Application of berberine or pharmaceutically acceptable salt thereof in preparation of pseudomonas aeruginosa quorum sensing system inhibitor

Abstract

The invention relates to the technical field of prevention and control of bacterial diseases, in particular to application of berberine or pharmaceutically acceptable salt thereof in preparation of a pseudomonas aeruginosa quorum sensing system inhibitor. The invention discloses berberine as a PQS quorum sensing system inhibitor of pseudomonas aeruginosa, and the berberine inhibits the expression of a pseudomonas aeruginosa gene pqsA, and inhibits the generation of pseudomonas aeruginosa pyocyanin, the formation of a biofilm and the movement ability, thereby weakening the toxicity and pathogenicity of the pseudomonas aeruginosa. In-depth study finds that the action target of the berberine is transcription activator protein PqsR of a PQS quorum sensing system. The berberine is proved to be capable of reducing the pathogenicity of the pseudomonas aeruginosa to Chinese cabbage and A549 cells, so that the berberine can be applied to prevention and treatment of pseudomonas aeruginosa diseases. Compared with a chemical antibacterial drug, berberine has the advantages of being wide in source, high in safety and not prone to generating bacterial drug resistance. The invention provides a new thought for preventing and treating pseudomonas aeruginosa diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial disease prevention and control, and in particular to application of berberine or a pharmaceutically acceptable salt thereof in the preparation of a Pseudomonas aeruginosa quorum sensing system inhibitor. Background Art

[0002] Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that is commonly found in various environments, including soil, water, and medical environments. It has attracted much attention because it can cause infections in immunocompromised individuals, including pneumonia, urinary tract infections, and sepsis. In particular, the unique structural and biochemical characteristics of Pseudomonas aeruginosa, such as the presence of an outer membrane permeability barrier and efflux pumps that actively expel a variety of antimicrobial agents, make Pseudomonas aeruginosa resistant to many antibiotics, making it a major threat to public health. New treatment options and strategies are urgently needed to address the challenges posed by this adaptive pathogen.

[0003] The quorum sensing (QS) system is a bacterial communication mechanism that regulates gene expression according to their density to coordinate group behavior. The quorum sensing system varies among different bacteria, so antimicrobial agents can be developed targeting the quorum sensing system of specific bacteria to achieve precise strikes against specific bacteria. In addition, quorum sensing inhibitors do not kill bacteria directly, but weaken their pathogenicity by interfering with the communication mechanism between bacteria. This non-bactericidal mechanism helps reduce bacterial resistance to antibiotics. The development of new antimicrobial drugs targeting the bacterial quorum sensing system has the characteristics of high specificity and is not easy to produce drug resistance.

[0004] Pseudomonas aeruginosa mainly achieves quorum sensing through several different signaling molecules, including N-acyl homoserine lactones (AHLs) and Pseudomonas quinolone signal (PQS). AHLs are mainly used in the Las and Rhl systems to regulate the expression of various virulence factors; while the PQS system uses 2-heptyl-3-hydroxy-4-quinolone as a signal to regulate specific gene expression. The relationship between the PQS system and pyocyanin and biofilm is particularly close. Pyocyanin is a toxic pigment produced by Pseudomonas aeruginosa, which has anti-biological activity and can cause damage to host cells. Research shows that the synthesis of PQS signals regulates the production of pyocyanin. When the bacterial density reaches a certain level, the concentration of PQS signals increases, which promotes the synthesis of pyocyanin, thereby enhancing the pathogenicity of bacteria. In addition, the PQS system also plays a key role in biofilm formation. Pseudomonas aeruginosa can form complex biofilm structures on host or environmental surfaces. This biofilm is composed of bacteria and the polymers they produce, which can effectively protect bacteria from the influence of the external environment and the attack of antibiotics. Therefore, in-depth study of the mechanism of the PQS system will provide new ideas for the development of new treatment strategies against Pseudomonas aeruginosa infections.

[0005] The CAS number of berberine is 2086-83-1, and its molecular formula is C 20 H 18 NO4, with a molecular weight of 336.37, and its chemical formula is shown below:

[0006]

[0007] Berberine (BBR) is a natural isoquinoline alkaloid with a molecular formula of C 20 H 18 NO4 + , and it is mainly extracted from plants such as Coptis chinensis, Phellodendron amurense, and Polygonum cuspidatum. Current research shows that berberine has a variety of biological activities, including antibacterial, anti-inflammatory, hypoglycemic, and anti-tumor effects. Existing research has confirmed that berberine shows inhibitory effects on a variety of bacteria, including Streptococcus oralis and Pseudomonas aeruginosa, indicating its potential as an antibiotic synergist against multi-drug resistant bacteria. However, there is currently no report on the study of berberine targeting the PqsR protein of Pseudomonas aeruginosa to inhibit the PQS quorum sensing system as an inhibitor of the Pseudomonas aeruginosa quorum sensing system. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a natural small molecule bacterial quorum sensing inhibitor for preventing and treating diseases caused by the Gram-negative bacterium Pseudomonas aeruginosa, providing more options for solving the problem of preventing and treating Pseudomonas aeruginosa diseases.

[0009] On the one hand, the present invention provides the use of berberine or a pharmaceutically acceptable salt thereof in the preparation of an inhibitor of the Pseudomonas aeruginosa quorum sensing system.

[0010] Further, in the above application, the inhibitor of the Pseudomonas aeruginosa quorum sensing system is used for preventing and / or treating Pseudomonas aeruginosa diseases.

[0011] Further, in the above application, berberine or a pharmaceutically acceptable salt thereof acts on the PQS system in the Pseudomonas aeruginosa quorum sensing system.

[0012] Further, in the above application, berberine or a pharmaceutically acceptable salt thereof inhibits the expression of the gene pqsA of the PQS system in the Pseudomonas aeruginosa quorum sensing system.

[0013] Further, in the above application, the action target of berberine or a pharmaceutically acceptable salt thereof is the PqsR protein of the PQS system in the Pseudomonas aeruginosa quorum sensing system.

[0014] Further, in the above application, berberine and its pharmaceutically acceptable salts inhibit the virulence and / or pathogenicity of Pseudomonas aeruginosa.

[0015] Further, in the above application, the virulence is the ability of Pseudomonas aeruginosa to produce pyocyanin.

[0016] Further, in the above application, the virulence is the ability of Pseudomonas aeruginosa to form biofilms.

[0017] Further, in the above application, the virulence is the motility of Pseudomonas aeruginosa, and the motility includes swarming motility, swimming motility and twitching motility.

[0018] Further, in the above application, the pathogenicity is the ability of Pseudomonas aeruginosa to infect plants or animals.

[0019] As used in the present invention, "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 prevents or reduces the enrichment of Pseudomonas aeruginosa, and cures or reduces the virulence and / or pathogenicity of Pseudomonas aeruginosa.

[0020] In the present invention, the term "pharmaceutically acceptable" means that it has no long-term harmful effects on the general health of the subject receiving treatment.

[0021] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of berberine and has no adverse effects biologically or otherwise. Pharmaceutically acceptable salts refer to converting the base group in the parent compound into a salt form, such as inorganic or organic acid salts of the base group (such as amino group). 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, 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, salicylic acid, etc.

[0022] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0023] The present invention provides a natural small molecule bacterial quorum sensing inhibitor - berberine, which is used for preparing a preparation for inhibiting the quorum sensing system of Pseudomonas aeruginosa. This discovery provides more options for solving the problem of drug resistance of Pseudomonas aeruginosa. Berberine can effectively interfere with the quorum sensing system of Pseudomonas aeruginosa, especially the PQS system, inhibit the expression of gene pqsA, thereby weakening the virulence and pathogenicity of Pseudomonas aeruginosa. This inhibitory effect is not only reflected in the production of pyocyanin by Pseudomonas aeruginosa, but also affects the formation of its biofilm and motility (including swarming motility, swimming motility and twitching motility). In addition, the application of berberine significantly reduces the pathogenicity of Pseudomonas aeruginosa to the host models of Chinese cabbage and human alveolar epithelial cells A549, showing its potential in plant and animal protection. Compared with chemical antibacterial agents, the plant natural product berberine has the advantages of wide source and high safety, and is not likely to cause bacteria to develop drug resistance. In addition, through in-depth research, the present invention reveals that the action target of berberine is the transcriptional activator protein PqsR protein of the PQS quorum sensing system, which provides a solid theoretical basis for its further development and application, and provides a new idea for the prevention and control of Pseudomonas aeruginosa diseases. Description of the Drawings

[0024] Figure 1 It is a schematic diagram of the effect of berberine at different concentrations on the expression of gene pqsA of the PQS quorum sensing system of Pseudomonas aeruginosa.

[0025] Figure 2 It is a schematic diagram of the effect of berberine on the Rhl system and Las system of the Pseudomonas aeruginosa quorum sensing system.

[0026] Figure 3 It is a schematic diagram of the effect of berberine on the growth of Pseudomonas aeruginosa.

[0027] Figure 4 It is that berberine follows the OD of the bacterial cells600 Schematic diagram of the effect of value change on the expression of pqsA gene in Pseudomonas aeruginosa.

[0028] Figure 5 It is a schematic diagram of the effect of berberine on pyocyanin synthesis in Pseudomonas aeruginosa. Among them, A is the detection result of β-galactosidase activity of the promoter transcriptional fusion strain of the pyocyanin synthesis gene operon phzA1B1C1D1E1F1G1 (phzA1) in Pseudomonas aeruginosa; B is the detection result of β-galactosidase activity of the promoter transcriptional fusion reporter strain of the pyocyanin synthesis gene operon phzA2B2C2D2E2F2G2 (phzA2); C is the quantitative result of pyocyanin in Pseudomonas aeruginosa under different drug treatments.

[0029] Figure 6 It is a schematic diagram of the effect of berberine on biofilm formation in Pseudomonas aeruginosa. Among them, A is the detection result of β-galactosidase activity of the promoter transcriptional fusion reporter strain of the important gene pelA that maintains the stability of biofilm in liquid environment in Pseudomonas aeruginosa; B is the surface area to volume ratio of the biofilm of Pseudomonas aeruginosa in liquid environment; C is the biofilm image formed under different drug treatment conditions under a laser scanning confocal microscope.

[0030] Figure 7 It is a schematic diagram of the effect of berberine on the motility of Pseudomonas aeruginosa. Among them, A is a schematic diagram and statistical chart of the effect of different drug treatments on the swarming motility of Pseudomonas aeruginosa; B is a schematic diagram and statistical chart of the effect of different drug treatments on the swimming motility of Pseudomonas aeruginosa; C is a schematic diagram and statistical chart of the effect of different drug treatments on the twitching motility of Pseudomonas aeruginosa.

[0031] Figure 8 It is a schematic diagram of the effect of berberine on the synthesis of PQS signal molecules in Pseudomonas aeruginosa. Among them, A is a schematic diagram of the growth effect of different drugs on Staphylococcus aureus under the drug concentration condition of 100 μg / ml; B is a schematic diagram of the growth of Staphylococcus aureus under different berberine concentration conditions; C is a thin layer chromatography detection chart of PQS and HHQ signal molecules in Pseudomonas aeruginosa under the treatment condition of 100 μg / ml berberine. Lanes 1, 2, 3, 4, and 5 are PQS standard, HHQ standard, signal molecule extract of Pseudomonas aeruginosa under dimethyl sulfoxide treatment, signal molecule extract of Pseudomonas aeruginosa under berberine treatment, and signal molecule extract of Pseudomonas aeruginosa under sinomenine treatment respectively; D is the detection result chart and statistical chart of the ability of Pseudomonas aeruginosa to antagonize Staphylococcus aureus under different drug treatment conditions.

[0032] Figure 9It is a schematic diagram of the effect of berberine on the expression of the quorum sensing system gene pqsA in Pseudomonas aeruginosa PqsR protein overexpressing strains.

[0033] Figure 10 It is a schematic diagram of the molecular docking of berberine with Pseudomonas aeruginosa PqsR protein and site-directed mutant PqsR protein.

[0034] Figure 11 It is a schematic diagram of the effect of berberine on the expression of the quorum sensing system gene pqsA in Pseudomonas aeruginosa point mutant PqsR protein complemented strains.

[0035] Figure 12 It is a schematic diagram of the change in the binding force between berberine and Pseudomonas aeruginosa PqsR protein and its mutants.

[0036] Figure 13 It is a schematic diagram of the effect of berberine on the pathogenicity of Pseudomonas aeruginosa. Among them, A is a schematic diagram and statistical chart of the detection of the infection ability of Pseudomonas aeruginosa on Chinese cabbage under different drug treatments; B is a graph of the analysis results of the survival rate of A549 cells under the infection of Pseudomonas aeruginosa after different drug treatments of Pseudomonas aeruginosa; C is a graph of the detection results of the invasion rate of Pseudomonas aeruginosa on cell A549 after different drug treatments of Pseudomonas aeruginosa. Specific implementation manners

[0037] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.

[0038] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings, but the examples given are not intended to limit the present invention.

[0039] In the following embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials can all be purchased on the market unless otherwise specified.

[0040] The sources of the test materials in the following embodiments are as follows:

[0041] The Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa pqsA gene mutant strains used in the present invention are from the Pathogenic Microbiology Laboratory of Yan'an University.

[0042] The berberine and sinomenine used in the present invention are purchased from Chenguang Biotechnology Co., Ltd. in Baoji City, among which the purity of berberine is 98% and the purity of sinomenine is 98%.

[0043] Using dimethyl sulfoxide (DMSO) as a solvent, berberine and sinomenine were prepared into a mother liquor of traditional Chinese medicine monomers with a concentration of 10 mg / mL. Through MIC (minimum inhibitory concentration) detection, it was found that the MIC (minimum inhibitory concentration) of berberine and sinomenine against Pseudomonas aeruginosa was 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.

[0044] The primers and related restriction enzyme site information used in this invention are shown in Table 1 below:

[0045] Table 1 Primers and Related Restriction Enzyme Site Information

[0046]

[0047]

[0048] Among them, the underlined sequence is the restriction enzyme site.

[0049] Example 1

[0050] This example is to construct a gene expression screening model for the QS system of Pseudomonas aeruginosa.

[0051] (1) Construction of transcriptional fusion strains

[0052] Step 1: Design primers pqsA F / pqsA R using Pseudomonas aeruginosa genomic DNA as a template, and use this primer pair to perform PCR amplification of the promoter sequence PpqsA of pqsA, with the DNA fragment size of 1105 bp;

[0053] Step 2: Perform double digestion of PpqsA and plasmid mini-CTX-lacZ with Sal I / Pst I;

[0054] Step 3: Purify the digested PpqsA and ligate it to the plasmid mini-CTX-lacZ, and transfer it into Escherichia coli TG1 competent cells by heat shock transformation method, and coat it on an LB plate containing tetracycline (Tetracycline Tc, 200 μg / mL, abbreviated as Tc200) for screening;

[0055] Step 4: After the obtained colonies were tested by PCR, the plasmid was extracted to obtain a recombinant vector, and the recombinant vector was digested and verified with Sal I / Pst I to obtain a recombinant vector mini-CTX-PpqsA-lacZ showing a positive result;

[0056] Step 5: Transform mini-CTX-PpqsA-lacZ into S17-1 competent cells to obtain S17-1 mini-CTX-PpqsA-lacZ;

[0057] 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. Then scrape the bacterial lawn on the plate, suspend it with liquid LB and dilute it for spreading on an LB double-antibiotic plate containing kanamycin (Km, 30 μg / mL, abbreviated as Km30) and Tc200. After the obtained colonies are streaked and cultured again on the double-antibiotic plate, a transcriptional fusion strain: PAO1 attB::mini-CTX-PpqsA-lacZ is obtained; Use the same method to obtain gene expression screening models for the other two quorum sensing systems (Rhl quorum sensing system and Las quorum sensing system) of Pseudomonas aeruginosa respectively: PAO1 attB::mini-CTX-PrhlI-lacZ and PAO1 attB::mini-CTX-PlasI-lacZ;

[0058] (2) Detection of gene expression of quorum sensing system under the action of berberine

[0059] Step 1: Using sinomenine and dimethyl sulfoxide as controls, incubate a berberine solution with a final concentration of 100 μg / mL together with the transcriptional fusion reporter strains PAO1 attB::mini-CTX-PpqsA-lacZ, PAO1 attB::mini-CTX-PrhlI-lacZ and PAO1 attB::mini-CTX-PlasI-lacZ at 37 °C and 200 rpm until OD 600 reaches 1.5;

[0060] Step 2: Detect the β-galactosidase activity of the transcriptional fusion reporter strain. The method for detecting the β-galactosidase activity of the transcriptional fusion reporter strain is to use o-nitrophenyl-β-D-galactoside (ONPG) as the substrate for determination: Pipette 20 - 100 μL of the bacterial liquid cultured to the logarithmic phase, and sequentially add 420 μL of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH = 7.0, 0.2% β-mercaptoethanol), 20 μL of chloroform, and 10 μL of 0.1% sodium dodecyl sulfate (SDS) to the bacterial liquid. After the mixture is rapidly mixed for 20 s, it is incubated at 30 °C for 1 h. After incubation, 100 μL of ONPG with a concentration of 4 mg / mL is added to the mixture for reaction. The reaction is terminated by adding 250 μL of 1 M Na2CO3, and the reaction time is recorded. Finally, the mixture is centrifuged at 14000×g for 3 min, and the supernatant is taken to detect the OD 420 and OD 550 , and then calculate the β-galactosidase activity in Miller units (MU) according to the following equation:

[0061] MU = 1,000×(OD 420 - 1.75×OD 550 ) / [OD 600 × volume (mL) × reaction time (min)];

[0062] The measurement results are as shown in Figure 1 . Compared with the sinomenine treatment group, berberine significantly inhibited the promoter activity of pqsA. Especially when the concentration of berberine was 100 μg / mL, its inhibitory effect on the promoter activity of pqsA was more significant. Using dimethyl sulfoxide as the control treatment group, the test results are as shown in Figure 2 . Compared with the expression of pqsA, berberine did not have a significant effect on the expression of rhlI and lasI. It indicates that berberine has a significant inhibitory effect on the PQS quorum sensing system of Pseudomonas aeruginosa.

[0063] Example 2

[0064] This example is a test experiment on the effect of berberine on the growth of Pseudomonas aeruginosa.

[0065] Using the wild strain PAO1 of Pseudomonas aeruginosa as the test strain, a total of three groups of experimental treatments were set up. The first group added the same volume of dimethyl sulfoxide solvent as the subsequent drugs, the second group added berberine with a final concentration of 100 μg / mL to the culture medium, and the third group added sinomenine with a final concentration of 100 μg / mL to the culture medium. Each group was set with three biological replicates. The specific test steps are as follows:

[0066] Step 1: Pick a single colony of Pseudomonas aeruginosa PAO1 and inoculate it into 5 mL of LB liquid medium containing 30 μg / mL of Km. Incubate the culture at 37°C with shaking at 200 rpm for 16 - 20 h until the stationary phase is reached;

[0067] Step 2: Add the Pseudomonas aeruginosa bacterial solution to 5 mL of LB liquid medium containing 30 μg / mL of Km and berberine (or sinomenine and the same volume of dimethyl sulfoxide) at a final concentration of 100 μg / mL according to a ratio of 1:100. Measure the initial OD 600 , using dimethyl sulfoxide solvent and sinomenine as controls;

[0068] Step 3: Incubate the transferred bacterial solution at 37°C with shaking at 200 rpm. Measure and record the OD of the bacterial solution every 2 h 600 , and culture for a total of 24 h.

[0069] The test results are as Figure 3 shown. Compared with the dimethyl sulfoxide and sinomenine control groups, berberine has no effect on the growth of Pseudomonas aeruginosa.

[0070] Example 3

[0071] This example is to test the effect of berberine on the expression of the pqsA gene in the QS system of Pseudomonas aeruginosa.

[0072] The specific steps for detecting the effect of berberine on the expression of the pqsA gene in the QS system of Pseudomonas aeruginosa at different growth stages are as follows:

[0073] Step 1: Incubate a berberine solution at a final concentration of 100 μg / mL with the transcriptional fusion strain PAO1attB::mini-CTX-PpqsA-lacZ. Similarly, use dimethyl sulfoxide solvent and sinomenine as negative controls. Incubate at 37°C with shaking at 200 rpm until the decline phase, and sample at OD 600 values of 0.75, 1.5, 2, 2.5, 3, and 3.5 for detecting the β-galactosidase activity under berberine treatment at different OD 600 values;

[0074] Step 2: Detect the β-galactosidase activity of the transcriptional fusion strain according to the detection method in step 2 of the detection of the quorum sensing system gene expression under the action of berberine in Example 1.

[0075] The expression of pqsA was detected at OD 600 values of 0.75, 1.5, 2, 2.5, 3, 3.5, respectively. The detection results are as Figure 4 shown. Compared with the dimethyl sulfoxide solvent and sinomenine treatment groups, under the action of berberine at a concentration of 100 μg / mL, until the decline phase (OD 600Before the value reached 3.5, the promoter activity of the Pseudomonas aeruginosa PAO1 quorum sensing system gene pqsA was inhibited. Compared with the dimethyl sulfoxide control, the activity of the pqsA promoter under the treatment of sinomenine in the control was always similar to that of the dimethyl sulfoxide treatment group, but berberine significantly inhibited the expression of pqsA throughout the growth cycle. When OD 600 reached 1.5, the inhibition of berberine on the enzyme activity of the pqsA promoter reached the maximum. When OD 600 reached 2.0, the expression of pqsA in both the dimethyl sulfoxide and berberine treatment groups tended to be stable. When OD 600 reached 3.5, Pseudomonas aeruginosa entered the decline phase, and the inhibition of berberine on the pqsA promoter of Pseudomonas aeruginosa disappeared. The above results may be due to the fact that as the bacteria grow and reproduce, a large number of action targets or corresponding competitive ligands of berberine in Pseudomonas aeruginosa are synthesized, interfering with its action effect.

[0076] Example 4

[0077] This example is a test for the effect of berberine on the synthesis of pyocyanin by Pseudomonas aeruginosa.

[0078] (1) Detection of the expression of pyocyanin synthesis genes under the action of berberine

[0079] For the detection of the expression of pyocyanin synthesis genes under the action of berberine, to detect the expression of the pyocyanin synthesis gene operons phzA1 and phzA2, by constructing PAO1 attB::mini-CTX-PphzA1-lacZ and PAO1 attB::mini-CTX-PphzA2-lacZ transcriptional fusion strains, the β-galactosidase activity of the transcriptional fusion strains was detected to judge the expression of the pyocyanin synthesis gene operons phzA1 and phzA2. The specific steps are as follows:

[0080] Refer to the method of Example 1 to construct the pyocyanin synthesis gene operons phzA1B1C1D1E1F1G1 (phzA1) and phzA2B2C2D2E2F2G2 (phzA2) transcriptional fusion strains PAO1attB::mini-CTX-PphzA1-lacZ; PAO1 attB::mini-CTX-PphzA2-lacZ;

[0081] The β-galactosidase activity of the transcriptional fusion strains was detected according to the detection method in step 2 of the detection of the quorum sensing system gene expression under the action of berberine in (2) of Example 1.

[0082] (2) Detection of pyocyanin production under the action of berberine

[0083] For the detection of pyocyanin production under the action of berberine, the test steps are specifically as follows:

[0084] Step 1: Pick a single colony of PAO1 into 5 mL of fresh LB liquid medium containing 30 μg / mL of Km, and culture it with shaking at 37 °C and 200 rpm for 16 - 20 h.

[0085] Step 2: Add the Pseudomonas aeruginosa bacterial solution into 5 mL of PB liquid medium containing 30 μg / mL of Km and berberine, sinomenine with a final concentration of 100 μg / mL or the same volume of dimethyl sulfoxide at a ratio of 1:100, and culture it with shaking at 37 °C and 200 rpm until the logarithmic phase. The preparation method of the PB liquid medium is as follows: Weigh 6 g of NaCl, 10 g of K2SO4, and 20 g of tryptone, dissolve them with distilled water, then make up the volume to 1 L with distilled water, and sterilize at 121 °C for 20 min.

[0086] Step 3: After the culture reaches the stationary phase, centrifuge and take the supernatant.

[0087] Step 4: Add 3 mL of chloroform to 5 mL of the supernatant, stir vigorously for 2 min, and then centrifuge at 8000 rpm for 10 min.

[0088] Step 5: Transfer the chloroform layer to a new test tube and mix it with 1 mL of 0.2 M hydrochloric acid.

[0089] (3) Step 6: After centrifuging at 8000 rpm for 10 min again, aspirate the top red solution and measure the OD. 520 . Calculate the content of pyocyanin according to the formula: OD 520 / OD 600 ×17.072.

[0090] The detection results of the effect of berberine on pyocyanin synthesis in Pseudomonas aeruginosa are as Figure 5 shown, where Figure 5 A shows that compared with the dimethyl sulfoxide treatment group, the expression level of phzA1 in the berberine treatment group decreased by 31.16%, and the difference reached an extremely significant level (***, p < 0.001); compared with the control sinomenine treatment group, the expression level of phzA1 in the berberine treatment group decreased by 28.85%, and the difference reached a significant level (**, p < 0.01). Figure 5 The result of B shows that the expression level of phzA2 in the berberine treatment group decreased by 34.59% compared with the dimethyl sulfoxide treatment group, and the difference reached a significant level (**, p < 0.01); it decreased by 26.74% compared with the control sinomenine treatment group. The difference also reached a significant level (**, p < 0.01). The results indicate that 100 μg / mL of berberine can inhibit the expression of pyocyanin synthesis genes in Pseudomonas aeruginosa by inhibiting the PQS system.

[0091] The test results of the effect of berberine on the production of pyocyanin by Pseudomonas aeruginosa PAO1 are as follows Figure 5 shown in C. Compared with the pyocyanin production of the dimethyl sulfoxide treatment group, the pyocyanin production of the berberine treatment group decreased by 33.97%, and the difference reached a significant level (**, p<0.01). Compared with the control traditional Chinese medicine sinomenine treatment group, it decreased by 34.81%, and the difference also reached a significant level (**, p<0.01). This indicates that berberine inhibits the production of pyocyanin by Pseudomonas aeruginosa.

[0092] Example 5

[0093] This example is a test on the effect of berberine on the biofilm formation of Pseudomonas aeruginosa.

[0094] Construct the planktonic biofilm synthesis gene pelA transcriptional fusion strain PAO1attB::mini-CTX-PpelA-lacZ according to the method of Example 1;

[0095] The β-galactosidase activity of the transcriptional fusion strain was detected according to the detection method of step 2 in the detection of the quorum sensing system gene expression under the action of berberine in Example 1.

[0096] The situation of planktonic biofilms was observed by laser confocal scanning microscopy (LSCM), and the specific operation steps are as follows:

[0097] Step 1: Transfer the pBBR-gfp-mut3 vector (green fluorescent protein expression vector) into Escherichia coli S17-1, and then conjugate with PAO1 on a TSB plate at 37°C for 48 h.

[0098] Step 2: Suspend the conjugated bacterial lawn with TSB medium, dilute it appropriately, and coat it on a TSB triple-antibody plate containing Km30, Tc200, and Gentamycin Gm (100 μg / mL, abbreviated as Gm100) to screen for strains labeled with green fluorescent protein (GFP).

[0099] Step 3: Immerse a sterile glass coverslip in a petri dish containing 5 mL of TSB medium. According to an inoculation amount of 1:100, transfer and culture the strains labeled with GFP green fluorescent protein in the petri dish until the stationary phase; culture with shaking at 37°C and 100 r / min for 72 h.

[0100] Step 4: Take out the coverslip, rinse it twice with PBS to remove non-adherent cells and culture medium, and air-dry the coverslip to obtain the biofilms of the strains labeled with green fluorescent protein.

[0101] Step 5. Observe the formation of biofilm under a laser confocal scanning microscope. Use the PAO1 strain without GFP labeling as a blank control (control).

[0102] The results are as Figure 6 shown in Figure 6 Figure A. Compared with the dimethyl sulfoxide treatment group, the expression level of pelA in the berberine treatment group decreased by 23.42%, and the difference reached a significant level (*, p < 0.05). Compared with the control group treated with sinomenine, a traditional Chinese medicine, the expression level of pelA decreased by 21.19%, and the difference reached a significant level (*, p < 0.05). This indicates that 100 μg / mL berberine can inhibit the expression of planktonic biofilm synthesis genes of Pseudomonas aeruginosa by inhibiting the PQS system; subsequently, the effect of berberine on the formation of planktonic biofilm of Pseudomonas aeruginosa PAO1 was explored. Figure 6 Figure B shows that the biofilms in the dimethyl sulfoxide treatment group and the sinomenine treatment group are relatively thick and continuous, while the thickness and density of the biofilm on the coverslip decrease and are sparsely distributed under the treatment of berberine. By analyzing the surface area to volume ratio of the biofilm on the coverslip in different treatment groups, the results are as Figure 6 shown in Figure C. The surface area to volume ratio of the berberine treatment group is significantly higher than that of the dimethyl sulfoxide treatment group, about 2 times higher (*, p < 0.05); compared with the sinomenine treatment group, the surface area to volume ratio is 1.8 times higher (*, p < 0.05). The results show that the biofilm structure of Pseudomonas aeruginosa becomes more dispersed and weak after treatment with berberine. This indicates that berberine has a significant effect on inhibiting the formation of planktonic biofilm of Pseudomonas aeruginosa, further supporting the potential of berberine to inhibit the formation of biofilm of Pseudomonas aeruginosa.

[0103] Example 6

[0104] This example is a test for the effect of berberine on the motility of Pseudomonas aeruginosa.

[0105] (1) Swarming motility detection test of Pseudomonas aeruginosa

[0106] Step 1. Add berberine at a final concentration of 100 μg / ml or sinomenine at a final concentration of 100 μg / ml or the same volume of dimethyl sulfoxide to the Swarming medium to prepare different Swarming plates. The preparation method of the Swarming medium is as follows: Weigh 3.0 g of agarose, 5 g of glucose, and 8 g of nutrient broth (NB), dissolve them with distilled water, and then make up the volume to 1 L with distilled water. Sterilize at 121 °C for 20 min.

[0107] Step 2. Pick a single colony of PAO1 into 5 mL of fresh LB liquid medium containing Km30, and culture it at 37 °C with shaking at 200 rpm for 16 - 20 h;

[0108] Step 3: Measure the OD of the bacterial solution 600 , dilute the bacterial solution until the OD of the diluted bacterial solution 600 = 0.5;

[0109] Step 4: Pipette 1 μL of the diluted bacterial solution and drop it in the center of the plate;

[0110] Step 5: After the bacterial solution penetrates the plate, place the plate in an incubator at 30 °C and incubate for 24 h. Observe the diffusion of the bacterial solution and measure its diameter. All experiments are set with 3 biological replicates;

[0111] (2) Pseudomonas aeruginosa swimming motility detection test

[0112] Step 1: Add berberine at a final concentration of 100 μg / mL or sinomenine at a final concentration of 100 μg / mL or the same volume of dimethyl sulfoxide to the Swimming medium to prepare different Swimming plates. The preparation method of the Swimming medium is as follows: Weigh 2.5 g of agarose, 5 g of NaCl and 10 g of peptone, dissolve them with distilled water, and then make up the volume to 1 L with distilled water. Sterilize at 121 °C for 20 min.

[0113] Step 2: Pick a single colony of PAO1 into 5 mL of fresh LB liquid medium containing Km30, and shake culture at 37 °C and 200 rpm for 16 - 20 h;

[0114] Step 3: Measure the OD of the bacterial solution 600 , dilute the bacterial solution until the OD of the diluted bacterial solution 600 = 0.5;

[0115] Step 4: Pipette 1 μL of the diluted bacterial solution and drop it in the center of the plate;

[0116] Step 5: After the bacterial solution penetrates the plate, place the plate in an incubator at 30 °C and incubate for 24 h. Observe the diffusion of the bacterial solution and measure its diameter. All experiments are set with 3 biological replicates;

[0117] (3) Pseudomonas aeruginosa twitching motility detection test

[0118] Step 1: Add berberine at a final concentration of 100 μg / mL or sinomenine at a final concentration of 100 μg / mL or the same volume of dimethyl sulfoxide to the Twitching medium to prepare different Twitching plates. Let the plates dry in a laminar flow hood before use. The preparation method of the Twitching medium is as follows: Weigh 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g / L of agar powder, dissolve them with distilled water, and then make up the volume to 1 L with distilled water. Sterilize at 121 °C for 20 min.

[0119] Step 2: Pick a single colony of PAO1 into 5 mL of fresh LB liquid medium containing 30 μg / mL of Km, and culture it at 37 °C with shaking at 200 rpm for 16 - 20 h.

[0120] Step 3: Dip the tip of a toothpick into the fresh Pseudomonas aeruginosa bacterial solution and pierce the Twitching plate, then incubate it upside down at 37 °C for 24 h.

[0121] Step 4: After the culture is completed, completely immerse the plate in Coomassie Brilliant Blue staining solution (0.05 g of Coomassie Brilliant Blue R250, 40 mL of methanol, 10 mL of acetic acid, mix well, and make up the volume to 100 mL with deionized water).

[0122] Step 5: Decolorize with 95% ethanol until the diffusion zone can be seen, and measure the diameter of the diffusion zone. All experiments are set with 3 biological replicates.

[0123] The detection results are as Figure 7 shown. Figure 7 As shown in A, compared with the dimethyl sulfoxide treatment group, the diameter of the Pseudomonas aeruginosa swarming motility in the berberine treatment group decreased by about 29.90%, and the difference reached a significant level (**, p < 0.01). Compared with the control sinomenine treatment group, the swarming motility diameter decreased by about 28.90%, and the difference reached a significant level (*, p < 0.05), indicating that 100 μg / mL berberine can significantly inhibit the swarming motility of Pseudomonas aeruginosa. Figure 7 As shown in B, compared with the dimethyl sulfoxide treatment group, the diameter of the Pseudomonas aeruginosa swimming motility in the berberine treatment group decreased by about 50.43%, and the difference reached a significant level (***, p < 0.001). Compared with the control sinomenine treatment group, the swimming motility diameter decreased by about 50.43%, and the difference reached a significant level (***, p < 0.001), indicating that 100 μg / mL berberine can significantly inhibit the swimming motility of Pseudomonas aeruginosa. Figure 7Figure C shows that compared with the dimethyl sulfoxide treatment group, the diameter of the twitching motility of Pseudomonas aeruginosa in the berberine treatment group decreased by about 16.67%, and the difference reached a significant level (*, p < 0.05). Compared with the control group treated with the traditional Chinese medicine sinomenine, the diameter of the twitching motility decreased by about 17.72%, and the difference reached a significant level (*, p < 0.05), indicating that 100 μg / mL berberine can significantly inhibit the twitching motility of Pseudomonas aeruginosa. The swimming, swarming, and twitching motilities of Pseudomonas aeruginosa depend on the normal functions of flagella, rhamnolipids, and pili, respectively. Among them, swimming motility depends on the activity of flagella, swarming motility requires the participation of rhamnolipids on this basis, and twitching motility is driven by pili. These three types of motilities are closely related to the pathogenicity of Pseudomonas aeruginosa and are all regulated by the PQS quorum sensing system. The above results show that berberine significantly reduces the motility of Pseudomonas aeruginosa by inhibiting the PQS quorum sensing system, further verifying the potential application value of berberine in anti-Pseudomonas aeruginosa infection.

[0124] Example 7

[0125] This example is a test for the effect of berberine on the synthesis of PQS signal molecules in Pseudomonas aeruginosa.

[0126] Using Staphylococcus aureus (ATCC29213) as the test strain, a total of three groups of experimental treatments were set up. The first group added the same volume of dimethyl sulfoxide solvent as the subsequent drugs, the second group added different final concentrations of berberine to the culture medium, and the third group added sinomenine with a final concentration of 100 μg / mL to the culture medium. Each group was set with three biological replicates.

[0127] (1) Test for the effect of berberine and sinomenine on the growth of Staphylococcus aureus

[0128] The steps for testing the effect of berberine and sinomenine on the growth of Staphylococcus aureus are as follows:

[0129] Step 1: Pick a single colony of Staphylococcus aureus (ATCC29213) into 5 mL of LB liquid medium and culture it at 37 °C and 200 rpm for 16 - 20 h.

[0130] Step 2: Add Staphylococcus aureus (ATCC29213) bacterial solution at a ratio of 1:100 to 5 mL of LB liquid medium containing berberine at final concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 100 μg / mL (or 100 μg / mL of sinomenine and the same volume of dimethyl sulfoxide), and detect the initial OD 600 ;

[0131] Step 3: Incubate the transferred bacterial solution at 37 °C with shaking at 200 rpm, and measure and record the OD of the bacterial solution every 1 h 600 , and incubate for a total of 12 h.

[0132] (2) Antagonistic test of Pseudomonas aeruginosa against Staphylococcus aureus

[0133] The steps of the antagonistic test of Pseudomonas aeruginosa against Staphylococcus aureus are as follows:

[0134] Step 1: Pick single colonies of PAO1 (abbreviated as PAO1 strain), Pseudomonas aeruginosa pqsA gene mutant strain (abbreviated as ΔpqsA strain), and Staphylococcus aureus (ATCC29213) into 5 mL of LB liquid medium, and culture overnight at 37 °C with shaking at 200 rpm.

[0135] Step 2: Add PAO1 and ΔpqsA bacterial solutions at a ratio of 1:100 to 5 mL of LB liquid medium containing Km30. Similarly, transfer Staphylococcus aureus at a ratio of 1:100 to 5 mL of LB and culture with shaking at 37 °C and 200 rpm until the OD 600 is 1.5;

[0136] Step 3: Spread the Staphylococcus aureus bacterial solution on an LB plate containing berberine or sinomenine at a working concentration of 40 μg / mL and the same volume of dimethyl sulfoxide. Respectively, pipette 1 μL of PAO1 bacterial solution and ΔpqsA bacterial solution onto the center of the plate and incubate in a 37 °C incubator upright for 12 h - 24 h, and measure the diameter of the inhibition zone. Each experiment is repeated 3 times.

[0137] (3) Thin-layer chromatography detection of PQS in Pseudomonas aeruginosa

[0138] The steps of the thin-layer chromatography detection of PQS in Pseudomonas aeruginosa are as follows:

[0139] Step 1: Pick a single colony of PAO1 into 5 mL of LB liquid medium containing Km30, and culture overnight at 37 °C with shaking at 200 rpm.

[0140] Step 2: Transfer the PAO1 bacterial solution to 100 mL of LB liquid medium at a ratio of 1:100, and culture it at 37 °C and 200 rpm until the stationary phase.

[0141] Step 3: Centrifuge at 10000 r / min for 30 min, collect the supernatant and filter it through a 0.22 μm microporous filter. Extract the filtrate twice with 3 volumes of acidified ethyl acetate (0.01% glacial acetic acid is added to ethyl acetate).

[0142] Step 4: Use a rotary evaporator to collect the extract and rotary evaporate it to concentrate. Dissolve the concentrated extract in methanol.

[0143] Step 5: Immerse a 20×20 cm silica gel 60F254 thin layer plate in a 5% (wt / vol) KH2PO4 solution for 30 minutes, and then activate it at 100 °C for 1 hour.

[0144] Step 6: Draw a faint line about 4 cm from the bottom of the activated silica gel thin layer plate with a pencil; label it as the sample extract.

[0145] Step 7: Take 1 μL of the dissolution solution and spot it on the GF254 TLC plate. After drying, perform chromatography in a chromatography tank using a mixed solution of dichloromethane:methanol = 95:5 by volume as the mobile phase.

[0146] Step 8: Observe and collect pictures using a UV analyzer at a wavelength of 312 nm after chromatography.

[0147] The test results are as Figure 8 shown, Figure 8 A and B in it show that sinomenine has no effect on the growth of Staphylococcus aureus at a concentration of 100 μg / mL, but berberine affects the growth of Staphylococcus aureus at a concentration of 100 μg / mL. To exclude the effect of berberine on the growth of Staphylococcus aureus, this study selected a concentration of 40 μg / mL for the antibacterial test. The test results are as Figure 8 shown in D in it. On the LB plate containing 40 μg / mL of berberine, the ΔpqsA strain has no antagonistic effect on Staphylococcus aureus, and the antagonistic antibacterial zone of PAO1 against Staphylococcus aureus decreases, indicating that the production of signal molecules used by the PAO1 strain to antagonize Staphylococcus aureus is inhibited. Statistical analysis of the antibacterial zone shows that compared with the dimethyl sulfoxide treatment group, the antibacterial zone of the berberine treatment group decreases by about 54.35%, and the difference reaches a significant level (**, p < 0.01). Compared with the control traditional Chinese medicine sinomenine treatment group, the antibacterial zone decreases by about 53.33%, and the difference reaches a significant level (*, p < 0.05). Detect the production of signal molecules (mainly PQS, HHQ) by the PAO1 strain after treatment with berberine, sinomenine, and dimethyl sulfoxide by thin layer method. The test results are as Figure 8As shown in C, lanes 1, 2, 3, 4, and 5 are: PQS standard; HHQ standard; signal molecule extract of Pseudomonas aeruginosa under dimethyl sulfoxide treatment conditions; signal molecule extract of Pseudomonas aeruginosa under berberine treatment conditions; signal molecule extract of Pseudomonas aeruginosa under sinomenine treatment conditions. The results show that after treatment with berberine, the signal molecule production of PAO1 is significantly reduced.

[0148] Example 8

[0149] This example is a test on the effect of berberine on the expression of the quorum sensing system gene pqsA in a Pseudomonas aeruginosa PqsR protein overexpressing strain.

[0150] (1) Construction and enzyme activity detection of a Pseudomonas aeruginosa PqsR protein overexpressing strain

[0151] Step 1: Design primers pqsR F / pqsR R using Pseudomonas aeruginosa genomic DNA as a template, and use these primers to PCR amplify a 1000bp sequence of the pqsR gene, namely the pqsR gene fragment;

[0152] Step 2: Perform double digestion on the pqsR gene fragment and plasmid pBBR1MCS-5 with Xho I / Pst I;

[0153] Step 3: Purify the digested pqsR gene fragment and ligate it to plasmid pBBR1MCS-5, transform it into Escherichia coli TG1 competent cells, and spread it on an LB plate containing Gm100 for screening;

[0154] Step 4: Screen positive clone strains by colony PCR, extract the recombinant vector among them, perform enzyme digestion verification on the recombinant vector with Xho I / PstI to obtain the recombinant vector pBBR1MCS-5-pqsR;

[0155] Step 5: Pick a single colony of PAO1 attB::mini-CTX-PpqsA-lacZ into 5 mL of LB liquid medium, culture it overnight at 37 °C and 200 rpm;

[0156] Step 6: Transfer the bacterial solution obtained in Step 5 at a ratio of 1:100 to 50 mL of fresh LB liquid medium, culture it at 37 °C and 200 rpm until the OD 600 is between 0.75 - 1.25, centrifuge at 4 °C and 8100×g for 8 min, and collect the bacterial cells;

[0157] Step 7: Resuspend the bacterial cells with 50 mL of pre-cooled 300 mM sucrose solution, centrifuge at 4 °C and 8100×g for 8 min, and collect the bacterial cells;

[0158] Step 8. Resuspend the cells with 25 mL of pre-chilled 300 mM sucrose solution, centrifuge at 8100×g for 8 min at 4°C, and collect the cells.

[0159] Step 9. Resuspend the cells with 0.5 mL of pre-chilled 300 mM sucrose solution, incubate on ice for 30 min to prepare electrocompetent cells, aliquot 100 μL per tube for use, and store the rest in a 4°C refrigerator.

[0160] Step 10. Add the recombinant vector pBBR1MCS-5-pqsR to the electrocompetent cells of PAO1attB::mini-CTX-PpqsA-lacZ, mix well, transfer to a sterile and pre-chilled electroporation cuvette for electroporation, and set the electroporation conditions as: 25 μF, 200 Ω, 1900 V, 3 - 5 ms.

[0161] Step 11. Transfer the electroporated cells into 5 mL of fresh LB liquid medium and culture at 37°C and 200 rpm for 3 - 5 h.

[0162] Step 12. Take an appropriate amount of the bacterial solution and spread it on 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 PAO1attB::mini-CTX-PpqsA-lacZ(pBBR1MCS-5-pqsR).

[0163] Step 13. Use the same method to obtain the empty vector control strain PAO1attB::mini-CTX-PpqsA-lacZ(pBBR1MCS-5).

[0164] Step 14. To detect the effect of different concentrations of berberine on the gene expression of the PQS quorum sensing system in the PqsR protein overexpression strain, use sinomenine, a traditional Chinese medicine monomer that has no effect on the PQS quorum sensing system of Pseudomonas aeruginosa, and dimethyl sulfoxide solvent as negative controls. Incubate different concentrations of berberine with PAO1attB::mini-CTX-PpqsA-lacZ(pBBR1MCS-5-pqsR) and its empty vector control strain attB::mini-CTX-PpqsA-lacZ(pBBR1MCS-5) respectively, and culture at 37°C and 200 rpm until OD 600 reaches 1.5.

[0165] Step 15. The β-galactosidase activity of the reporter strain was detected according to the detection method of step 2 in the detection of the gene expression of the quorum sensing system under the action of berberine in Example 1.

[0166] The test results are as follows Figure 9 As shown, in the strain with normal expression of pqsR, as the concentration of berberine gradually increased, the activity of the pqsA promoter gradually decreased, and the maximum inhibitory efficiency was achieved at a concentration of 100 μg / mL. After overexpressing pqsR, as the concentration of berberine increased, the activity of the pqsA promoter did not change. However, whether pqsR was normally expressed or overexpressed, the activity of the pqsA promoter in the control group did not change significantly. The above results indicate that berberine reduces the expression of the pqsA gene by targeting and inhibiting the PqsR protein.

[0167] Example 9

[0168] This example is a molecular docking experiment of berberine and the PqsR protein.

[0169] Use the PubChem website to search for the molecular structure of berberine and its Compound CID, and its Compound CID: 2353. The protein information of PqsR was obtained by downloading from the PDB database, and the PDB ID is 4jvc. The molecular docking of berberine and the PqsR protein was performed using AutoDock Vina 1.1.2 software.

[0170] The docking results are as follows Figure 10 As shown, the berberine molecule can interact with the PqsR protein. It can be seen from the figure that the small molecule berberine has hydrophobic interactions with TYR-258, ILE-263, and ILE-236 on the PqsR protein, and has a hydrogen bond interaction with THR-265 on the PqsR protein. According to relevant literature, a negative binding affinity indicates the possibility of binding between the small molecule and the protein, and a value less than -6 kcal / mol indicates a greater possibility of binding. The docking software shows that the binding affinity score of berberine and PqsR is -8.1 kcal / mol, suggesting that berberine and the PqsR protein have a high binding affinity.

[0171] Example 10

[0172] This example is a test experiment on the effect of berberine on the expression of the pqsA gene in the complemented strain of the point mutant PqsR protein of Pseudomonas aeruginosa.

[0173] (1) Site-directed mutagenesis experiment of the PqsR protein of Pseudomonas aeruginosa

[0174] Step 1: Design primers for the 258th, 236th, and 263rd amino acid residues of the PqsR protein respectively using the genomic DNA of Pseudomonas aeruginosa as a template, such as pqsRF1 / pqsRR2, pqsR Y258G F2 / pqsR Y258G R1, and use this primer to PCR amplify the 1999 bp sequence of the pqsR gene;

[0175] Step 2: Double digest the amplified pqsR fragment and the plasmid pUC18T-mini-TN7T-Gm-lacZ with Hind III / Pst I.

[0176] Step 3: Purify the digested pqsR fragment and ligate it to the plasmid pUC18T-mini-TN7T-Gm-lacZ, then transform it into competent Escherichia coli TG1 cells, and spread it on an LB plate containing Ampicillin (Amp, 100 μg / mL, abbreviated as Amp100) for screening.

[0177] Step 4: After PCR verification of the obtained colonies, extract the plasmid to obtain the recombinant vector, and perform double digestion verification of the recombinant vector with Hind III / Pst I to obtain the recombinant vector pUC18T-mini-TN7T-pqsR-Gm-lacZ showing a positive result.

[0178] Step 5: Pick a single colony of the ΔpqsR-mini-CTX-pqsA-lacZ strain, place it in 5 mL of TSB medium containing Km30Tc200, and culture it at 37 °C and 200 rpm for 16 - 20 h.

[0179] Step 6: Transfer the recombinant vector and the pTNS3 plasmid into ΔpqsR-mini-CTX-pqsA-lacZ by electroporation.

[0180] Step 7: Screen the following reporter strains through a quadruple antibiotic TSB plate containing Km30, TC200, Amp100, and Gm100, including:

[0181] ΔpqsR attB::pqsA'lacZ-pUC18T-mini-Tn7T-Gm

[0182] ΔpqsR attB::pqsA'lacZ-pUC18T-mini-Tn7T-pqsR-Gm

[0183] ΔpqsR attB::pqsA'lacZ-pUC18T-mini-Tn7T-pqsR T265L -Gm

[0184] ΔpqsR attB::pqsA'lacZ-pUC18T-mini-Tn7T-pqsR I236L -Gm

[0185] ΔpqsR attB::pqsA'lacZ-pUC18T-mini-Tn7T-pqsR I263L-Gm

[0186] ΔpqsR attB::pqsA'lacZ-pUC18T-mini-Tn7T-pqsR Y258G -Gm

[0187] Step 8: The β-galactosidase activity of the reporter strain was detected according to the detection method in Example 1.

[0188] To further verify the interaction between berberine and PqsR protein, the present invention constructed transcriptional fusion reporter strains with site-directed mutations at four amino acid sites, including THR-265, ILE236, ILE263, and TYR-258, and detected the effect of adding 100 μg / mL berberine on the β-galactosidase activity of these reporter strains according to Example 1. The test results are as Figure 11 shown. When the mini-Tn7T-Gm empty plasmid was complemented in the ΔpqsR-mini-CTX-pqsA-lacZ strain, no β-galactosidase activity was detected in the successfully constructed transcriptional fusion reporter strain; when the mini-Tn7T-pqsR-Gm, mini-Tn7T-pqsR T265L -Gm, mini-Tn7T-pqsR I236L -Gm, mini-Tn7T-pqsR I263L -Gm, mini-Tn7T-pqsR Y258G -Gm plasmids were complemented respectively, β-galactosidase activity could still be detected in the successfully constructed transcriptional fusion reporter strains, indicating that PqsR T265L 、PqsR I236L 、PqsR I263L 、PqsR Y258G still regulates the transcriptional activation of pqsA. Compared with the transcriptional fusion reporter strain complemented with wild-type pqsR, the mutation of ILE263 enhanced the expression of pqsA. It is speculated that changing the ILE residue to the LEU residue leads to a change in the structure of the PqsR protein, making the conformation of its active site more likely to bind to the pqsA sequence. However, after treatment with berberine, the expression activity of pqsA in the transcriptional fusion reporter strain complemented with pqsR I236L was still inhibited by berberine, indicating that ILE263 may not be the direct action target of berberine. In addition, compared with the transcriptional fusion reporter strain complemented with wild-type pqsR, the mutations of THR-265, ILE236, and TYR-258 all weakened the expression of pqsA. After treatment with berberine, the transcriptional fusion reporter strain complemented with pqsR T265LThe expression activity of pqsA in the transcriptional fusion reporter strain was inhibited, which was similar to the result of complementing wild-type pqsR, indicating that THR-265 might not be the direct target of berberine either. However, after separately complementing pqsR I236L and pqsR Y258G genes, the expression of pqsA was not inhibited, suggesting that ILE236 and TYR-258 are the key sites for the interaction between berberine and PqsR protein. These results are similar to those of Example 9. Using the PqsR I236L protein after point mutation and berberine for molecular docking again, the results showed that after mutation, the binding affinity value between PqsR protein and berberine increased from -8.1 kcal / mol to -6.816 kcal / mol, indicating that the ILE-236 site is the key binding site between PqsR protein and berberine.

[0189] Example 11

[0190] This example is a test for detecting the binding ability between berberine and PqsR protein.

[0191] (1) Preparation of PqsR protein

[0192] Step 1: Design primers for the soluble part of PqsR protein using Pseudomonas aeruginosa genomic DNA as a template, such as pqsR F / pqsR R, pqsR I236L F / pqsR I236L R, and use these primers to PCR amplify a 654-bp sequence;

[0193] Step 2: Perform double digestion on the amplified pqsR gene fragment and plasmid pET28b with Nde I / Sal I.

[0194] Step 3: Purify the digested fragments and then ligate them to the plasmid, transform them into Escherichia coli TG1 competent cells, and spread them on an LB plate containing Km30 for screening;

[0195] Step 4: After the obtained colonies are tested by PCR, extract the plasmid to obtain the recombinant vector, perform enzyme digestion verification on the recombinant vector with Nde I / Sal I, and obtain the recombinant vector pET28b-pqsR with a positive result; pET28b-pqsR I236L ;

[0196] Step 5: Transform the obtained recombinant vector into Escherichia coli Transetta to obtain the expression vector Transetta-pET28b-pqsR with a His tag; Transetta-pET28b-pqsR I236L .

[0197] Step 6: Use the ultrasonic disruption method to disrupt the bacterial cells that have been successfully expressed after induction with 0.5 mM IPTG at 16°C, and obtain the cell lysate supernatant containing the target protein.

[0198] Step 7: Take a His purification column, first wash it with ddH2O, and then equilibrate the column with a lysis buffer at a low flow rate.

[0199] Step 8: Filter the cell lysate supernatant in Step 6 and pass it through the column to adsorb the His fusion protein onto the packing material.

[0200] Step 9: Add the washing buffer until no protein is eluted.

[0201] Step 10: Elute with the elution buffer, collect the protein sample, and after detecting by SDS-PAGE, dialyze the protein sample overnight at 4°C to obtain the PqsR protein.

[0202] The buffers used for His protein purification are as follows:

[0203] Lysis buffer (pH 8.0): 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole.

[0204] Washing buffer (pH 8.0): 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole.

[0205] Elution buffer (pH 8.0): 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole.

[0206] Dialysis buffer (pH 8.0): 50 mM NaH2PO4, 300 mM NaCl, 10% glycerol.

[0207] (2) Microfluidic thermal shift detection assay

[0208] Step 1: Dilute the purified PqsR protein and the PqsR I236L protein and add the NT-647-NHS dye, and incubate in the dark for 30 min for staining.

[0209] Step 2: Equilibrate the column with the Assay Buffer, and the Assay Buffer is 0.05% Tween, 20 mM HEPES.

[0210] Step 3: Load the incubated and stained protein onto the column, discard the waste liquid, wash away the excess dye, and recover the labeled protein.

[0211] Step 4: Prepare 2 mM berberine using Assay buffer, perform 2-fold serial dilution on it, add the labeled protein sample, and finally obtain samples containing berberine at different working concentrations. The working concentrations of berberine are: 250 μM, 125 μM, 62.5 μM, 31.3 μM, 15.6 μM, 7.81 μM, 3.91 μM, 1.95 μM, 0.977 μM, 0.488 μM, 0.244 μM, 0.122 μM, 0.061 μM, 0.0305 μM.

[0212] Step 5: Sampling using a capillary, detecting the binding index using a microfluidic thermal shift assay instrument, and plotting the binding curve using the Kd model in MO.Affinity Analysis.

[0213] To further explore the key binding site between PqsR protein and berberine, the binding ability between the purified protein and berberine was detected using a microfluidic thermal shift assay instrument combined with fluorescence labeling method. The test results are as Figure 12 shown. There is binding between the purified PqsR protein and berberine, and the binding constant Kd = 44.41 ± 32.11 μM. After mutating the 236th isoleucine residue of PqsR protein to leucine residue, the mutant protein cannot bind to berberine. Therefore, the 236th isoleucine residue of PqsR protein is the direct binding site between berberine and PqsR protein. This is similar to the results of Example 9 and Example 10. The above results indicate that berberine inhibits the expression of pqsA and its downstream genes by directly binding to the important transcriptional regulatory protein PqsR of the PQS system of Pseudomonas aeruginosa.

[0214] Example 12

[0215] This example is a test on the effect of berberine on the pathogenicity of Pseudomonas aeruginosa

[0216] (1) Test on the effect of berberine on the pathogenicity of Pseudomonas aeruginosa infecting Chinese cabbage

[0217] Using dimethyl sulfoxide as the solvent control and sinomenine as the control traditional Chinese medicine, the infectivity of Pseudomonas aeruginosa on Chinese cabbage leaves after adding berberine was detected. The specific experimental steps are as follows:

[0218] Step 1: Pick a single colony of Pseudomonas aeruginosa PAO1 into a 5 mL fresh LB liquid medium containing Km30, 100 μg / mL berberine, 100 μg / mL sinomenine, or the same volume of dimethyl sulfoxide, and shake culture at 37 °C and 200 rpm for 16 - 20 h;

[0219] Step 2: Aspirate the bacterial liquid, centrifuge at 5500 rpm for 5 min, discard the supernatant, then wash the bacteria with an equal volume of 10 mM MgSO4, and repeat twice;

[0220] Step 3. Dilute the bacterial solution with 10 mM MgSO4 to OD 600 = 1.5;

[0221] Step 4. Select fresh Chinese cabbage leaves, disinfect the leaves with 0.2% sodium hypochlorite, and air dry;

[0222] Step 5. Inoculate 10 μL of the treated bacterial solution at the same position on the Chinese cabbage stem, incubate at 30 °C for 3 d, observe the rotted area of the Chinese cabbage, and calculate it using Image J software; perform 3 replicates for each group of experiments.

[0223] (2) Cell infection experiment of berberine on Pseudomonas aeruginosa infecting A549 cells

[0224] Using dimethyl sulfoxide as the solvent control and sinomenine as the control traditional Chinese medicine, detect the survival rate of A549 cells by Pseudomonas aeruginosa under the treatment of adding berberine. The specific steps are as follows:

[0225] Step 1. Take out the cryopreserved cells from liquid nitrogen, suspend them with the medium, and place them in a carbon dioxide incubator for culturing until the cells adhere to the wall.

[0226] Step 2. Take out the cell culture dish (100 mm) with confluent monolayer cells from the carbon dioxide incubator, in the ultra-clean workbench, aspirate the medium in the bottle, digest the cells with trypsin, collect the cells, and prepare a cell suspension.

[0227] Step 3. Transfer the cell suspension to a 96-well culture dish. After the cells adhere completely, take out the cell culture, and gently rinse each well with 1×PBS.

[0228] Step 4. Add bacteria to each well for infection, and incubate in a carbon dioxide incubator for 8 h.

[0229] Step 5. Take out the culture in each well, rinse with 1xPBS, add CCK8 solution, and incubate in a carbon dioxide incubator for 4 h.

[0230] Step 5. Take out the 96-well culture dish, detect the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, calculate the cell survival rate (%), and set 3 replicates for each group of experiments;

[0231] Cell survival rate (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0232] As: Absorbance of the experimental well (cells infected with bacteria treated with drugs, medium, cck-8 solution);

[0233] Ac: Absorbance of the control well (cells not infected with bacteria, medium, cck-8 solution)

[0234] Ab: Absorbance of blank wells (including culture medium, cck-8 solution, without cells)

[0235] (3) Cell invasion assay of berberine on Pseudomonas aeruginosa invading A549 cells

[0236] Using dimethyl sulfoxide as the solvent control and sinomenine as the control traditional Chinese medicine, the effect of berberine treatment on the invasion of Pseudomonas aeruginosa into A549 cells was detected. The specific steps are as follows:

[0237] Step 1: Take out the cryopreserved cells from liquid nitrogen, suspend them with culture medium and place them in a carbon dioxide incubator until the cells adhere to the wall.

[0238] Step 2: Take out the cell culture dish (100mm) with confluent monolayer cells from the carbon dioxide incubator. In the laminar flow hood, aspirate the culture medium in the bottle, digest the cells with trypsin, collect the cells and prepare a cell suspension.

[0239] Step 3: Transfer the cell suspension to a 96-well culture dish. After the cells adhere completely, take out the cell culture, and gently rinse each well with 1×PBS.

[0240] Step 4: Add Gm10 and incubate in a carbon dioxide incubator for 1 h.

[0241] Step 5: Take out the culture, rinse with 1xPBS and add pre-cooled Triton-x100 and incubate for 20 min.

[0242] Step 6: Suspend the bacteria in each well with fresh LB medium, dilute and spread on a Km30 plate for counting, and set 3 replicates for each group of experiments.

[0243] The test results of the infection ability of Pseudomonas aeruginosa on Chinese cabbage leaves are as Figure 13 shown in A. Compared with the dimethyl sulfoxide treatment group, after berberine treatment, the rotted area caused by the infection of Pseudomonas aeruginosa on Chinese cabbage decreased by about 64.75%, and the difference reached a significant level (*, p < 0.05). Compared with the control traditional Chinese medicine sinomenine, the rotted area of the berberine treatment group decreased by about 64.11%, and the difference reached a significant level (*, p < 0.05), indicating that berberine can reduce the pathogenicity of Pseudomonas aeruginosa to Chinese cabbage.

[0244] The test results of the survival rate of A549 cells after adding berberine treatment are as Figure 13As shown in B, compared with the dimethyl sulfoxide and the positive control sinomenine-treated groups, the survival rate of A549 cells infected with Pseudomonas aeruginosa after berberine treatment was significantly increased by about 2-fold, and the difference reached a significant level (**, p<0.01). Under the treatment of berberine, the relative invasion rate of Pseudomonas aeruginosa to A549 cells is shown in Figure 13 As shown in C, compared with the dimethyl sulfoxide-treated group and the positive control sinomenine-treated group, the invasion ability of Pseudomonas aeruginosa after berberine treatment was significantly decreased by about 1.3-fold, and the difference reached a significant level (**, p<0.01), indicating that berberine can significantly inhibit the invasion ability of Pseudomonas aeruginosa to A549 cells. The above results show that berberine can reduce the pathogenicity of Pseudomonas aeruginosa to A549 cells.

[0245] Based on the above examples, although the monomer berberine screened in the present invention has no significant effect on the normal growth of Pseudomonas aeruginosa. However, berberine can significantly inhibit the PQS quorum sensing system of Pseudomonas aeruginosa in a concentration-dependent manner, and its inhibitory effect is most obvious at 100 μg / mL. Further studies have shown that berberine can not only inhibit the expression of virulence factors such as pyocyanin and biofilm formation, but also significantly weaken the motility of Pseudomonas aeruginosa, including swarming, swimming and twitching motility.

[0246] Further studies on the mechanism of action of berberine were carried out. Through gene overexpression and molecular docking experiments, it was found that berberine binds to the transcriptional activator protein PqsR of the PQS quorum sensing system, thereby reducing the production of PQS signals and inhibiting the expression of PQS system-related genes. Further, a microscale thermophoresis (MST) experiment was carried out using in vitro purified PqsR protein, and the results confirmed that PqsR is the direct target protein of berberine, and the key site of its binding site is the 236th isoleucine residue.

[0247] In addition, the protective effect of berberine on the host was also verified. The results showed that berberine can reduce the pathogenicity of Pseudomonas aeruginosa to plants (such as Chinese cabbage) and animal cell models (such as human alveolar epithelial cells), and significantly reduce the infection damage suffered by the host. These results fully demonstrate that berberine is an effective compound for inhibiting the pathogenicity of Pseudomonas aeruginosa.

[0248] In summary, berberine can effectively inhibit the PQS quorum sensing system of Pseudomonas aeruginosa without affecting its normal growth, reduce its pathogenicity to plant and animal models, and has a protective effect on the host. Chemical antibacterial drugs are prone to cause the emergence of bacterial drug resistance, while natural products derived from plants have attracted much attention due to their rich resources and high safety. At present, the number of quorum sensing inhibitors (QSIs) with clear action targets is limited. Through a series of experiments, the present invention found that the action target of berberine is the PqsR protein, which provides strong support for the development and application of drugs for the treatment of Pseudomonas aeruginosa infection or adjuvant therapy by studying this target. In addition, berberine, as a quorum sensing inhibitor, has broad application prospects in the development and preparation of drugs for the treatment of Pseudomonas aeruginosa infection and adjuvant therapy.

[0249] As described above, the basic principles, main features and advantages of the present invention are preferably described. The above embodiments and the description are only for describing 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 should fall within the protection scope determined by the present invention.

Claims

1. Use of berberine or a pharmaceutically acceptable salt thereof in the preparation of an inhibitor of the Pseudomonas aeruginosa quorum sensing system.

2. The use according to claim 1, wherein the inhibitor of the Pseudomonas aeruginosa quorum sensing system is used for preventing and / or treating Pseudomonas aeruginosa diseases.

3. The application according to claim 1, characterized in that, The berberine or a pharmaceutically acceptable salt thereof acts on the PQS system in the Pseudomonas aeruginosa quorum sensing system.

4. The application according to claim 3, characterized in that, The berberine or a pharmaceutically acceptable salt thereof inhibits the expression of the gene pqsA of the PQS system in the Pseudomonas aeruginosa quorum sensing system.

5. The application according to claim 3, wherein The action target of the berberine or a pharmaceutically acceptable salt thereof is the PqsR protein of the PQS system in the Pseudomonas aeruginosa quorum sensing system.

6. The application according to any one of claims 1-5, characterized in that, The berberine and its pharmaceutically acceptable salts inhibit the virulence and / or pathogenicity of Pseudomonas aeruginosa.

7. The application according to claim 6, characterized in that, The virulence is the ability of Pseudomonas aeruginosa to produce pyocyanin.

8. The application according to claim 6, characterized in that, The virulence is the ability of Pseudomonas aeruginosa to form biofilms.

9. The application according to claim 6, characterized in that, The virulence is the motility of Pseudomonas aeruginosa, and the motility includes swarming motility, swimming motility and twitching motility.

10. The application according to claim 6, characterized in that, The pathogenicity is the ability of Pseudomonas aeruginosa to infect plants or animals.

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