pqsA. When a candidate compound can inhibit luminescence, it can be identified as a candidate compound for QSI.

[0072] By adding exogenous signaling molecules and co-culturing, QSIs can be screened, and it can be determined whether a candidate QSI exerts its effect by inhibiting the synthesis of signaling molecules or interfering with the binding of signaling molecules to regulatory proteins. Simultaneously, the precise target of the compound can be determined, i.e., which of the three QS systems the candidate compound acts on. The co-culture refers to the mixed culture of the QS inhibitor screening system, the mutant, and the compound. Specifically, the protease-deficient mutants ΔlasA, ΔlasB, and ΔaprA are mixed with the QS inhibitor screening system at a 1:1 ratio by bacterial mass, and then the compound is added, followed by co-culture at 30-37°C. The exogenous addition of QS signaling molecules refers to the mixed culture of the QS inhibitor screening system, the compound, and exogenous QS signaling molecules. Specifically, commercially available N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is added to the Las screening system (strain) culture process; N-butanoyl-L-homoserine lactone (C4-HSL) is added to the Rhl screening system (strain) culture process; and 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS) are added to the Pqs screening system (strain) culture process.

[0073] 2.2 Construction of mutants and reporter

[0074] 2.2.1 Construction of protease-deficient mutants—as signaling molecule donor bacteria

[0075] There are four main genes controlling proteases in *Pseudomonas aeruginosa*: lasA, lasB, aprA, and PIV, which regulate elastase A, elastase B, alkaline protease, and protease IV, respectively. In the co-culture system, this invention uses PAO1 as the signal molecule donor. Since the verification method of the screening system uses protease as the detection indicator, it is necessary to reduce the protease yield to minimize interference from PAO1's own protease. Therefore, the lasA, lasB, and aprA genes were knocked out sequentially. The specific knockout steps are as follows:

[0076] First, the lasA gene was knocked out using homologous recombination on the PAO1 strain to obtain the mutant ΔlasA. The primers used in the knockout process are detailed in Table 2. To verify the success of the knockout, the verification primers used were lasA 1-up and lasA 2-down. Using PAO1 as a template, 2760 bp should be amplified, and using ΔlasA as a template, 976 bp should be amplified. The PCR verification diagram is shown below. Figure 2As shown in Figure A, the results indicate that the lasA gene deletion / knockout was successful. Knockout of the lasB gene based on ΔlasA yielded the mutant ΔlasAΔlasB. The verification primers were lasB 1-up and lasB 2-down. Using ΔlasA as a template, 2026 bp was amplified, and using ΔlasAΔlasB as a template, 1351 bp was amplified. The PCR verification diagram is shown below. Figure 2 As shown in Figure B, the results indicate that the lasB gene deletion / knockout was successful. Further knockout of the aprA gene based on ΔlasAΔlasB yielded the mutant ΔlasAΔlasBΔaprA. The verification primers were aprA 1-up and aprA 2-down. Using ΔlasAΔlasB as a template, 1814 bp was amplified, and using ΔlasAΔlasBΔaprA as a template, 564 bp was amplified. The PCR verification diagram is shown below. Figure 3 As shown in Figure A, the results indicate that the aprA gene deletion / knockout was successful.

[0077] 2.2.2 Construction of the Las screening system

[0078] To retain only the Las system within the QS system, this study used a gene knockout method to obtain a quintuple mutant with only the receptor gene lasR retained. First, the rhlR gene was knocked out in strain JP2, resulting in the JP2ΔrhlR deletion mutant. The validation primers were rhlR validation primer up and rhlR validation primer down. Using JP2 as a template, 640 bp was amplified, but no amplification was achieved using JP2ΔrhlR as a template. The PCR validation diagram is shown below. Figure 3 As shown in B. Next, pqsA was knocked out from JP2ΔrhlR to obtain the JP2ΔrhlRΔpqsA deletion mutant. The validation primers were pqsA validation primer-up and pqsA validation primer-down. Using JP2ΔrhlR as a template, 2168 bp was amplified, and using JP2ΔrhlRΔpqsA as a template, 614 bp was amplified. The PCR validation diagram is shown below. Figure 4 A. Finally, pqsR was knocked out from JP2ΔrhlRΔpqsA to obtain JP2ΔrhlRΔpqsAΔpqsR (with only lasR). The verification primers were pqsR full-size up and pqsR full-size down. Using JP2ΔrhlRΔpqsA as a template, 999bp was amplified, and using JP2ΔrhlRΔpqsAΔpqsR as a template, 4000bp was amplified. PCR verification is as follows. Figure 4 B.

[0079] 2.2.3 Construction of the Rhl screening system

[0080] In constructing the QSI screening system, to retain only the Rhl system within the QS system, this paper describes a five-mutant variant of the single-recipient receptor gene rhlR obtained through gene knockout. First, the pqsA gene was knocked out in the JP2ΔlasIΔrhlI strain, resulting in the JP2ΔpqsA deletion mutant. Validation primers were pqsA validation primer up-2 and pqsA validation primer down. Using JP2 as a template, 644bp was amplified, but no amplification was achieved using JP2ΔpqsA as a template. PCR validation is as follows. Figure 4 C. The lasR mutation was obtained by knocking out lasR from JP2ΔpqsA, resulting in the deletion mutant JP2ΔpqsAΔlasR. The validation primers were lasR validation primer 3-up and lasR validation primer down-modified. Using JP2ΔpqsA as a template, 963 bp was amplified, and using JP2ΔpqsAΔlasR as a template, 297 bp was amplified. PCR validation was performed as follows. Figure 5 A. Finally, pqsR was knocked out from JP2ΔpqsAΔlasR to obtain JP2ΔpqsAΔlasRΔpqsR (with only rhlR). The verification primers were pqsR full-size up and pqsR full-size down. Using JP2ΔpqsAΔlasR as a template, 999bp was amplified, and using JP2ΔpqsAΔlasRΔpqsR as a template, 4000bp was amplified. PCR verification was as follows. Figure 5 B.

[0081] 2.2.4 Construction of the Pqs Filtering System

[0082] In constructing the QSI screening system, to retain only the Pqs system within the QS system, this paper obtained a five-mutant variant of the single-retainer receptor gene pqsR through gene knockout. Based on JP2ΔrhlRΔpqsA, the lasR gene was knocked out to obtain the mutant JP2ΔrhlRΔpqsAΔlasR (single-retainer pqsR). The validation primers were lasR validation primer 3-up and lasR validation primer down-modified. Using JP2ΔrhlRΔpqsA as a template, 963 bp should be amplified, and using JP2ΔrhlRΔpqsAΔlasR as a template, 297 bp should be amplified. The PCR validation diagram is shown below. Figure 5 As shown in C.

[0083] 2.3 Feasibility Validation of Quorum Sensing Inhibitor Screening Methods

[0084] 2.3.1 Principle Verification

[0085] Two strains were co-cultured: protease-deficient mutants ΔlasA, ΔlasB, and ΔaprA were co-cultured with single-strain lasR and single-strain rhlR at a 1:1 volume ratio. The former provided signaling molecules. Through the binding of the signaling molecule (AHL) to the receptor (lasR / rhlR), the Las and Rhl systems were activated, respectively, further regulating the expression of protease-related genes and producing proteases.

[0086] like Figure 6 As shown, the single-strain lasR and single-strain rhlR do not produce proteases. However, co-culturing them with strains ΔlasA, ΔlasB, and ΔaprA activates the Las and Rhl systems, respectively, increasing protease production and thus significantly enhancing the degree of milk hydrolysis. In contrast, strains ΔlasA, ΔlasB, and ΔaprA produce less protease and have a less pronounced hydrolytic effect, thus not interfering with the screening system. Co-culturing restores protease production, completely hydrolyzing defatted milk and clarifying the mixed solution. Therefore, this method is proven to be feasible.

[0087] Studies have shown that proteases are regulated by two systems, Las and Rhl, while the Pqs system does not participate in protease production. Figure 6 As shown, mutants that activate the single-residual Pqs system do not produce proteases, therefore proteases cannot be used as a detection indicator to indicate the effect of QSI on the Pqs system. Therefore, the luminescent reporter pKD-pqsA was introduced into the single-residual pqsR knockout mutant, forming a luminescent reporter strain JP2ΔrhlRΔpqsAΔlasR (PKD-pqsA, single-residual pqsR) containing the luminescent reporter plasmid pKD-pqsA. The Pqs system was activated by co-culturing with a strain that produces the signaling molecule (providing the signaling molecule PQS), thereby activating the expression of the pqsA gene, and the Pqs system was measured at the transcriptional level.

[0088] 2.3.2 Drug Validation

[0089] 2.3.1 The screening system used in this study was validated and determined to be feasible. It was then further validated using drugs to confirm the operability of the screening system.

[0090] As is well known, furanone C-30 is currently a widely accepted and reported QSI, therefore furanone C-30 was chosen to calibrate the QSI screening system constructed in this study. Simultaneously, five previously reported QSIs were randomly selected for testing to corroborate the newly constructed QSI screening system.

[0091] 2.3.2.1, Furanone C-30 (studied using the QS inhibitor screening system and co-culture of mutants and compounds)

[0092] Two synthetic derivatives of natural furanones (furanone C-30 and furanone C-56, lacking alkyl side chains) exhibit antagonistic activity against the QS system of *Pseudomonas aeruginosa*, while natural furanone compounds do not show inhibitory activity against the QS system. Synthetic furanone C-30 can reduce QS signaling based on acyl-homoserine lactones and alleviate the virulence of *Pseudomonas aeruginosa*. Furanone C-30 inhibits LasR and RhlR by competing with AHL, thereby interfering with protein folding and solubility, and the inhibition of RhlR is independent of LasR. This study determined the effects of the compound on the Las and Rhl systems using milk hydrolysis experiments at concentrations of 20 μM and 40 μM of furanone C-30. Results are as follows... Figure 7 As shown, 40 μM furanone C-30 inhibited the production of proteases in both the Las and Rhl systems (OD). 440 The values ​​were significantly higher than those of the control group, indicating that furanone C-30 has an inhibitory effect on both the Las and Rhl systems, a result consistent with literature reports.

[0093] 2.3.2.2, Baicalin (studied using the QS inhibitor screening system and co-culture of mutants and compounds)

[0094] Baicalin is a natural bioactive compound extracted from the traditional Chinese medicine Scutellaria baicalensis, belonging to the flavonoid class of compounds. Baicalin can dose-dependently inhibit the motility of polysaccharide (PA), exotoxin A, elastase, rhamnolipids, and other related toxicological factors. Sub-MIC baicalin treatment downregulates the expression of QS-related genes such as lasI, lasR, rhlI, rhlR, pqsR, and pqsA. Baicalin inhibits not only AHL-mediated QS but also the Pqs system. The baicalin concentration used in this study was 250 μg / mL.

[0095] like Figure 8 As shown, 250 μg / mL baicalin inhibited the production of proteases in both the Las and Rhl systems (OD200). 440 The values ​​were all significantly higher than those of the control group, indicating that baicalin inhibited both the Las and Rhl systems.

[0096] 2.3.2.3, Phlorin (studied using the QS inhibitor screening system and co-culture of mutants and compounds)

[0097] Phlorizin is an extract from the root bark of apple trees and belongs to the flavonoid class of compounds. Phlorizin inhibits the luminescence of *E. coli* strains carrying both lasR and lasB-luxCDABE, as well as those carrying rhlR and rhlA-luxCDABE. Phlorizin is a dual LasR / RhlR inhibitor. The phlorizin concentration used in this study was 40 mM.

[0098] like Figure 9 As shown, 40 mM phlorizin inhibits the production of proteases in the Las and Rhl systems (OD200). 440 The values ​​were significantly higher than those of the control group, indicating that phlorizin has an inhibitory effect on both the Las and Rhl systems.

[0099] 2.3.2.4, Phyllanthin (studied using the QS inhibitor screening system and co-culture of mutants and compounds)

[0100] Populin is a flavonoid compound found in fruits, medicinal herbs, and various plants such as passionflower. Populin inhibits pyocyanin, elastase, biofilm formation, and protease activity in the flavonoid polymorphon (PA), affecting plexus motility. It is a novel flavonoid compound with hydroxyl groups at positions 5 and 7 of its A-ring skeleton. Structure-activity relationship analysis showed that the presence of these two hydroxyl groups in the A-ring is essential for inhibiting the LasR and RhlR systems. This indicates that populin can inhibit both the Las and Rhl systems. The concentration of populin used in this study was 4 mg / mL.

[0101] like Figure 10 As shown, 4 mg / mL succinate significantly inhibited the production of protease in the Rhl system (OD200). 440 The value was significantly higher than that of the control group. It did not inhibit the Las system but instead promoted the increase of protease production, indicating that apigenin has an inhibitory effect on the Rhl system and a promoting effect on the Las system.

[0102] 2.3.2.5, Allicin (studied using a QS inhibitor screening system and a mixed culture method of compound and exogenous QS signaling molecule (C4-HSL))

[0103] Allicin (diallyl thiosulfinate) is an important bioactive compound extracted from the edible plant garlic. Garlic extract is the only QSI that has been tested in humans. Component analysis of garlic extract identified ajoene as the most active QSI. However, studies have shown that synthetic ajoene exhibits lower in vitro activity than crude garlic extract. Allicin significantly inhibits the production of signaling molecules C4-HSL and PQS, and also inhibits the production of toxicity factors related to PA, such as rhamnolipids, elastase, pyocyanin, and pyoverdine. Therefore, allicin acts on both the Rhl and Pqs QS systems. The allicin concentrations used in this study were 125 μM and 250 μM.

[0104] like Figure 11 As shown, 125 μM allicin exhibited a significant inhibitory effect on the production of proteases in the Rhl system (OD). 440The value was significantly higher than that of the control group. However, it did not inhibit the Las system but instead promoted the increase of protease production, indicating that 125 μM allicin inhibited the Rhl system but promoted the Las system.

[0105] 2.3.2.6, Tea polyphenols (studied using the QS inhibitor screening system and co-culture of mutants and compounds)

[0106] Tea polyphenols are an aqueous extract of the non-fermented leaves of the tea plant. Tea contains various components, including catechins, caffeine, fluorides, and other undefined compounds. These components may contribute to human health, exhibiting antioxidant, anticancer, and antibacterial activities. Tea polyphenols reduce the production of purple pigment in *Pseudomonas aeruginosa*, and also decrease the production of PA-related virulence factors such as proteases, pyocyanin, and rhamnolipids, as well as biofilm formation. They may also reduce mortality in *C. elegans* and decrease the incidence of *Pseudomonas aeruginosa* infection in a mouse model of wound resection. The tea polyphenol concentration used in this study was 3.125 mg / mL, dissolved in PBS buffer.

[0107] like Figure 12 As shown, 3.125 mg / mL tea polyphenols did not significantly inhibit the production of proteases in the Las and Rhl systems (OD200). 440 The values ​​were almost identical to those of the control group, indicating that allicin had no effect on either the Las or Rhl systems.

[0108] 2.4 New QSI – N-14

[0109] The screening method presented in this study has been validated through its principles and by verification with six previously reported QSIs, demonstrating its feasibility. Therefore, this study utilizes this screening system to screen for unknown compounds, aiming to identify novel QSIs.

[0110] Many amide-based chemically synthesized drugs have been reported as QSIs, such as N-(2-pyrimidinyl)butyramide (a structural analog of C11, C4-HSL), phenylalanine-arginine-β-naphthylamide and benzamide-benzimidazole compounds, and 2-difluoromethylpyridine compounds. Pyridine amides have bactericidal activity, and no such QSIs have been reported to date. Therefore, this study synthesized 20 pyridine amide chemically synthesized compounds for preliminary screening. As a result, N-14 was selected as a novel QSI (chemical name: N-5-(1-methylpyrrolidone-2-yl)pyridin-2-ylbut-3-yneamide, chemical structure shown in [reference needed]) without affecting growth. Figure 13 A).

[0111] 2.4.1 Effect of N-14 on the growth of Pseudomonas aeruginosa

[0112] First, a necessary prerequisite for screening accurate QSIs is that they do not inhibit cell production; if growth is inhibited, the QS system will also be inhibited. Therefore, this study first examined the effect of compound N-14 on the growth of PAO1. Figure 13 As shown in Figure B, compound N-14 does not inhibit the growth of PAO1 cells at concentrations of 80 μg / mL and below, therefore it does not exert selective pressure on the bacteria. Figure 13 As shown in C, D, and E, the effect of compound N-14 on the growth of knockout mutants of the three single-residue QS systems was examined. Neither compound N-14 nor compound N-14 affected the growth of Pseudomonas aeruginosa at concentrations of 60 μg / mL and 80 μg / mL.

[0113] 2.4.2 Impact of N-14 on the QS System

[0114] This study aimed to screen for QSIs from 20 chemically synthesized compounds, and therefore the novel QSI screening system designed in this paper was used for the experiment.

[0115] like Figure 14 As shown in A and B, 60 μg / mL of compound N-14 significantly inhibited the production of proteases in both the Las and Rhl systems (OD). 440 The values ​​were all significantly higher than those of the control group, indicating that compound N-14 can inhibit the Las and Rhl systems. Figure 14 As shown in Figure C, compound N-14 at concentrations of 60 μg / mL and 80 μg / mL significantly inhibited the expression of gene pqsA, indicating that compound N-14 also has an inhibitory effect on the Pqs system.

[0116] 2.4.3 Effects of N-14 on biofilms

[0117] Studies have shown that biofilms are one of the important virulence factors produced by paclobutrazol (PA). The presence of biofilms allows bacteria to colonize wound surfaces, and the virulence factors encapsulated in the biofilm accumulate, leading to host pathogenicity. Simultaneously, biofilms are crucial for bacterial resistance to antibiotics. Therefore, detecting the effect of N-14 on biofilm is also a key indicator. This paper uses two concentrations of compound N-14, 60 μg / mL and 80 μg / mL, to determine the biofilm composition. Figure 15 As shown, compound N-14 has a significant inhibitory effect on the production of biofilms.

[0118] 2.4.4 Influence of Molecular Docking Prediction N-14 on the QS System

[0119] The above results confirm that N-14 is a novel QSI that acts on both the Las and Rhl QS systems. Therefore, this paper considers using computer molecular docking to assist in determining the above results, providing a strong basis for further identifying the target of N-14.

[0120]

[0121] The reference method was used to perform molecular docking between N-14 and the protein LasR. The results showed that N-14 mainly binds to the amino acids Leu, Tle, Ala, and Ile of the LasR protein, and also binds to the amino acids Thr and Tyr through hydrogen bonds (see [reference]). Figure 16 D). Compared to C 12 The total binding energy of -HSL (as above (1), CAS: 137173-46-7) to LasR is -8.578 kcal / mol, and the total binding energy of N-14 to LasR is -7.784 kcal / mol. Furthermore, in the references, the PQS signaling molecule docks with the PqsR protein. The amino acids that N-14 binds to the PqsR protein include Phe, Pro, Ile, and Ala, and it also binds to Leu via hydrogen bonds (see...). Figure 17 (D). The results showed that the total binding energy of PQS to PqsR was -6.187 kcal / mol, and the total binding energy of N-14 to PqsR was -3.542 kcal / mol. These results confirm that N-14 binds more strongly to LasR protein than to PqsR. Figure 17 The diagram shows the molecular docking of N-14 with PqsR; A and C are 3D diagrams, while B and D are 2D diagrams. A and B represent the docking results of PQS with PqsR, while C and D represent the docking results of N-14 with PqsR. In summary, N-14 can bind to both LasR and PqsR, therefore, N-14 may be able to act on the Las, Rhl, and Pqs systems.

[0122] 3. Discussion and Conclusion

[0123] This invention presents a novel QSI screening method using gene knockout. This method offers several advantages, such as accurately identifying which quorum sensing system a compound acts on while simultaneously determining whether it is a QSI. It is simple to operate, has a fast detection speed, and the use of co-culture saves costs. Furthermore, by adding exogenous signaling molecules and co-culturing, the mechanism of action of QSIs can be determined: whether they inhibit the synthesis of QS signaling molecules or affect the interaction between QS signaling molecules and regulatory proteins. By centrifuging co-culture media with and without the analyte, the supernatant is collected, and the amount of signaling molecules or protease in the supernatant is compared to preliminarily determine whether the analyte inhibits the synthesis of signaling molecules. By adding commercially available signaling molecules exogenously to a single-component Las / Rhl screening system, the amount of protease produced in the supernatant of the culture media with and without the analyte can be compared to determine whether the mechanism of action of the signaling molecules is affected. The co-culture method used in this study reduces experimental costs for initial screening and is of great significance for the promotion of this technology.

[0124] This invention also optimizes the detection sensitivity and accuracy. Literature reports that the detection of protease hydrolysis often uses the skim milk agar plate method with a ratio of 5%-10%. This involves adding an appropriate amount of supernatant after culture to a perforated plate and incubating at 37°C for 24-48 hours. The protease yield is determined by the diameter of the transparent area of ​​the hydrolysis zone. However, this method has the following drawbacks: (1) It is time-consuming, requiring too much time and extending the entire experimental cycle; (2) The results are inaccurate. The supernatant after centrifugation is not completely sterilized. During the incubation process on the nutrient-rich milk plate, a small number of bacteria remaining in the supernatant will regrow under suitable conditions, leading to inaccurate final results. The simple optimization of this invention involves replacing the skim milk plates with a skim milk solution, using a 2% skim milk concentration. The supernatant after centrifugation is added to the 2% skim milk solution, and the mixture is thoroughly mixed in an EP tube at a milk-to-supernatant ratio of 7:3. The tube is then incubated in a 37°C water bath for 1-2 hours. The milk hydrolysis can be observed visually, or the absorbance can be measured using a spectrophotometer for comparison. Compared to previous methods, this significantly reduces experimental time, saves materials, and eliminates the impact of secondary bacterial growth, while also enabling high-throughput screening. This aligns with the design philosophy of this study: simple, convenient, and rapid. Furthermore, the experiment uses QS-regulated proteases as indicators, resulting in significantly improved sensitivity of enzymatic reactions compared to traditional methods, less susceptibility to external environmental factors, and significantly improved accuracy.

[0125] This invention uses the well-known QSI—furanone C-30—to calibrate a novel screening system. Five compounds were randomly selected from previously reported QSIs for testing, further demonstrating the accuracy and feasibility of the screening system. Literature reports indicate that furanone C-30's mechanism of action involves competing with natural signaling molecules and subsequently blocking AHL receptors, thereby inhibiting the QS system. It has also been demonstrated that furanone C-30 independently inhibits LasR and RhlR. Using furanone C-30 in the QSI screening method presented in this paper, the results showed that furanone C-30 acts independently on the Las and Rhl systems. The effect of furanone C-30 on the Pqs system was detected using a microplate reader, revealing that furanone C-30 also inhibits the Pqs system. Therefore, it can be concluded that furanone C-30 has inhibitory effects on all three systems: Las, Rhl, and Pqs.

[0126] In summary, this study aimed to design a simple and rapid QSI screening method, which can significantly reduce the screening time and improve accuracy. Validation of five reported QSI compounds in the literature fully demonstrates the feasibility of the QSI screening method presented in this study. Furthermore, this study successfully screened a novel chemically synthesized QSI—N-14—based on the new screening method. This novel QSI acts on the Las, Rhl, and Pqs QS systems. As is well known, drug resistance in Pseudomonas aeruginosa is becoming increasingly serious, and inhibiting quorum sensing systems is one of the most researched areas. Rapidly screening for effective QSIs is urgently needed. The experimental results show that the method presented in this paper can achieve the expected goals, providing a new approach to QSI screening and theoretical support for accelerating the discovery of true QSIs.

[0127] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

Claims

1. A method for screening for a quorum-sensing inhibitor while simultaneously determining its target, characterized by, The QS inhibitor screening system comprises: A QS inhibitor screening system is constructed, and the QS inhibitor screening system takes a protease or a luminescent reporter as a screening marker; A mutant with a deleted protease gene is constructed, which provides QS signal molecules during co-culture; QS system inhibitors are screened by co-culture or exogenous addition of QS signal molecules, and the target of the compound is determined; The co-culture refers to mixed culture of the QS inhibitor screening system, the mutant with a deleted protease gene, and the compound. The exogenous addition of QS signal molecules refers to mixed culture of the QS inhibitor screening system, the compound, and exogenous QS signal molecules. The QS inhibitor screening system includes a Las screening system, a Rhl screening system, and a Pqs screening system containing a luminescent reporter pKD- pqsA -1. The construction method of the Las screening system is as follows: by a homologous recombination method, JP2 is sequentially subjected to rhlR , pqsA , pqsR gene knockout, and finally the strain JP2Δ rhlR Δ pqsA Δ pqsR that can be used for screening Las system inhibitors is obtained. The JP2 is a strain in which PAOl is knocked out lasI , rhlI two genes The construction method of the Rhl screening system is as follows: by homologous recombination method, JP2 is sequentially subjected to pqsA , lasR , pqsR gene knockout, and finally the strain JP2Δ pqsA Δ lasR Δ pqsR can be used for screening Rhl screening system inhibitors. The construction method of the Pqs screening system is as follows: by a homologous recombination method, JP2 is sequentially subjected to gene knockout rhlR , pqsA , lasR , and finally JP2Δ rhlR Δ pqsA Δ lasR pKD- pqsA is introduced into JP2Δ lasR Δ rhlR Δ pqsA , and the Pqs screening system is obtained. Protease deletion mutants are mutants obtained on the basis of the PAOl strain ΔlasAΔlasBΔaprA Protease deletion mutants ΔlasAΔlasBΔaprA Cocultured with QS inhibitor screening system and inhibitor respectively to verify whether the inhibitor can play an inhibitory role and determine the target.

2. The method for screening quorum-sensing inhibitors and simultaneously determining their target according to claim 1, wherein, According to the protease production of the QS inhibitor screening system after co-culture or exogenous addition of QS signal molecules, it is determined whether the compound is a quorum sensing inhibitor and whether it acts on the Las system or the Rhl system. In addition, it is determined whether the compound acts on the Pqs system by the inhibition of the luminescent reporter of the Pqs screening system.

3. The method for screening quorum-sensing inhibitors and simultaneously determining their target according to claim 1, wherein the bacteria are selected from the group consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella enterica, Vibrio cholerae, and Bacillus subtilis. The co-culture method is as follows: Protease deletion mutant Δ lasA Δ lasB Δ aprA Mixed with QS inhibitor screening system strain, then add compound, co-culture at 30-37°C.

4. The method for screening quorum-sensing inhibitors and simultaneously determining their target according to claim 3, wherein, The exogenous addition of QS signal molecules is as follows: During the culture of the QS inhibitor screening system, different concentrations of signal molecules are directly added to the culture medium. N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is added to the culture process of the Las screening system; N-butanoyl-L-homoserine lactone (C4-HSL) is added to the culture process of the Rhl screening system; 2-heptyl-4-quinolone (HHQ) or / and 2-heptyl-3-hydroxy-4-quinolone (PQS) is added to the culture process of the Pqs screening system.

5. The method for screening quorum-sensing inhibitors and simultaneously determining their target according to claim 1, wherein, The screened quorum sensing inhibitor is a pyridine amide compound.

6. The method for screening quorum-sensing inhibitors and simultaneously determining their target according to claim 5, wherein, The pyridine amide compound is N-14, and the structural formula is as follows: 。

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