Multi-target quorum quenching enzyme preparation and its preparation method and application

Abstract

The present application belongs to the technical field of quorum quenching enzyme preparation, and particularly relates to a multi-target quorum quenching enzyme preparation and a preparation method and application thereof, such as being mainly used for inhibiting the virulence factors of Pseudomonas aeruginosa, and including acyl homoserine lactone acyltransferase AiiO protein and 3-hydroxy-4-oxoquinolone 2,4-dioxygenase AqdC protein, and the mass ratio of the two is 20:(1-25); such as being mainly used for inhibiting the virulence factors and the synthesis of biological membranes of Pseudomonas aeruginosa, and further including deferiprone, and the mass ratio of AiiO, AqdC and deferiprone is 20:(1-25):(0.695-2.78), the present application can target the Las, Rhl and PQS of Pseudomonas aeruginosa, the yield and stability of the prepared AqdC protein are both high, the two types of enzymes are used in combination after the regulation amount, and the virulence factors regulated by different quorum sensing pathways in Pseudomonas aeruginosa are effectively inhibited; the regulation amount of deferiprone is used in combination with the two types of quorum quenching enzymes, and the virulence and biological membranes of Pseudomonas aeruginosa can be effectively inhibited in a comprehensive manner.

Description

Technical Field

[0001] This invention belongs to the field of quorum sensing quenching enzyme preparation technology, specifically relating to multi-target quorum sensing quenching enzyme preparations, their preparation methods, and applications. Background Technology

[0002] Quorum sensing refers to the phenomenon where microorganisms secrete specific signaling molecules and sense changes in their concentrations, thereby responding to changes in cell density. Quorum sensing systems and their secreted signaling molecules can participate in the regulation of various virulence factors and biofilm formation in pathogenic bacteria, and are closely related to their virulence and pathogenicity. Currently, various quorum sensing systems have been discovered in Gram-negative pathogens, and their corresponding signaling molecules are mainly N-acyl homoserine lactones (AHLs) and quinolones. Different quorum sensing systems and signaling molecules can regulate different virulence factors. Current quorum sensing quenching enzyme preparations contain quorum sensing quenching enzymes with single components or functions, which can only degrade one type of signaling molecules (AHLs) to inhibit quorum sensing. They cannot effectively inhibit complex quorum sensing systems and the multiple virulence factors they regulate. In addition, pathogens often coexist, such as in bacterial respiratory tract infections where multiple pathogens often coexist. There is intra- and inter-population quorum sensing and communication between pathogens, which may induce symbiotic bacteria to exert their pathogenicity through signaling molecules. Inhibiting a single type of quorum sensing system in a pathogen may not achieve a good therapeutic effect.

[0003] *Pseudomonas aeruginosa* is one of the most prevalent opportunistic pathogens causing infections in patients with burns, trauma, immunodeficiency, or pulmonary fibrosis. *P. aeruginosa* can induce host disease through the secretion of various virulence factors, such as pyocyanin, fluorescein, extracellular proteases, and biofilm formation. For example, elastase can cause tissue damage and inflammation, promoting pathogen invasion and colonization. Pyocyanin disrupts the host's defense mechanisms, leading to cystic fibrosis. fluorescein is an important sidecarrier for *P. aeruginosa*, regulating toxin secretion and production, and disrupting the host's iron environment. Currently, the widespread use of broad-spectrum antibiotics and the high rate of drug resistance in *P. aeruginosa* pose significant challenges to the clinical control of *P. aeruginosa* infections. Therefore, developing novel antibacterial agents targeting *P. aeruginosa* and its coexisting flora is of great importance for the prevention and control of *P. aeruginosa* and its coexisting flora infections in clinical practice. Pseudomonas aeruginosa possesses a complex quorum sensing cascade regulatory system. The Las and Rhl quorum sensing systems are regulated by AHL-like signaling molecules 3-oxo-C12-HSL and C4-HSL, respectively, while the PQS quorum sensing system is regulated by the signaling molecule PQS. The Las and Rhl quorum sensing systems primarily regulate the synthesis of virulence factors such as proteases and pyocyanin, while the PQS quorum sensing system primarily regulates the formation of virulence factors such as green luciferin. Inhibiting the complex quorum sensing system of Pseudomonas aeruginosa is an effective way to achieve comprehensive inhibition of multiple virulence factors. Furthermore, since iron is an essential nutrient for pathogenic bacteria, limiting the concentration of available iron ions can also inhibit biofilm synthesis, serving as an alternative strategy to antibiotic treatment.

[0004] Existing patents and research findings only contain a single category of quorum sensing quenching enzymes, for example:

[0005] (1) The research results “Intervention study of targeted specific AHL lactonease on Pseudomonas aeruginosa infection in the lungs” used paraoxonase to degrade the Pseudomonas aeruginosa signal molecule 3-oxo-C12-HSL, thereby inhibiting the quorum sensing system and pathogenicity of the bacterium.

[0006] (2) The patent “An N-acyl homoserine lactonease and its drug” uses a single acyl homoserine lactonease AiiK to inhibit the biofilm, virulence factor extracellular protease and pyocyanin of Pseudomonas aeruginosa.

[0007] (3) The patent “A quorum sensing quenching enzyme OLB-26 and its encoding gene and application” mainly provides an amino acid sequence of a fused quorum sensing quenching enzyme and determines its enzyme activity characteristics. The fusion protein fuses the encoding genes of two quorum sensing quenching enzymes AiiO and AiiA that degrade different AHL signaling molecules.

[0008] (4) The Chinese literature “Cloning and Expression of Quorum Sensing Quenching Enzyme and Its Effect on Pseudomonas aeruginosa” uses a single N-acyl homoserine lactone enzyme AiiA to act on Pseudomonas aeruginosa, inhibiting its biofilm synthesis, virulence factor pyocyanin and total protease formation. Summary of the Invention

[0009] To overcome the shortcomings of the prior art, the concept of this invention is as follows:

[0010] This invention provides two quorum sensing quenching enzymes: acylhomoserine lactone acyltransferase and 3-hydroxy-4-oxoquinolone 2,4-dioxygenase. The acylhomoserine lactone acyltransferase used in this invention can degrade the signal molecules 3-oxo-C12-HSL and C4-HSL of the *Pseudomonas aeruginosa* Las and Rhl quorum sensing systems, as well as AHL-type signal molecules (AHL with acyl side chains of 4-14 cm in length, with or without 3-oxo or 3-hydroxy substitution) in other pathogenic bacteria. The dioxygenase can degrade the signal molecule PQS of the PQS system. This invention, by preparing high-yield and highly stable dioxygenases, and then by controlling the dosage and combining the two enzymes, effectively inhibits the synthesis of virulence factors. Furthermore, by controlling the dosage and auxiliary addition of deferoxone at a sub-inhibitory concentration, it effectively inhibits the formation of *Pseudomonas aeruginosa* biofilms. This formulation can effectively inhibit the pathogenicity regulated by the complex quorum sensing system of Pseudomonas aeruginosa, preventing it from developing drug resistance. It can also inhibit coexisting Gram-negative bacteria of Pseudomonas aeruginosa that cause respiratory and burn infections, and that can synthesize AHL or respond to AHL to promote virulence and biofilm formation.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] Multi-target quorum sensing quenching enzyme preparations are mainly used to inhibit the virulence factors of Pseudomonas aeruginosa, including acylhomoserine lactone acyltransferase AiiO protein and 3-hydroxy-4-oxoquinolone 2,4-dioxygenase AqdC protein, with a mass ratio of 20:(1-25); and also mainly used to inhibit the virulence factors and biofilm synthesis of Pseudomonas aeruginosa, including deferoxone (DFP), with a mass ratio of AiiO protein, AqdC protein and deferoxone of 20:(1-25):(0.695-2.78).

[0013] Furthermore, the AqdC protein is prepared by IPTG-induced expression, comprising the following steps:

[0014] The plasmid pET28b(+)::his8-aqdC was used. IChemical or electrochemical transformation of *Escherichia coli* BL21(DE3) was performed using the recombinant engineered strain *E. coli* BL21(DE3)-pET28b(+)::his8-aqdC. I IPTG-induced expression of the quorum sensing quencher enzyme AqdC was induced. 1% of the recombinant engineered bacteria were inoculated into LB broth containing 50 μg / mL kanamycin and activated at 37°C and 180 rpm for 10 h. 1% of the activated bacterial culture was then transferred to LB broth containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until the bacterial density reached an OD value. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.2 mM to induce the expression of the target protein. Incubate at 16℃ and 180 rpm for 20-24 h to purify the target protein.

[0015] Protein purification: Collect bacterial cells and centrifuge the bacterial suspension at 4°C for 10 min. Discard the supernatant and resuspend the bacterial precipitate thoroughly in binding buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM β-mercaptoethanol). Dissolve the bacterial suspension using an ultrasonic homogenizer. Centrifuge the disrupted suspension at 4°C for 40 min. Transfer the supernatant to a nickel-bound affinity chromatography column. Shake the column on ice for 40 min, then discard the column buffer. Add 2-3 times the loading volume of washing buffer (WB) to the column twice, discarding the column buffer again. Add 15 mL of elution buffer (EB) to the column, collecting the column buffer (WB: 20 ​​mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 50 mmol / L imidazole; elution buffer (EB: 20 ​​mmol / L...). Tris-HCl (pH 8.0), 150 mmol / L NaCl, and 300 mmol / L imidazole were used. The target protein eluted with elution buffer was concentrated by ultrafiltration, desalted and purified using a gel chromatography column at a flow rate of 2 mL / min and a pressure of 0.4 MPa. The eluent at the peak position of the target protein was collected and analyzed by polyacrylamide gel electrophoresis to determine the concentration of the purified target protein. The protein solution was concentrated by ultrafiltration, and 10% sterile glycerol was added. The solution was then aliquoted and frozen.

[0016] Furthermore, the quorum sensing quencher enzyme AqdC protein is prepared by a lactose self-induction method, comprising the following steps:

[0017] E. coli BL21(DE3)-pET28b(+)::his8-aqdC I After activation in 10 mL of high-density proliferation liquid medium containing 50 μg / mL kanamycin, the culture medium was transferred at an inoculum rate of 3‰ to 200 mL of lactose self-induction medium containing 50 μg / mL kanamycin in 1L graduated shake flasks. The volume of lactose self-induction medium was 200 mL. The culture was carried out at 37°C and 180 rpm until the cell density OD reached 200 mL. 600 Once the pH reaches 0.6-0.8, cool to 20-25℃ and culture in a shake flask at 250 rpm for 24 h. The protein purification method is the same as the purification method described above.

[0018] The application of multi-target quorum sensing quenching enzyme preparations can be used to inhibit a variety of Gram-negative pathogens that cause respiratory and burn infections, including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, and other coexisting bacteria that can synthesize AHL or respond to AHL to promote virulence and biofilm.

[0019] Furthermore, the coexisting bacterial community is a Pseudomonas aeruginosa community, specifically in its application in inhibiting the complex quorum sensing system of Pseudomonas aeruginosa and the inhibition of its regulated virulence factors and biofilm synthesis.

[0020] The combined use of AqdC and AiiO proteins to inhibit the virulence factors of Pseudomonas aeruginosa is as follows:

[0021] After culturing Pseudomonas aeruginosa in 12-well plates for 8 hours, purified AqdC and AiiO protein solutions were added to the wells. 10-250 μg of AqdC protein and 200 μg of AiiO protein were added per mL of culture medium and cultured for 24 hours.

[0022] The combined use of AqdC, AiiO protein, and deferoxone to inhibit the virulence factors and biofilm synthesis of Pseudomonas aeruginosa is as follows:

[0023] After culturing Pseudomonas aeruginosa in 12-well plates for 8 hours, purified AqdC and AiiO protein solutions and deferoxone solution were added to the wells. 10-250 μg of AqdC protein, 200 μg of AiiO protein and 50-200 μM deferoxone solution were added per mL of culture medium and cultured for 24 hours.

[0024] Compared with existing technologies, the present invention has the following advantages:

[0025] Existing technologies employ single-class quorum sensing quenching enzymes, which cannot effectively inhibit the complex quorum sensing system of *Pseudomonas aeruginosa*, thus failing to comprehensively suppress the synthesis of virulence factors. The multi-target quorum sensing quenching enzyme preparation proposed in this invention can specifically inhibit the Las, Rhl, and PQS quorum sensing systems of *P. aeruginosa*. The prepared AqdC protein exhibits high yield and stability. The combined use of the two enzyme classes, after dosage adjustment, effectively inhibits virulence factors regulated by different quorum sensing pathways in *P. aeruginosa*, including total extracellular proteases, pyocyanin, and fluorescein. The dosage adjustment of deferiphenone, combined with the two quorum sensing quenching enzymes, effectively avoids biofilm increase caused by iron release, effectively inhibiting biofilm formation in *P. aeruginosa*. Therefore, it comprehensively and effectively inhibits both the virulence and biofilm of *P. aeruginosa*, and also effectively inhibits *P. aeruginosa* and its coexisting Gram-negative pathogens. Attached image description:

[0026] Figure 1(a) shows the results of AqdC protein lactose self-induction expression and purification, where M: Blue Plus II protein marker; 1: uninduced whole protein; 2: induced whole protein; 3: supernatant after centrifugation of fermentation broth; 4: intracellular supernatant after cell disruption; 5: intracellular precipitate after cell disruption; 6: protein purified by affinity chromatography; 7: concentrated protein after desalting.

[0027] Figure 1(b) shows the specific activity stability of the quorum sensing quencher enzyme AqdC;

[0028] Figure 2 The effect of quorum sensing quencher enzyme AqdC on virulence factors of Pseudomonas aeruginosa;

[0029] Figure 3 The effect of quorum sensing quencher enzyme AiiO on virulence factors of Pseudomonas aeruginosa;

[0030] Figure 4 The inhibitory effect of the combined use of quorum sensing quenching enzymes AiiO and AqdC on the virulence factors of Pseudomonas aeruginosa;

[0031] Figure 5 A comparison of the inhibitory effects of quorum sensing quenching enzymes alone and in combination on Pseudomonas aeruginosa virulence factors;

[0032] Figure 6 The inhibitory effect of quorum sensing quenching enzymes AqdC and AiiO combined with deferoxone on the virulence factors of Pseudomonas aeruginosa;

[0033] Figure 7 The inhibitory effect of quorum sensing quenching enzymes AqdC and AiiO combined with deferoxone on Pseudomonas aeruginosa biofilm was investigated. Detailed Implementation

[0034] To better understand the content of this invention, specific implementation examples will be used to further illustrate the invention below. The following embodiments are based on the technology of this invention and include detailed implementation methods and operating steps; however, the scope of protection of this invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the experimental equipment, materials, reagents, etc., used are all commercially available.

[0035] Example 1: Expression, purification, and specific activity and stability determination of quorum sensing quencher enzyme AqdC

[0036] Using recombinant engineered E. coli BL21(DE3)-pET28b(+)::his8-aqdC I IPTG-induced expression of the quorum sensing quencher enzyme AqdC was performed. 1% of the recombinant engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and activated at 37°C and 180 rpm for 10 h. 1% of the activated bacterial culture was then transferred to LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until the bacterial density reached an OD value. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.2mM to induce the expression of the target protein. Incubate at 16℃ and 180rpm for 20-24h to purify the target protein.

[0037] Protein purification: Collect bacterial cells, centrifuge the bacterial suspension at 4°C for 10 min, discard the supernatant, and resuspend the bacterial precipitate thoroughly in binding buffer. Binding buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM β-mercaptoethanol; use an ultrasonic disruptor to disrupt the bacterial suspension. Centrifuge the disrupted bacterial suspension at 4°C for 40 min, transfer the supernatant to a nickel-binding affinity chromatography column, and vortex the column on ice for 40 min, then discard the column pass liquid. Add 2-3 times the loading volume of washing buffer (WB) to the column twice, discarding the column pass liquid. Add 15 mL of Elution buffer (EB) to the column, collecting the column pass liquid. Washing buffer (WB): 20 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 50 mmol / L imidazole. Elution buffer (EB): 20 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 300 mmol / L imidazole. The target protein eluted with the Elution buffer was concentrated by ultrafiltration, desalted and purified using gel chromatography, and the eluent at the elution peak was collected. The target protein was then analyzed by polyacrylamide gel electrophoresis to determine the concentration of the purified protein. The purified protein solution was concentrated by ultrafiltration, 10% sterile glycerol was added, and the solution was aliquoted and frozen.

[0038] In addition to inducing protein expression via IPTG as described above, lactose autoinduction can also be used to induce protein expression. E. coli BL21(DE3)-pET28b(+)::his8-aqdC I After activation in 10 mL of high-density proliferation liquid medium containing 50 μg / mL kanamycin, the culture was transferred at a 3‰ inoculation rate to 200 mL of lactose self-induction medium containing 50 μg / mL kanamycin in 1L graduated shake flasks. The volume of the lactose self-induction medium was 200 mL. The culture was carried out at 37℃ and 180 rpm until the cell density reached OD0.05. 600 Once the pH reaches 0.6-0.8, cool to 20-25℃ and culture in a shake flask at 250 rpm for 24 h. The protein purification method is the same as the purification method described above.

[0039] High-density proliferation medium (1L): 5g glucose, 3.55g disodium hydrogen phosphate, 3.4g potassium dihydrogen phosphate, 2.68g ammonium chloride, 0.71g sodium sulfate, 0.5g magnesium sulfate heptahydrate, 0.3g trisodium citrate, 2.5g sodium succinate hexahydrate, 1mL ferric chloride (0.1M), add deionized water to make up to volume, pH 7.0, sterilize by moist heat at 120℃ for 20min.

[0040] Lactose self-induction medium (1L): 10g peptone, 5g yeast extract, 3.56g disodium hydrogen phosphate, 3.4g potassium dihydrogen phosphate, 2.68g ammonium chloride, 0.71g sodium sulfate, 0.5g magnesium sulfate heptahydrate, 20mL glycerol, 0.5g glucose, 2g lactose, 0.3g citrate, 5.4g succinate, 1mL ferric chloride (0.1M), add deionized water to make up to volume, pH 7.0, sterilize by moist heat at 120℃ for 20min.

[0041] The specific activity and stability of AqdC protein were determined as follows: In Tris-HCl buffer (pH 8.0), with a final PQS concentration of 45 μM, 1.7 μM of AqdC protein was added to a total reaction volume of 2 mL. The OD of the reaction mixture was measured using a microplate reader. 377 The change in absorbance was used to calculate the remaining amount of PQS in the system, in order to characterize the enzyme activity of AqdC in catalyzing the degradation of PQS. The PQS concentration was calculated as follows: A = εbc, where A (absorbance) and ε = 10.89 × 10⁻⁶. 3 M -1 cm -1 b (optical path length, cm), c (concentration). The enzyme activity of AqdC protein is defined as the amount of enzyme required to decompose 1 μmol of PQS in 1 minute at 30°C, defined as one unit of enzyme activity (U). The specific activity of AqdC protein is defined as the enzyme activity per milligram of protein, expressed in U / mg. AqdC protein was incubated at 37°C, and samples were taken periodically to determine its specific activity and stability.

[0042] The results showed that IPTG induced AqdC protein expression, yielding 9.3 mg of purified AqdC protein per liter of fermentation broth. Lactose self-induction was used to induce AqdC protein expression, and the results were shown in Figure 1(a) by polyacrylamide gel electrophoresis. Lactose self-induction significantly increased the expression level of the target protein AqdC, yielding 20.1 mg of purified AqdC protein per liter of fermentation broth, approximately twice that of IPTG-induced expression. Figure 1(b) shows the specific activity and stability of purified AqdC after incubation at 37℃. The protein exhibited good stability within 16 hours.

[0043] Example 2: Expression and purification of quorum sensing quencher enzyme AiO

[0044] The recombinant protein AiiO was expressed using the constructed Escherichia coli BL21(DE3)-pET28a(+)-aiiO. The protein expression and purification methods were the same as in Example 1.

[0045] Example 3: Inhibition of Pseudomonas aeruginosa virulence factors by the combined use of quorum sensing quenching enzymes AqdC and AiiO.

[0046] Pseudomonas aeruginosa was cultured overnight at 37°C and 180 rpm. The overnight culture was diluted to OD using sterile LB medium. 600 The bacterial culture was diluted 1% and then transferred to sterile LB medium in 12-well plates. After incubation at 37°C for 8 hours, purified AqdC and AiiO protein solutions were added to the plates. In the protein-only experiments, the experimental groups received 10, 100, and 250 μg of AqdC protein per mL of culture medium; the experimental groups received 10, 100, and 200 μg of AiiO protein per mL of culture medium; in the combined AqdC and AiiO experiments, the experimental groups received 10, 100, and 250 μg of AqdC protein and 200 μg of AiiO protein per mL of culture medium. The control group received sterile Tris-HCl buffer containing 10% glycerol, with the buffer volume matching that of the experimental groups. Each group was tested in triplicate. Virulence factors were measured after 24 hours of incubation.

[0047] Pyrosisin assay: Transfer the culture medium from the well plate to a 2 mL centrifuge tube, centrifuge at 4°C for 5 min, collect the supernatant, add 1 mL chloroform, vortex to mix, and centrifuge at 4°C for 5 min. Discard the supernatant, retaining the lower organic phase. Add 250 μL of 0.2 M HCl to the organic phase, vortex to mix, and centrifuge at 4°C for 5 min. Transfer 150 μL of the upper inorganic phase to a 96-well plate and measure the OD using a microplate reader. 520 To characterize the content of pyocyanin.

[0048] Green fluorescein assay: Transfer the culture medium from the well plate to a 2 mL centrifuge tube, centrifuge at 4°C for 5 min, and transfer 100 μL of the supernatant to a 2 mL centrifuge tube. Dilute with 900 μL of 50 mM Tris-HCl (pH 7.4). Transfer 100 μL of the diluted solution to a white opaque 96-well plate and measure the fluorescence intensity at 460 nm under 400 nm excitation light using a microplate reader to characterize the green fluorescein content.

[0049] Extracellular total protease assay: Transfer the culture medium from the plate to a 2 mL centrifuge tube, centrifuge at 4°C for 5 min, transfer 50 μL of the supernatant to a 2 mL centrifuge tube, add 400 μL of 1.25% skim milk, and incubate at 37°C for 7 min. Transfer 150 μL of the reaction solution to a 96-well plate and measure the OD. 600 The result is the turbidity of the remaining protein in skim milk. The activity of total extracellular protease is characterized by 1 / residual protein measurement.

[0050] Statistical analysis was performed using SPSS software. Data represent the mean ± standard deviation from three independent biological replicates. ANOVA and Tukey's HSD test were used for data analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The control group's pyocyanin and fluorophore levels were set to 1, and the control group's total extracellular protease activity level was also set to 1, indicated by black lines. Data from the experimental groups were normalized.

[0051] The virulence factors of Pseudomonas aeruginosa were measured after 24 hours of culture, and the results are as follows: Figure 2 As shown in the figure. Analysis revealed that when the AqdC addition reached 250 μg / mL, total protease and pyocyanin were significantly inhibited; AqdC showed a good inhibitory effect on green fluorescein, with highly significant inhibitory effects observed at addition levels ranging from 10 to 250 μg / mL. After the addition of AqdC protein, the expression of the three virulence factors decreased compared to the control group. At an AqdC addition level of 250 μg / mL, the pyocyanin level was 74.89% of the control group, the green fluorescein level was 23.53% of the control group, and the extracellular total protease level was 95.21% of the control group.

[0052] Combination Figure 3 It was found that the expression of pyocyanin was inhibited when the concentration of AiiO protein was 200 μg / mL. AiiO protein showed a highly significant inhibitory effect on total extracellular proteases, and this inhibitory effect gradually increased with increasing AiiO protein concentration. The inhibitory effects of AiiO protein at concentrations of 100 and 200 μg / mL on fluorophore were very significant. At an AiiO concentration of 200 μg / mL, the level of pyocyanin was 76.31% of that in the control group, the level of fluorophore was 55.58% of that in the control group, and the level of total extracellular proteases was 60.40% of that in the control group.

[0053] The combined use of 10-250 μg / mL AqdC and 200 μg / mL AiiO, combined with... Figure 4Several different concentrations of combined formulations were found to inhibit all three virulence factors. The protein addition combination of AiiO 200 μg / mL + AqdC 250 μg / mL showed a particularly significant inhibitory effect on all three virulence factors. With this protein addition combination, the levels of pyocyanin, fluorophore, and total extracellular protease were 40.73% and 8.96% of the control group, respectively, and 55.95% of the control group. These results were significantly superior to the inhibitory effects of the two quorum sensing quenchers used alone.

[0054] Comparing the inhibitory effects of the above-mentioned addition methods on virulence factors, it can be found that AiiO equal to 200 μg / mL + AqdC10-250 μg / mL or greater is the optimal combination of the two types of quorum sensing quenching enzymes. The combined addition of the two types of quorum sensing quenching enzymes is beneficial for the comprehensive inhibition of the two quorum sensing systems in Pseudomonas aeruginosa, and compared to the addition of quorum sensing quenching enzymes alone, which can only inhibit one type of signal molecule, it has a more significant inhibitory effect on multiple virulence factors. Figure 5 ).

[0055] Example 4: Inhibition of virulence factors and biofilms in Pseudomonas aeruginosa by the combined use of quorum sensing quenching enzymes and deferoxone.

[0056] Deferoxone (DFP) was prepared into a 12.8 mM stock solution using sterile water and sterilized using a 0.2 μm sterile filter membrane for later use. After culturing *Pseudomonas aeruginosa* in 12-well plates for 8 hours, 250 μg of purified AqdC protein, 200 μg of AiiO protein, and a sub-inhibitory concentration of DFP solution (final concentration 100 μM) were added to each well of *P. aeruginosa* culture. The buffer volume added to the control group was the same as that added to the experimental group, with three replicates per group. Virulence factors and biofilm were measured after 24 hours of culture. The methods for virulence factor determination and data analysis were the same as in Example 3.

[0057] Biofilm assay: Discard the suspended cells in the wells after culture. Gently wash the inner wall of the wells with PBS until no residual suspended cells remain. Add an equal volume of culture medium to each well of ice-cold methanol for fixation for 30 min. Discard the methanol and, after it has completely evaporated, add an equal volume of culture medium to each well of 0.2% crystal violet solution and stain for 30 min. Discard the crystal violet solution and gently wash the inner wall of the wells with deionized water until no residual crystal violet remains. After the wells are dry, add an equal volume of culture medium to each well of 33% glacial acetic acid and measure the OD using a microplate reader. 595 To characterize the biofilm content.

[0058] The data represent the mean ± standard deviation from three independent biological replicates. The control group biofilm measurement level was set to 1, represented by the black line, and the experimental group data were normalized.

[0059] The combination of AqdC 250 μg / mL + AiiO 200 μg / mL with a sub-inhibitory concentration of 100 μM deferone was selected to determine virulence factors and biofilm formation. Figure 6 , 7 It was found that the combination of the three drugs also had a good inhibitory effect on virulence factors. In addition, the inhibitory effect on biofilm was significantly improved, with biofilm reaching 70.04% of the control group.

[0060] In the combined use of two quorum sensing quenching enzymes, either the two quorum sensing quenching enzymes can be added separately as described in this invention, or a fusion protein expressed and purified using recombinant bacteria can be added. The fusion protein is obtained as follows: Gene fragments of the two quorum sensing quenching enzymes, AqdC and AiiO, are prepared separately by PCR amplification. Overlap PCR is performed using the purified PCR products as templates to prepare a fusion gene encoding AqdC and AiiO. The two gene fragments can be linked together using a linker peptide of a certain length. The purified fusion gene fragment is then inserted into the multiple cloning site of the vector pET28a(+) or pET28b(+) to obtain a recombinant plasmid. The recombinant plasmid is then chemically or electrochemically transformed into *Escherichia coli* BL21(DE3) to obtain recombinant bacteria. The fusion protein can be expressed and purified according to the recombinant strain culture method and protein expression and purification method described in this invention.

[0061] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of a multi-target quorum sensing quenching enzyme preparation in the preparation of a reagent for inhibiting virulence factors of Pseudomonas aeruginosa, characterized in that, The multi-target quorum sensing quenching enzyme preparation is composed of acylhomoserine lactone acyltransferase AiiO protein and 3-hydroxy-4-oxoquinolone 2,4-dioxygenase AqdC protein, with a mass ratio of 20:(1-25); the virulence factors are extracellular total protease, pyocyanin, and green fluorescein.

2. The application of a multi-target quorum sensing quenching enzyme preparation in the preparation of reagents for inhibiting virulence factors and biofilms of Pseudomonas aeruginosa, characterized in that, The multi-target quorum sensing quenching enzyme preparation is composed of acylhomoserine lactone acyltransferase AiiO protein, 3-hydroxy-4-oxoquinolone 2,4-dioxygenase AqdC protein, and deferiphenone, with a mass ratio of 20:(1-25):(0.695-2.78); the virulence factors are extracellular total protease, pyocyanin, and green fluorescein.

3. The application as described in claim 1 or 2, characterized in that, The AqdC protein was prepared by IPTG-induced expression, including the following steps: The plasmid pET28b(+)::his8-aqdC was used. I Chemical or electrochemical transformation of E. coli Escherichia coli BL21(DE3), using recombinant engineered bacteria E. coli BL21 (DE3)-pET28b(+)::his8-aqdC I IPTG-induced expression of the quorum sensing quencher enzyme AqdC was induced. 1% of the recombinant engineered bacteria were inoculated into LB broth containing 50 μg / mL kanamycin and activated at 37°C and 180 rpm for 10 h. 1% of the activated bacterial culture was then transferred to LB broth containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until the bacterial density reached an OD value. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.2 mM to induce the expression of the target protein. Incubate at 16℃ and 180 rpm for 20-24 h to purify the target protein. The AqdC protein can also be prepared by a lactose-inducible method, including the following steps: Will E. coli BL21 (DE3)-pET28b(+)::his8-aqdC I After activation in 10 mL of liquid medium containing 50 μg / mL kanamycin, the culture was transferred at an inoculation rate of 3‰ to 200 mL of lactose self-induction medium containing 50 μg / mL kanamycin in 1 L graduated shake flasks. The volume of lactose self-induction medium was 200 mL. The culture was carried out at 37 °C and 180 rpm until the cell density reached OD. 600 Once the pH reaches 0.6-0.8, cool the temperature to 20-25℃ and culture in a shake flask at 250 rpm for 24 h using a baffle to purify the target protein.

4. The application as described in claim 1, characterized in that, The specific procedure for inhibiting virulence factors is as follows: After culturing Pseudomonas aeruginosa in a 12-well plate for 8 h, purified AqdC and AiiO protein solutions are added to the well plate. 10-250 μg of AqdC protein and 200 μg of AiiO protein are added per mL of culture medium and cultured for 24 h.

5. The application as described in claim 2, characterized in that, The specific procedure for inhibiting virulence factors and biofilms is as follows: After culturing Pseudomonas aeruginosa in 12-well plates for 8 h, purified AqdC and AiiO protein solutions and deferoxone solution are added to the well plates. 10-250 μg of AqdC protein, 200 μg of AiiO protein and 50-200 μM deferoxone solution are added per mL of culture medium and cultured for 24 h.

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