Application of isopsoralen chalcone in preparation of preparation for inhibiting pseudomonas aeruginosa quorum sensing system

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of isopsoralen chalcone in preparation of a pseudomonas aeruginosa quorum sensing system inhibiting preparation. The invention discloses isobavachalcone which can effectively interfere with a quorum sensing system of pseudomonas aeruginosa, and particularly can inhibit a PQS system in the quorum sensing system of the pseudomonas aeruginosa, inhibit expression of a gene pqsA and weaken toxicity and pathogenicity of the pseudomonas aeruginosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to the application of isobavachalcone in the preparation of a preparation for inhibiting the quorum sensing system of Pseudomonas aeruginosa. Background Art

[0002] Pseudomonas aeruginosa is a Gram-negative bacterium that widely exists in natural and artificial environments. Due to the abuse of antibiotics, the emergence of multi-drug resistant and even pan-drug resistant Pseudomonas aeruginosa has brought great challenges to clinical treatment. Research shows that the pathogenicity and drug resistance of Pseudomonas aeruginosa are related to quorum sensing (QS). The quorum sensing system regulates the formation of pathogenic bacterial biofilms by mediating the expression of pathogenic genes and controls its pathogenicity. An efficient quorum sensing inhibitor (QSI) is expected to achieve the control of the pathogenicity and biofilm of pathogenic bacteria. Pseudomonas aeruginosa has three quorum sensing systems, namely the LAS, RHL, and PQS systems. Among them, the LAS system consists of LasI and LasR. LasI is the synthase of the signal molecule, synthesizing N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), and LasR is the receptor of this signal molecule. Similar to the LAS system, the RHL system consists of RhlI and RhlR. RhlI is the synthase of the signal molecule, synthesizing N-butyryl-L-homoserine lactone (C4-HSL), and RhIR is the receptor of this signal molecule. In the PQS system, PhnA, PhnB, PqsA, PqsB, PqsC, PqsD, PqsE, and PqsH jointly catalyze the synthesis of two signal molecules, 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), and PqsR is the receptor of these signal molecules. The three quorum sensing systems, LAS, RHL, and PQS, jointly regulate the production of virulence factors of Pseudomonas aeruginosa, such as extracellular protease LasA, elastase LasB, alkaline protease, hydrogen cyanide, exotoxin A, rhamnolipid, pyocyanin, and biofilm.

[0003] The CAS number of isobavachalcone is 20784-50-3, and its molecular formula is C 20 H 20 O 4, with a molecular weight of 324.38, and its chemical formula is as follows:

[0004]

[0005] Isobavachalcone is a chalcone / dihydrochalcone compound isolated and identified from the dried ripe fruits of the leguminous plant Psoralea corylifolia Linn., namely Psoralea corylifolia. In current pharmaceutical research, isobavachalcone is an Akt inhibitor, which can inhibit platelet aggregation, can be used as an inhibitor of Epstein-Barr virus early antigen (EBV-EA) induction, has a strong inhibitory effect on the promotion of skin tumors, can be used as a strong inhibitor of MMP-2, and has DNA strand breakage (cleavage) activity. Currently, there is no report on the work of isobavachalcone inhibiting the Pseudomonas aeruginosa PQS quorum sensing system. Summary of the Invention

[0006] The object of the present invention is to provide a natural small molecule bacterial quorum sensing inhibitor for the treatment of diseases caused by Gram-negative bacterium Pseudomonas aeruginosa, and to provide more options for solving the problem of Pseudomonas aeruginosa drug resistance. In this regard, the present invention provides the application of isobavachalcone in the preparation of a preparation for inhibiting the Pseudomonas aeruginosa quorum sensing system to meet this need in the art.

[0007] On the one hand, the present invention relates to the application of isobavachalcone or a pharmaceutically acceptable salt thereof in the preparation of a preparation for inhibiting the Pseudomonas aeruginosa quorum sensing system.

[0008] Further, in the application provided by the present invention, isobavachalcone or a pharmaceutically acceptable salt thereof acts on the PQS system in the Pseudomonas aeruginosa quorum sensing system.

[0009] Further, in the application provided by the present invention, isobavachalcone or a pharmaceutically acceptable salt thereof inhibits the expression of the gene pqsA in the Pseudomonas aeruginosa quorum sensing system.

[0010] Further, in the application provided by the present invention, the related genes of the PQS system are pqsA, pqsB, pqsC, pqsD and pqsE.

[0011] Further, in the application provided by the present invention, the action target of isobavachalcone or a pharmaceutically acceptable salt thereof is the PqsR protein.

[0012] On the other hand, the present invention relates to the application of isobavachalcone or a pharmaceutically acceptable salt thereof in the preparation of a preparation for preventing and treating Pseudomonas aeruginosa, which includes: isobavachalcone or a pharmaceutically acceptable salt thereof inhibits the virulence and / or pathogenicity of Pseudomonas aeruginosa.

[0013] Furthermore, in the application provided by the present invention, the virulence includes: the ability of Pseudomonas aeruginosa to produce pyocyanin.

[0014] Furthermore, in the application provided by the present invention, the virulence includes: the motility of Pseudomonas aeruginosa.

[0015] Specifically, the motility of Pseudomonas aeruginosa includes: swarming motility, swimming motility, and twitching motility.

[0016] Furthermore, in the application provided by the present invention, the pathogenicity includes: the ability of Pseudomonas aeruginosa to infect plants.

[0017] Furthermore, in the application provided by the present invention, the virulence includes: the ability of Pseudomonas aeruginosa to infect animals.

[0018] As used herein, "prevention and treatment" means that in the presence of possible Pseudomonas aeruginosa or factors leading to the occurrence of Pseudomonas aeruginosa infection, after use, it can prevent or reduce the enrichment of Pseudomonas aeruginosa, and cure or reduce the virulence and / or pathogenicity of Pseudomonas aeruginosa.

[0019] In the present invention, the term "pharmaceutically acceptable" means that it has no long-term harmful effects on the general health of the subject being treated.

[0020] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of isobavachalcone and has no adverse effects biologically or otherwise. Pharmaceutically acceptable salts refer to converting the basic group in the parent compound into a salt form, such as inorganic or organic acid salts of basic groups (such as amino groups). Generally, the parent compound is reacted with conventional types of acids in a solvent system for preparation. Inorganic acids generally include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid, etc.; organic acids generally include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, or salicylic acid, etc.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0022] The present invention provides a natural small - molecule bacterial quorum - sensing inhibitor, isobavachalcone, for preparing a preparation for inhibiting the quorum - sensing system of Pseudomonas aeruginosa. This discovery provides more options for solving the problem of Pseudomonas aeruginosa drug resistance. Isobavachalcone can effectively interfere with the quorum - sensing system of Pseudomonas aeruginosa, especially the PQS system, inhibit the expression of the gene pqsA, thereby weakening the virulence and pathogenicity of Pseudomonas aeruginosa. This inhibitory effect is not only reflected in the regulation of bacterial biofilm formation, but also directly affects the motility of Pseudomonas aeruginosa (including swarming motility, swimming motility and twitching motility) and the synthesis of pyocyanin. In addition, the application of isobavachalcone significantly reduces the pathogenicity of Pseudomonas aeruginosa to host models such as Chinese cabbage and Caenorhabditis elegans, showing its potential in plant and animal protection. Compared with chemical antibacterial agents, the plant natural product isobavachalcone has the advantages of wide sources and high safety, and is not likely to cause the generation of bacterial drug resistance. More importantly, through in - depth research, the present invention reveals that the action target of isobavachalcone is the transcriptional activator protein PqsR of the PQS quorum - sensing system, which provides a solid theoretical basis for its further development and application. Therefore, isobavachalcone, as a Pseudomonas aeruginosa quorum - sensing inhibitor, has great potential for developing into a therapeutic drug or adjuvant drug for solving Pseudomonas aeruginosa infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a schematic diagram of the effect of isobavachalcone on the gene expression of the PQS, RHL and LAS systems of Pseudomonas aeruginosa.

[0025] Among them, A is the effect of 20 μg / mL isobavachalcone on the expression of the QS gene pqsA; B is the effect of 20 μg / mL isobavachalcone on the expression of the QS gene lasI; C is the effect of 20 μg / mL isobavachalcone on the expression of the QS gene rhlI.

[0026] Figure 2 It is a schematic diagram of the effect of isobavachalcone on the growth of Pseudomonas aeruginosa. Among them, A is the effect of 20 μg / mL isobavachalcone on the growth of Pseudomonas aeruginosa; B is the effect of 100 μg / mL isobavachalcone on the growth of Pseudomonas aeruginosa

[0027] Figure 3Schematic diagram of the effects of different concentrations of isobavachalcone on the gene expression of the PQS system in Pseudomonas aeruginosa. Among them, A shows the changes in the expression level of pqsA in the wild-type strain of Pseudomonas aeruginosa with the increase in the concentration of isobavachalcone; B shows the half-inhibitory concentration (IC50) of isobavachalcone on the expression level of pqsA in the wild-type strain of Pseudomonas aeruginosa.

[0028] Figure 4 Schematic diagram of the effects of different concentrations of isobavachalcone on the gene expression of the PQS system in the PqsR protein overexpressing strain. Among them, A shows the changes in the expression levels of pqsA in the wild-type strain and the pqsR overexpressing strain of Pseudomonas aeruginosa with the increase in the concentration of isobavachalcone. B shows the half-inhibitory concentration (IC50) of isobavachalcone on the expression level of pqsA in the wild-type strain of Pseudomonas aeruginosa; C shows the half-inhibitory concentration (IC50) of isobavachalcone on the expression level of pqsA in the pqsR overexpressing strain of Pseudomonas aeruginosa.

[0029] Figure 5 Schematic diagram of the molecular docking of isobavachalcone and PqsR protein. Among them, A is the 3D diagram. B is the enlarged partial 3D diagram. C is the 2D interaction diagram.

[0030] Figure 6 Analysis of potential interacting amino acid residue targets of isobavachalcone on PqsR. Among them, A shows the expression of pqsA after site-directed mutation of PqsR (with or without isobavachalcone); B is the schematic diagram of the molecular docking of isobavachalcone and PqsR I236L

[0031] Figure 7 Schematic diagram of the effects of isobavachalcone on the motility of Pseudomonas aeruginosa. Among them, A is swarming motility; B is swimming motility; C is twitching motility.

[0032] Figure 8 Schematic diagram of the effects of isobavachalcone on pyocyanin synthesis in Pseudomonas aeruginosa. Among them, A and B show the expression levels of pyocyanin synthesis genes (phzA1 and phzA2) in the wild-type strain of Pseudomonas aeruginosa after treatment with traditional Chinese medicine monomers. C and D show the quantitative determination of pyocyanin in the wild-type strain of Pseudomonas aeruginosa after treatment with traditional Chinese medicine monomers.

[0033] Figure 9 Schematic diagram of the effects of isobavachalcone on the pathogenicity of Pseudomonas aeruginosa. Among them, A and B show the effects of treatment with traditional Chinese medicine monomers on the infection of Chinese cabbage by Pseudomonas aeruginosa; C shows the effect of treatment with traditional Chinese medicine monomers on the infection of Caenorhabditis elegans by Pseudomonas aeruginosa. Detailed implementation methods

[0034] ​Next, the technical solutions of the present invention will be described in conjunction with embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The % in the following embodiments is the mass percentage unless otherwise specified. The ratios in the following embodiments are mass ratios unless otherwise specified.

[0035] The sources of the test materials in the following embodiments are as follows:

[0036] Pseudomonas aeruginosa PAO1 used in the present invention is derived from the Pathogenic Microbiology Laboratory of Yan'an University; other related strains are constructed based on PAO1 and used to explain the present invention.

[0037] Isobavachalcone and eupatilin used in the present invention are purchased from Chenguang Biotechnology Co., Ltd. in Baoji City. Using DMSO as a solvent, isobavachalcone and eupatilin are prepared into a mother liquor of traditional Chinese medicine monomers with a concentration of 4096 μg / mL. Through MIC (minimum inhibitory concentration) detection, the present invention finds that the MIC (minimum inhibitory concentration) of isobavachalcone and eupatilin against Pseudomonas aeruginosa is greater than 256 μg / mL, indicating that these two traditional Chinese medicine monomers do not affect the growth of Pseudomonas aeruginosa within a specific concentration range (<256 μg / mL) and will not cause selective pressure on Pseudomonas aeruginosa.

[0038] The primers and related restriction enzyme site information used in the present invention are shown in the following table:

[0039]

[0040]

[0041]

[0042] Among them, the underlined sequences are restriction enzyme sites.

[0043] Example 1: Construction of a gene expression screening model for the QS system of Pseudomonas aeruginosa

[0044] Experiment for constructing transcriptional fusion strains.

[0045] Step 1: The present invention designs primers pqsA F / pqsA R using Pseudomonas aeruginosa genomic DNA as a template, and uses this primer pair to perform PCR amplification of the promoter sequence PpqsA of pqsA, and the size of the DNA fragment is 1105 bp;

[0046] Step 2: Double-digest PpqsA and plasmid mini-CTX-lacZ with Sal I / Pst I;

[0047] Step 3: Purify the digested PpqsA and ligate it to the plasmid mini-CTX-lacZ, then transform it into competent Escherichia coli TG1 cells. Spread the cells on an LB plate containing tetracycline hydrochloride (Tetracycline Tc, 200 μg / mL, abbreviated as Tc200) for screening.

[0048] Step 4: After PCR testing of the obtained colonies, extract the plasmid to obtain the recombinant vector. Digest the recombinant vector with Sal I / Pst I for verification, and obtain the recombinant vector mini-CTX-PpqsA-lacZ showing a positive result.

[0049] Step 5: Transform mini-CTX-PpqsA-lacZ into S17-1 to obtain S17-1 mini-CTX-PpqsA-lacZ.

[0050] Step 6: Conjugate the obtained recombinant strain S17-1 mini-CTX-PpqsA-lacZ with Pseudomonas aeruginosa PAO1 on an LB plate at 37 °C for 48 h. Suspend the conjugated bacterial lawn with liquid LB, appropriately dilute it, and spread it on an LB double-antibiotic plate containing kanamycin (Kanamycin Km, 30 μg / mL, abbreviated as Km30) and Tc200. After re-streaking the obtained colonies on the double-antibiotic plate for culture, obtain the transcriptional fusion strain (i.e., the gene expression screening model of the Pseudomonas aeruginosa PQS system): PAO1 attB::mini-CTX-PpqsA-lacZ; Use the same method to obtain the gene expression screening models of the other two quorum sensing systems (RHL quorum sensing system and LAS quorum sensing system) of Pseudomonas aeruginosa: PAO1 attB::mini-CTX-PrhlI-lacZ and PAO1 attB::mini-CTX-PlasI-lacZ.

[0051] The results are as Figure 1 shown. Compared with the DMSO solvent and the isoeupatorin control treatment group, isobavachalcone at a concentration of 100 μg / mL significantly inhibited the promoter activity of pqsA, and the inhibition effect exceeded 50%, and this difference reached a significant level (****, p < 0.0001). In addition, compared with pqsA, isobavachalcone did not have a significant effect on the expression of rhlI and lasI, indicating that isobavachalcone only has a significant inhibitory effect on the PQS quorum sensing system of Pseudomonas aeruginosa.

[0052] Example 2: Effect of isobavachalcone on the growth of Pseudomonas aeruginosa

[0053] Growth curve analysis experiment under the action of isobavachalcone.

[0054] Growth curve analysis experiment under the action of isobavachalcone. The strain was inoculated into liquid LB medium and transferred to fresh liquid LB medium at a ratio of 1:100 after overnight culture. The treatment groups were added with 20 μg / mL and 100 μg / mL of isobavachalcone respectively, and the control groups were added with 20 μg / mL and 100 μg / mL of eupatilin and DMSO respectively. They were cultured with shaking at 37 °C and 220 r / min, and samples were taken at 2-h intervals to measure OD 600 , and the growth curve was plotted. An appropriate amount of antibiotic was added to the medium as needed.

[0055] Using the wild strain PAO1 of Pseudomonas aeruginosa as the test strain, a total of three groups of experimental treatments were set. The first group was added with isobavachalcone at final concentrations of 20 μg / mL and 100 μg / mL in the medium respectively, the second group was added with eupatilin at final concentrations of 20 μg / mL and 100 μg / mL in the medium respectively, and the third group was added with the same volume of DMSO solvent in the medium. Each group was set with three biological replicates. The results are as Figure 2 shown in Figure 2 A and

[0056] Example 3: Effects of different concentrations of isobavachalcone on the gene expression of the PQS quorum sensing system in Pseudomonas aeruginosa Detection experiment on the gene expression of the PQS quorum sensing system under the action of different concentrations of isobavachalcone.

[0057] Detection experiment on the gene expression of the PQS quorum sensing system under the action of different concentrations of isobavachalcone. To detect the effects of different concentrations of isobavachalcone on the PQS system, the present invention incubated different concentrations of isobavachalcone with the PAO1::mini-CTX-PpqsA-lacZ strain according to the following steps, and analyzed the expression of the pqsA promoter by detecting the activity of β-galactosidase;

[0058] Step 1: Using the traditional Chinese medicine monomer eupatilin and DMSO solvent that have no effect on the PQS quorum sensing system of Pseudomonas aeruginosa as negative controls, incubate different concentrations of isobavachalcone solution with the transcriptional fusion strain PAO1attB::mini-CTX-PpqsA-lacZ, and culture at 37 °C and 200 rpm until OD 600 reaches 2.0;

[0059] Step 2. To detect the β-galactosidase activity of the reporter strain, 2-nitrophenyl-β-D-galactopyranoside (ONPG) was used as the substrate for the assay: Pipette 20 - 100 μL of the bacterial liquid cultured to the logarithmic phase, and successively add 420 μL of Z buffer (60 mM / L Na 2 HPO 4 、40 mM / L NaH 2 PO 4 、10 mM / L KCl、1 mM / L MgSO 4 、pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform, and 10 μL of 0.1% sodium dodecyl sulfate (SDS). After the mixture was rapidly mixed for 20 s, it was incubated at 30 °C for 1 h. After incubation, 100 μL of ONPG with a concentration of 4 mg / mL was added to the mixture for reaction. The reaction was terminated by adding 250 μL of 1 M Na 2 CO 3 , and the reaction time was recorded. The OD 420 and OD 550 of the mixture were detected, and then the β-galactosidase activity was calculated in Miller units (MU) according to the following equation:

[0060] MU = 1,000 × (OD 420 - 1.75 × OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0061] The results were as Figure 3 shown. The pqsA promoter activity decreased with the increase in the concentration of isobavachalcone, showing concentration-dependent inhibition, and the IC 50 was 8.611 μg / mL. The inhibition efficiency of isobavachalcone on pqsA expression reached the maximum at 16 μg / mL. Therefore, to ensure the inhibition of the PQS system activity of Pseudomonas aeruginosa by isobavachalcone, the concentration of isobavachalcone was 20 μg / mL and OD 600 was 2.0 for the following studies.

[0062] Example 4: Effects of different concentrations of isobavachalcone on the expression of PQS quorum sensing system genes in PqsR protein overexpressing strains

[0063] Detection experiment on the expression of PQS quorum sensing system genes in PqsR protein overexpressing strains with different concentrations of isobavachalcone.

[0064] Step 1: Design primers pqsR F / pqsR R using Pseudomonas aeruginosa genomic DNA as a template, and PCR amplify the 999bp sequence of gene pqsR with these primers;

[0065] Step 2: Perform double digestion on the pqsR gene fragment and plasmid pUCP24 with Xho I / Pst I;

[0066] Step 3: Purify the digested pqsR gene fragment and ligate it to plasmid pUCP24, transform it into Escherichia coli TG1 competent cells, and spread it on an LB plate containing gentamicin (Gentamicin Gm, 100μg / mL, abbreviated as Gm100) for screening;

[0067] Step 4: Obtain positive clone strains through colony PCR screening, extract the recombinant vector therein, perform enzyme digestion verification on the recombinant vector with Xho I / Pst I, and obtain the recombinant vector pUCP24-pqsR;

[0068] Step 5: Pick a single colony of PAO1 attB::mini-CTX-PpqsA-lacZ into 5 mL of LB liquid medium, and culture it overnight at 37°C with 200 rpm;

[0069] Step 6: Transfer the bacterial liquid obtained in Step 5 to 50 mL of fresh LB liquid medium at a ratio of 1:100, culture it at 37°C with 200 rpm until the OD 600 is between 0.75 - 1.25, centrifuge at 4°C and 8100 rpm for 8 min, and collect the bacterial cells;

[0070] Step 7: Resuspend the bacterial cells with 50 mL of pre-cooled 300 mM sucrose solution, centrifuge at 4°C and 8100 rpm for 8 min, and collect the bacterial cells;

[0071] Step 8: Resuspend the bacterial cells with 25 mL of pre-cooled 300 mM sucrose solution, centrifuge at 4°C and 8100 rpm for 8 min, and collect the bacterial cells;

[0072] Step 9: Resuspend the bacterial cells with 0.5 mL of pre-cooled 300 mM sucrose solution, ice-bath for 30 min to prepare electrocompetent cells, and aliquot 100 μL per tube for later use;

[0073] Step 10: Take an appropriate amount of recombinant vector pUCP24-pqsR and add it to the electrocompetent cells of PAO1 attB:mini-CTX-PpqsA-lacZ, mix well and transfer it to a sterile and pre-cooled electroporation cuvette for electroporation. The electroporation conditions are set as: 25 μF, 200 Ω, 1900 V, 3 - 5 ms;

[0074] Step 11: Transfer the electrotransformed cells into 5 mL of fresh LB liquid medium and culture at 37 °C with 200 rpm for 3 - 5 h;

[0075] Step 12: Take an appropriate amount of the bacterial liquid and spread it on a TSB medium containing Km30, Tc200, and Gm100 for resistance screening. After culturing at 37 °C for 2 d, streak the obtained colonies on a new TSB medium containing Km30, Tc200, and Gm100 to obtain the pqsR gene overexpression strain PAO1 attB::mini-CTX-PpqsA-lacZ (pUCP24-pqsR);

[0076] Step 13: Use the same method to obtain the empty vector control strain PAO1 attB::mini-CTX-PpqsA-lacZ (pUCP24);

[0077] Step 14: To detect the effect of different concentrations of isobavachalcone on the gene expression of the PQS quorum sensing system in the PqsR protein overexpression strain, the present invention uses the traditional Chinese medicine monomer eupatilin and DMSO solvent, which have no effect on the PQS quorum sensing system of Pseudomonas aeruginosa, as negative controls. Incubate different concentrations of isobavachalcone with PAO1 attB::mini-CTX-PpqsA-lacZ (pUCP24-pqsR) and its empty vector control strain respectively, and culture at 37 °C with 200 rpm until OD 600 reaches 2.0;

[0078] Step 15: Detect the β-galactosidase activity of the reporter strain according to Step 2 of Method 3: To detect the β-galactosidase activity of the reporter strain, use 2-nitrophenyl-β-D-galactoside (ONPG) as the substrate for determination: Pipette 20 - 100 μL of the bacterial liquid cultured to the logarithmic phase, and successively add 420 μL of Z buffer (60 mM / L Na 2 HPO 4 、40 mM / L NaH 2 PO 4 、10 mM / L KCl、1 mM / L MgSO 4 、pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform, and 10 μL of 0.1% sodium dodecyl sulfate (SDS). After quickly mixing the mixture for 20 s, incubate at 30 °C for 1 h. After incubation, add 100 μL of ONPG with a concentration of 4 mg / mL to the mixture for reaction. Terminate the reaction by adding 250 μL of 1 M Na 2 CO 3 and record the reaction time, and detect the OD 420 and OD 550, and then calculate the β-galactosidase activity in Miller units (MU) according to the following equation: MU = 1,000 × (OD 420 - 1.75 × OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0079] Since the PQS system is jointly determined by the biosynthetic gene cluster of the PQS signal molecule (pqsABCDE and pqsH) and the PQS receptor protein PqsR. And in the presence of the PQS signal molecule and the HHQ signal molecule, the PqsR regulatory protein will bind to the pqsA promoter region, thereby promoting the synthesis of the PQS signal molecule. Therefore, the present invention speculates that isobavachalcone may inhibit the expression of pqsA by targeting and inhibiting PqsR. To verify this speculation, the present invention amplified the pqsR gene by Method 4, further overexpressed pqsR on the basis of PAO1 attB::mini-CTX-PpqsA-lacZ, and then detected the effect of isobavachalcone on the pqsA promoter activity in the pqsR overexpressing strain by Method 3. The results are as Figure 4 shown. In the pqsR normally expressing strain, as the concentration of isobavachalcone gradually increases, the pqsA promoter activity gradually decreases, and the maximum inhibition efficiency is reached when the concentration is 16 μg / mL. After overexpressing pqsR, as the concentration of isobavachalcone increases, the pqsA promoter activity does not change, and the IC 50 increases from 9.585 μg / mL to 88156 μg / mL. However, whether pqsR is normally expressed or overexpressed, the pqsA promoter activity in the control group does not change significantly. The above results confirm the speculation of the present invention, that is, isobavachalcone reduces the expression of the pqsA gene by targeting and inhibiting PqsR.

[0080] Example 5: Molecular docking experiment of isobavachalcone with PqsR protein

[0081] Molecular docking experiment.

[0082] The crystal structure of the PqsR protein used in the molecular docking experiment was downloaded from the PDB database, with the PDB ID of 4jvd. The 3D structure of the small molecule isobavachalcone was constructed using Chem3D 14.1 and energy minimized under the MMFF94 force field.

[0083] The present invention uses AutoDock Vina 1.1.2 software for molecular docking. Before the docking starts, PyMol 2.5.4 is used to process the receptor protein, including removing water molecules, salt ions, and small molecules. Subsequently, the docking box is set to enclose the entire protein structure. In addition, ADFRsuite 1.0 is used to convert all the processed small molecules and receptor proteins into the PDBQT format required for docking by AutoDock Vina 1.1.2. During docking, the parameters are kept at the default settings. The docking conformation with the highest score in the output is considered by us as the binding conformation. Finally, PyMol 2.5.4 and Discovery studio are used for visual analysis of the docking results.

[0084] The docking results are as Figure 5 shown, the interaction diagram of the small molecule isobavachalcone and the PqsR protein. It can be seen from Figure A that isobavachalcone binds in the middle groove of the PqsR protein. We can see from the detailed interaction diagrams in Figures B - C that the small molecule has hydrophobic interactions with LEU-197, MET-224, LEU-208, ALA-168, VAL-170, and ILE-236 on the PqsR protein. In addition, it can be observed that the small molecule has a hydrogen bond interaction with LEU207 on the protein, a Pi-Sigma interaction between ILE-263 and ILE236, and a Pi-Pi T-shaped interaction with TYR258. Hydrogen bond interaction is one of the strongest non-covalent interactions, indicating that LEU207 is an amino acid that plays an important role in the binding of the small molecule and the protein. And the binding affinity score of isobavachalcone and PqsR is -8.1 kcal / mol, suggesting strong binding potential.

[0085] Example 6: Key amino acid residue sites where isobavachalcone binds to the PqsR protein

[0086] Method for site-directed mutagenesis of key amino acid residue sites of the PqsR protein.

[0087] Implementation method for mutagenesis of amino acid residue sites of the PqsR protein. To construct the site-directed mutagenesis complementary vector pUC18T-mini-Tn7T-PqsR A168L -Gm, we replaced the conserved amino acid residue alanine at the 168th position on PqsR with leucine. Primer pairs pUC18T upF / pqsR A168L upR and pqsR A168LThe front and rear DNA sequences of the pqsR gene were amplified with lowF / pUC18T and lowR, and the upstream and downstream sequences were ligated by overlap PCR to form a gene sequence. The product of overlap PCR was inserted into the Hind III / Sac I site of the pUC18T-mini-Tn7T-Gm plasmid to obtain the site-directed mutagenesis recombinant plasmid pUC18T-mini-Tn7T-PqsR A168L -Gm. Site-directed mutagenesis vectors at other sites were obtained using the same method.

[0088] To further verify the interaction between isobavachalcone and PqsR, we constructed site-directed mutagenesis complementary vectors for these amino acid sites (ALA-168, VAL-170, LEU-197, LEU-208, LEU-207, MET-224, ILE236, ILE-263, and TYR-258). Then, these vectors were transformed into △pqsR::pMini-CTX-PpqsA-lacZ to detect the expression of the pqsA gene, as Figure 5 shown in A. The results showed that compared with the complementary wild-type pqsR, the expression of the pqsA gene was hardly detectable in □pqsR::pMini-CTX-PpqsA-lacZ when complemented with pqsR V170A 、pqsR L197A 、pqsR L207A 、pqsR L208A and pqsR Y258A , indicating that these sites are key active sites related to the activation of transcription by PqsR. Compared with the complementary wild-type pqsR, although the expression of pqsA decreased after complementation with pqsR A168L 、pqsR M224L 、pqsR I263L , the expression of pqsA was still inhibited after adding isobavachalcone, indicating that ALA-168, MET-224, and ILE-263 are not the action sites of isobavachalcone. In contrast, the expression of pqsA in the pqsR I236L complemented strain decreased but was not affected by isobavachalcone, indicating that ILE-236 is the key site for the interaction between isobavachalcone and PqsR. After site-directed mutagenesis, molecular docking was performed between the PqsR I236L protein and isobavachalcone. The results showed that after mutation, the binding affinity value between PqsR and isobavachalcone increased from -8.1 kcal / mol to -6.927 kcal / mol, indicating that the ILE-236 site is the main interaction and binding site between the PqsR protein and isobavachalcone.

[0089] Example 7: Effect of Isobavachalcone on the Motility of Pseudomonas aeruginosa

[0090] Pseudomonas aeruginosa motility detection experiment.

[0091] The motility of Pseudomonas aeruginosa includes swarming motility, swimming motility and twitching motility. The culture medium used for the swarming motility experiment is 0.6% agar, 0.8% nutrient broth, 0.5% glucose, and 20 μg / mL isobavachalcone is added to the treatment group, while 20 μg / mL isoeupatorin and DMSO are added to the control group. The culture medium for the swimming motility experiment is 0.3% agar, 0.5% NaCl and 1% peptone, and 20 μg / mL isobavachalcone is added to the treatment group, while 20 μg / mL isoeupatorin and DMSO are added to the control group. The culture medium for the twitching motility experiment is an LB agar plate, and 20 μg / mL isobavachalcone is added to the treatment group, while 20 μg / mL isoeupatorin and DMSO are added to the control group. After incubation at 37°C for 24 h, the diameters of the swarming motility and swimming motility bacterial lawns are measured. The bacteria are inoculated with a stab through the LB medium to observe their twitching motility. After incubation at 37°C for 48 h, it is stained with crystal violet. After removing the LB agar layer, the twitching motility traces of the colonies on the plate are washed with PBS, and the diameter of the diffusion circle at the bottom of the twitching motility is measured.

[0092] To explore the effect of isobavachalcone on the motility of Pseudomonas aeruginosa, the results are as Figure 7 shown. Compared with the DMSO and isoeupatorin control treatment groups, the swarming motility diameter of Pseudomonas aeruginosa in the 20 μg / ml isobavachalcone treatment group decreased by approximately 54.3%, as shown in Figure 7 A, and this difference reached a significant level (***, p < 0.001), indicating that 20 μg / ml isobavachalcone can significantly inhibit the swarming motility of Pseudomonas aeruginosa; compared with the DMSO and isoeupatorin control treatment groups, the swimming motility diameter of Pseudomonas aeruginosa in the 20 μg / ml isobavachalcone treatment group decreased by approximately 41.6%, as shown in Figure 7 B, and this difference reached a significant level (***, p < 0.001), indicating that 20 μg / ml isobavachalcone can significantly inhibit the swimming motility of Pseudomonas aeruginosa; compared with the DMSO and isoeupatorin control treatment groups, the twitching motility diameter of Pseudomonas aeruginosa in the 20 μg / ml isobavachalcone treatment group decreased by approximately 35.8%, as shown in Figure 7 C, and this difference reached a significant level (**, p < 0.01), indicating that 20 μg / mL isobavachalcone can significantly inhibit the twitching motility of Pseudomonas aeruginosa.

[0093] Example 8: Effect of isobavachalcone on pyocyanin synthesis in Pseudomonas aeruginosa

[0094] Pyocyanin production detection experiment.

[0095] For the pyocyanin production detection experiment, in PB medium (0.14% NaCl, 1% K2SO4, 2% tryptone), at 37°C, 20 μg / mL of isobavachalcone, eupatilin and DMSO were added to the culture medium and cultured for 24 h, and then centrifuged to obtain the supernatant. 3 ml of chloroform was added to 5 ml of the supernatant, and after vigorously rotating and mixing for 2 min, it was centrifuged at 8000 rpm for 10 min. The chloroform layer was transferred to a fresh tube and mixed with 1 ml of 0.2 M HCl. After centrifugation again, the OD of the HCl phase was measured. 520 . Using the formula OD 520 / OD 600 ×17.072 to calculate the pyocyanin content (μg / mL).

[0096] Experiment for detecting the expression of pyocyanin synthesis genes under the action of isobavachalcone.

[0097] Step 1: The transcriptional fusion strains of the pyocyanin synthesis gene operons phzA1B1C1D1E1F1G1 (phzA1) and phzA2B2C2D2E2F2G2 (phzA2) constructed through the "transcriptional fusion strain construction experiment" were PAO1attB::mini-CTX-PphzA1-lacZ; PAO1 attB::mini-CTX-PphzA2-lacZ;

[0098] Step 2: Detect the β-galactosidase activity of the reporter strain according to Step 2 in the "Experiment for Detecting the Gene Expression of the PQS Quorum Sensing System under the Action of Different Concentrations of Isobavachalcone": To detect the β-galactosidase activity of the reporter strain, it was measured using 2-nitrophenyl-β-D-galactoside (ONPG) as the substrate: Pipette 20 - 100 μL of the bacterial liquid cultured to the logarithmic phase, and successively add 420 μL of Z buffer (60 mM / L Na 2 HPO 4 、40 mM / L NaH 2 PO 4 、10 mM / L KCl、1 mM / L MgSO 4 、pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform and 10 μL of 0.1% sodium dodecyl sulfate (SDS). After the mixture was rapidly mixed for 20 s, it was incubated at 30°C for 1 h. After incubation, 100 μL of ONPG with a concentration of 4 mg / mL was added to the mixture for reaction. The reaction was terminated by adding 250 μL of 1 M Na 2 CO 3 , and the reaction time was recorded, and the OD 420 and OD 550, and then calculate the β-galactosidase activity in Miller units (MU) according to the following equation: MU = 1,000×(OD 420 -1.75×OD 550 ) / [OD 600 ×volume (mL)×reaction time (min)];

[0099] Pyocyanin plays an important role in the infection of Pseudomonas aeruginosa, and its synthesis is regulated by quorum sensing QS. It is usually used as a marker to evaluate QS behavior. In this study, the effects of isobavachalcone on the expression of pyocyanin-related synthesis genes phzA1 and phzA2 in Pseudomonas aeruginosa were further analyzed by lacZ transcriptional fusion. The results are as Figure 8 shown. Compared with the DMSO and isoschaftoside control treatment groups, the addition of 20 μg / mL isobavachalcone had a significant inhibitory effect on the expression levels of pyocyanin synthesis genes phzA1 and phzA2, and this difference reached a significant level (**, p < 0.01, ****, p < 0.0001). At the same time, 20 μg / mL isobavachalcone also significantly inhibited the production of pyocyanin in Pseudomonas aeruginosa, and this difference reached a significant level (****, p < 0.0001).

[0100] Example 9: Effects of isobavachalcone on the pathogenicity of Pseudomonas aeruginosa

[0101] Chinese cabbage infection experiment.

[0102] To detect the effects of isobavachalcone on the pathogenicity of Pseudomonas aeruginosa, a Chinese cabbage infection experiment was conducted. PAO1 was cultured overnight in LB liquid medium, and then the culture was harvested. It was washed twice with 10 mmol / L MgSO4 and then diluted to 10 8 CFU / mL. After disinfecting the surface of Chinese cabbage with 0.1% H 2 O 2 , 10 μL of the diluted bacterial suspension (containing DMSO, isobavachalcone or isoschaftoside) was injected into the midvein of Chinese cabbage leaves with a syringe. The specimens were stored at 30 °C for 4 days, and the rotted area of the Chinese cabbage was observed and analyzed.

[0103] Caenorhabditis elegans infection experiment.

[0104] For the nematode infection experiment, NGM plates (0.3% NaCl, 1 mM MgSO 4 , 1 mM CaCl 2, 5 μg / mL cholesterol, 100 μg / mL FUDR, and 2% agar). The synchronized L4-stage nematodes were transferred onto NGM plates, and the survival rate of the nematodes was recorded every 12 h for 5 consecutive days.

[0105] To explore the effect of isobavachalcone on the phytopathogenicity of Pseudomonas aeruginosa, in this invention, DMSO was used as the solvent control, and eupatilin was used as the control traditional Chinese medicine. After adding isobavachalcone, the infectivity of Pseudomonas aeruginosa to Chinese cabbage leaves was examined. The results are as Figure 9 shown in A and B. Compared with the DMSO and eupatilin control treatment groups, the rotted area caused by the infection of Pseudomonas aeruginosa on Chinese cabbage in the 20 μg / ml isobavachalcone treatment group decreased by approximately 43.6%, and this difference reached a significant level (***, p < 0.001), indicating that isobavachalcone can reduce the pathogenicity of Pseudomonas aeruginosa to Chinese cabbage.

[0106] To explore the effect of isobavachalcone on the animal pathogenicity of Pseudomonas aeruginosa, in this invention, DMSO was used as the solvent control, and eupatilin was used as the control traditional Chinese medicine. According to the "Caenorhabditis elegans infection experiment", the effect of isobavachalcone on the infectivity of Pseudomonas aeruginosa to Caenorhabditis elegans was detected. The results are as Figure 9 shown in C. Compared with the DMSO and eupatilin control treatment groups, after treatment with isobavachalcone, the virulence of Pseudomonas aeruginosa to Caenorhabditis elegans was significantly weakened, and this difference reached a significant level (**, p < 0.01), indicating that isobavachalcone can reduce the pathogenicity of Pseudomonas aeruginosa to Caenorhabditis elegans.

[0107] The traditional Chinese medicine monomer isobavachalcone screened in this invention does not affect the normal growth of Pseudomonas aeruginosa. However, preliminary experiments showed that isobavachalcone has a significant inhibitory effect on the PQS quorum sensing system of Pseudomonas aeruginosa. Further research results showed that the inhibitory effect of isobavachalcone on the PQS quorum sensing system of Pseudomonas aeruginosa was concentration-dependent, and the inhibitory effect reached the maximum at 16 μg / mL. Through gene overexpression and molecular docking experiments, it was found that the drug action target of isobavachalcone is the transcriptional activator protein PqsR of the PQS quorum sensing system. Isobavachalcone can bind to the PqsR protein, inhibit the activity of the pqsA promoter, and thus inhibit the PQS system of Pseudomonas aeruginosa. Moreover, this traditional Chinese medicine monomer can also significantly weaken the motility of Pseudomonas aeruginosa, including swarming motility, swimming motility, and twitching motility. Finally, the research also showed that isobavachalcone can reduce the pathogenicity of Pseudomonas aeruginosa to Chinese cabbage and Caenorhabditis elegans.

[0108] As described above, isobavachalcone can inhibit the PQS quorum sensing system without affecting the normal growth of Pseudomonas aeruginosa, and can reduce the pathogenicity of this bacterium to plant and animal models, protecting their hosts. Chemical antibacterial agents are likely to cause drug resistance in Pseudomonas aeruginosa, and natural plant products have attracted people's attention due to their wide sources and high safety. This invention also proves that the target of isobavachalcone is the PqsR protein, which greatly supports the application value of isobavachalcone. Therefore, isobavachalcone is a promising quorum sensing inhibitor for solving Pseudomonas aeruginosa infection problems and has the potential to be developed into a therapeutic drug or adjuvant for treating Pseudomonas aeruginosa infection.

[0109] As described above, the basic principles, main features and advantages of the present invention have been preferably described. The above embodiments and the description are only descriptions of the preferred embodiments of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. Use of isopsoralea corylifolia chalcone or a pharmaceutically acceptable salt thereof in the preparation of a preparation for inhibiting the quorum sensing system of Pseudomonas aeruginosa.

2. The use according to claim 1, characterized in that: Isopsoralea corylifolia chalcone or a pharmaceutically acceptable salt thereof acts on the PQS system in the quorum sensing system of Pseudomonas aeruginosa.

3. The use according to claim 2, characterized in that: Isopsoralea corylifolia chalcone or a pharmaceutically acceptable salt thereof inhibits the expression of gene pqsA in the quorum sensing system of Pseudomonas aeruginosa.

4. The use according to claim 2, characterized in that: The relevant genes of the PQS system are pqsA, pqsB, pqsC, pqsD and pqsE.

5. The use according to claim 2, characterized in that: The target of isopsoralea corylifolia chalcone or its pharmaceutically acceptable salt is PqsR protein.

6. Use of isopsoralea corylifolia chalcone or a pharmaceutically acceptable salt thereof in the preparation of a Pseudomonas aeruginosa control preparation, characterized in that: Isopsoralea corylifolia chalcone or a pharmaceutically acceptable salt thereof inhibits the virulence and / or pathogenicity of Pseudomonas aeruginosa.

7. The use according to claim 6, characterized in that: The virulence includes: the ability of Pseudomonas aeruginosa to produce pyocyanin.

8. The use according to claim 6, characterized in that: The virulence includes: the motility of Pseudomonas aeruginosa.

9. The use according to claim 6, characterized in that: The pathogenicity includes: the ability of Pseudomonas aeruginosa to infect plants.

10. The use according to claim 6, characterized in that: The virulence includes: the ability of Pseudomonas aeruginosa to infect animals.

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