Application of 4-imidazole formaldehyde in preparation of pseudomonas aeruginosa infection inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of 4-imidazole formaldehyde in a pseudomonas aeruginosa quorum sensing inhibitor. It is found for the first time that 4-imidazole formaldehyde can inhibit quorum sensing of pseudomonas aeruginosa, reduce QS system regulation gene expression and virulence factor yield and remarkably reduce infection caused by QS system regulation gene expression and virulence factor yield; the drug application designed on the basis of bacterial metabolism can avoid the drug resistance of a conventional bacteriostatic agent, and a new strategy is provided for prevention and treatment of pseudomonas aeruginosa infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of 4-imidazolecarboxaldehyde in the preparation of a Pseudomonas aeruginosa infection inhibitor. Background Art

[0002] Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, frequently causes a variety of acute and chronic infections in immunocompromised patients, burn victims, and intubated hospital patients. Furthermore, P. aeruginosa exhibits unique resistance mechanisms, rendering commonly used antibiotics ineffective. Virulence factors such as pyocyanin, proteases, and rhamnolipids play a key role in acute P. aeruginosa infection. Proteases such as elastase and proteolytic enzymes degrade functional proteins such as host cell junctional proteins and immunoglobulins; pyocyanin disrupts redox homeostasis, causing metabolic disturbances within the cell; and rhamnolipids disrupt cell polarity, facilitating early bacterial invasion. During chronic infection, P. aeruginosa forms a stable biofilm structure, which reduces the effectiveness of antibiotics and contributes to resistance. Furthermore, motility mediated by type IV pili and flagella, such as swimming, swarming, and gliding, is associated with initial attachment and acute infection. The pathogenicity and increasing drug resistance of Pseudomonas aeruginosa make its treatment increasingly difficult. The World Health Organization has listed Pseudomonas aeruginosa as a pathogen in urgent need of new drug development.

[0003] The pathogenicity and drug resistance of Pseudomonas aeruginosa are primarily regulated by the quorum sensing (QS) system. The QS system regulates bacterial behavior by sensing intercellularly secreted signaling molecules, primarily regulating virulence factor production, biofilm formation, and motility. Targeting the QS system, quorum sensing inhibitors are being developed to disrupt the QS system by degrading signaling molecules or competing with receptor proteins. This reduces the production of virulence factors associated with bacterial pathogenicity and drug resistance, thereby reducing the pathogenicity of P. aeruginosa and ultimately treating bacterial infections. Compared to traditional antimicrobial drugs, quorum sensing inhibitors do not impose stress on bacteria, potentially preventing the development of drug resistance. Therefore, targeting the quorum sensing system to treat P. aeruginosa infections could serve as an alternative to antibiotics.

[0004] 4-Imidazolecarboxaldehyde, a pharmaceutical intermediate, is commonly used to prepare C17,20-lyase inhibitors for androgen-dependent prostate cancer and in the synthesis of antimalarial drugs. However, there are no reports on its effects on the QS system or its ability to inhibit infection. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides the use of 4-imidazolecarboxaldehyde in the preparation of a Pseudomonas aeruginosa infection inhibitor.

[0006] The present invention provides application of 4-imidazolecarboxaldehyde in preparing a Pseudomonas aeruginosa infection inhibitor.

[0007] The present invention discovered for the first time that 4-imidazolecarboxaldehyde can inhibit the expression of QS system regulatory genes lasR, lasI, rhlR, rhlI, pqsR, and pqsA, reduce the secretion of virulence factors pyocyanin, proteolytic enzymes, and rhamnolipids, and inhibit the motility of Pseudomonas aeruginosa. This drug application based on bacterial metabolism design can circumvent the drug resistance of conventional antibacterial agents and provide a new strategy for the prevention and treatment of Pseudomonas aeruginosa infection.

[0008] Furthermore, the Pseudomonas aeruginosa infection inhibitor inhibits infection by inhibiting the quorum sensing ability of Pseudomonas aeruginosa.

[0009] Furthermore, the inhibiting of the quorum sensing ability of Pseudomonas aeruginosa includes inhibiting the production of virulence factors and inhibiting the motility of Pseudomonas aeruginosa.

[0010] Furthermore, the virulence factors include pyocyanin, proteolytic enzymes and rhamnolipids.

[0011] Furthermore, the athletic ability includes swimming ability, clump movement ability and crawling ability.

[0012] The present invention also provides a Pseudomonas aeruginosa infection inhibitor, wherein the active ingredient of the Pseudomonas aeruginosa infection inhibitor includes 4-imidazolecarboxaldehyde.

[0013] Furthermore, the concentration of 4-imidazole carboxaldehyde in the Pseudomonas aeruginosa infection inhibitor is 80 μg / mL to 160 μg / mL.

[0014] Furthermore, the dosage form of the Pseudomonas aeruginosa infection inhibitor is any one of an emulsifiable concentrate, a suspension concentrate, a microemulsion, a suspoemulsion, an aqueous emulsion, and a dispersible oil suspension.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered that 4-imidazole carboxaldehyde can be used as a quorum sensing inhibitor to reduce the expression of genes regulating the QS system of Pseudomonas aeruginosa and the production of virulence factors, thereby reducing the infection caused by the bacteria. The specific effects are as follows: (1) 4-Imidazolecarboxaldehyde inhibits the QS system regulatory genes of Pseudomonas aeruginosa lasR 、 lasI 、 rhlR 、 rhlI 、 PcqI 、 pq expression.

[0016] (2) 4-Imidazolecarboxaldehyde inhibited the production of virulence factors such as pyocyanin, rhamnolipids, and proteolytic enzymes, which are closely related to acute infection with Pseudomonas aeruginosa.

[0017] (3) 4-Imidazolecarboxaldehyde inhibited the formation of biofilm associated with chronic infection with Pseudomonas aeruginosa.

[0018] (4) 4-Imidazole carboxaldehyde inhibited the clustering, swimming, and gliding abilities associated with the initial attachment of Pseudomonas aeruginosa infection.

[0019] (5) 4-Imidazole carboxaldehyde reduced the rot area of ​​cabbage infected with Pseudomonas aeruginosa and increased the survival rate of fruit flies, showing an anti-pathogenic effect.

[0020] The present invention provides a new approach and method for treating Pseudomonas aeruginosa infection, reducing bacterial drug resistance, and developing a novel QS system inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the effect of 4-imidazolecarboxaldehyde on the growth of Pseudomonas aeruginosa.

[0022] Figure 2 The effect of 4-imidazole carboxaldehyde on the expression of core regulatory genes of the QS system of Pseudomonas aeruginosa is shown in Figure 2, where A is lasR Gene expression statistics, B is rhlR Gene expression statistics, C is PcqI Gene expression statistics, D is lasI Gene expression statistics, E is rhlI Gene expression statistics, F is pq Gene expression statistics graph.

[0023] Figure 3 Graph showing the effect of 4-imidazole carboxaldehyde on the production of virulence factors, wherein A is a graph showing the results of proteolytic enzymes, B is a statistical graph showing the quantitative analysis of proteolytic enzymes, C is a graph showing the results of pyocyanin, D is a statistical graph showing the quantitative analysis of pyocyanin, E is a graph showing the results of rhamnolipids, and F is a statistical graph showing the quantitative analysis of rhamnolipids.

[0024] Figure 4 Graph showing the effect of 4-imidazole carboxaldehyde on biofilm formation, where A is the biofilm result graph and B is the biofilm quantitative analysis statistical graph.

[0025] Figure 5 Graph showing the effect of 4-imidazole formaldehyde on the motility of Pseudomonas aeruginosa PAO1, where A is a swimming motility graph, B is a statistical graph showing the quantitative analysis of swimming motility, C is a clustering graph, D is a statistical graph showing the quantitative analysis of clustering motility, E is a gliding graph, and F is a statistical graph showing the quantitative analysis of gliding motility.

[0026] Figure 6 The figure shows the effect of 4-imidazole carboxaldehyde on the rot area of ​​cabbage, where A is the cabbage rot result diagram, B is the quantitative statistical analysis diagram of cabbage rot, and C is the statistical diagram of the number of bacteria (expressed in CFU) in 1 mg of cabbage.

[0027] Figure 7 This is a statistical chart of Drosophila viability.

[0028] Figure 8 The statistical diagram shows the effect of 4-imidazole formaldehyde alone on the viability of Drosophila. * express P <0.05, ** express P <0.01, *** express P <0.001. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0030] The Pseudomonas aeruginosa PAO1, PAO1-pKD-lasI, PAO1-pKD-lasR, PAO1-pKD-rhlI, PAO1-pKD-rhlR, PAO1-pKD-pqsA and PAO1-pKD-pqsR used in the experiments of the present invention have been disclosed in the following document: “WANG S, FENG Y, HAN X, et al. Inhibition of Virulence Factors and Biofilm Formationby Wogonin Attenuates Pathogenicity of Pseudomonas aeruginosa PAO1 viaTargeting pqs Quorum-Sensing System [J]. Int J Mol Sci, 2021, 22(23).” The applicant promises to distribute the biological material to the public within twenty years from the date of application and provide a way to obtain the biological material.

[0031] Obtain address: Northwest University, No. 229, Taibai North Road, Beilin District, Xi'an City, Shaanxi Province.

[0032] Example 1: Effect of 4-imidazolecarboxaldehyde on the growth of Pseudomonas aeruginosa.

[0033] Experimental method: Pick a monoclonal colony of Pseudomonas aeruginosa PAO1 in 5mL fresh liquid LB and culture it overnight at 37℃ and 200r / min. Inoculate the overnight cultured bacteria into liquid LB medium at a volume fraction of 1% and activate it for 3h. Add 5μL of the activated bacteria to 95μL liquid LB medium and 96-well plates containing LB liquid medium with 4-imidazole formaldehyde concentrations of 160μg / mL and 80μg / mL, respectively. Use LB medium containing DMSO with a final concentration of 1% by volume as the experimental control group. Use a multifunctional microplate reader to detect OD every 30min. 600 , continuous monitoring for 24 hours.

[0034] Experimental results: Figure 1 4-Imidazolecarboxaldehyde at experimental concentrations of 160 μg / mL and 80 μg / mL had no significant effect on the growth of Pseudomonas aeruginosa within 24 hours, indicating that the compound does not cause survival pressure on the bacteria.

[0035] Example 2: Effect of 4-imidazolecarboxaldehyde on the expression of key regulatory genes in the QS system.

[0036] Experimental methods: Gene expression was detected using fusion luminescent Pseudomonas aeruginosa strains containing the target gene promoter and the luminescent cluster enzyme luxCDABE. The fusion luminescent strains containing the target gene promoter and the luminescent cluster enzyme luxCDABE were PAO1-pKD-lasI, PAO1-pKD-lasR, PAO1-pKD-rhlI, PAO1-pKD-rhlR, PAO1-pKD-pqsA, and PAO1-pKD-pqsR. The key regulatory genes of the QS system in Pseudomonas aeruginosa were determined based on the effect of 4-imidazole formaldehyde on the chemiluminescence of the fusion luminescent strains containing the target gene promoter and the luminescent cluster enzyme luxCDABE. lasI 、 lasR 、 rhlI 、 rhlR 、 pq 、 PcqI The specific method is as follows:

[0037] Monoclonal colonies of the P. aeruginosa fusion luminescent strains PAO1-pKD-lasI, PAO1-pKD-lasR, PAO1-pKD-rhlI, PAO1-pKD-rhlR, PAO1-pKD-pqsA, and PAO1-pKD-pqsR were selected and incubated in fresh liquid LB containing 300 μg / mL trimethoprim (Tmp) and cultured at 37°C at 200 rpm for 12 hours. The cultured strains were transferred to fresh liquid LB for activation for 3 hours. Five μL of the activated bacterial suspension was then transferred to a 96-well plate containing 95 μL of liquid LB medium and 4-imidazole carboxaldehyde at concentrations of 160 μg / mL and 80 μg / mL, respectively. Luminescence was monitored every 30 minutes for 24 hours using a multi-function microplate reader to assess the effect of 4-imidazole carboxaldehyde on the expression of genes regulated by the QS system.

[0038] Experimental results: The results are as follows Figure 2 As shown in the figure, 4-imidazolecarboxaldehyde at the experimental concentrations significantly inhibited the luminescence of QS system-regulated genes in a concentration-dependent manner during the first 16 hours, but no significant inhibition was observed after 16 hours. These results demonstrate that 4-imidazolecarboxaldehyde, at concentrations that do not affect P. aeruginosa growth, has a transcriptional inhibitory effect on QS system-regulated genes.

[0039] Example 3: Effect of 4-imidazolecarboxaldehyde on the production of virulence factors regulated by the QS system.

[0040] 1. Proteinase: Pick a monoclonal colony of Pseudomonas aeruginosa PAO1 and place it in 5 mL of fresh liquid LB medium. Culture it at 37°C and 200 r / min overnight for activation. Transfer the activated bacterial solution to a fresh LB medium containing 4-imidazole formaldehyde at a concentration of 160 μg / mL and 80 μg / mL at a volume fraction of 1%. The control group is a liquid LB medium without 4-imidazole formaldehyde. Culture it at 37°C and 200 r / min for 12 hours. The cultured bacterial solution is centrifuged at 8000 r / min for 5 minutes to obtain the supernatant. Add 200 μL of the supernatant to 800 μL of 2% milk, respectively, and place it in a 37°C water bath. Observe the turbidity of the liquid every 30 minutes. Observe the turbidity of the liquid according to the OD value. 440 Determine the content of proteolytic enzymes.

[0041] 2. Pyocyanin: A single colony of Pseudomonas aeruginosa PAO1 was selected and cultured in 5 mL of fresh liquid LB at 37°C and 200 rpm until logarithmic phase. The culture was transferred at a 1% inoculum volume into fresh Pseudomonas Broth medium (Merck, Z699101) containing 160 μg / mL and 80 μg / mL 4-imidazole carboxaldehyde, respectively. A control group was treated with fresh Pseudomonas Broth medium containing 0 μg / mL 4-imidazole carboxaldehyde. The culture was incubated at 37°C and 200 rpm for 16 hours. The culture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was obtained. The supernatant was thoroughly mixed with an equal volume of chloroform and centrifuged at 8000 rpm for 10 minutes to obtain the upper organic phase. The upper organic phase was transferred to a new centrifuge tube, 1 mL of 0.2 mol / L HCl solution was added, and the mixture was thoroughly mixed. After standing and stratifying, measure the OD of the upper HCl solution 520 The yield of pyocyanin was determined based on the OD value.

[0042] 3. Rhamnolipid: Pick a monoclonal colony of Pseudomonas aeruginosa PAO1 and place it in 5 mL of fresh liquid LB. Cultivate at 37°C and 200 rpm until the logarithmic phase. Adjust the bacterial solution to OD 600 After the concentration of the culture medium was 0.2, 2 μL of the solution was spotted onto the center of a solid culture medium plate containing 160 μg / mL and 80 μg / mL of hexadecyltrimethylammonium bromide. A control group used liquid LB medium without 4-imidazole carboxaldehyde. The plates were incubated at 37°C for 3 days. The rhamnolipid production was determined by the size of the transparent zone surrounding the colony.

[0043] 4. Experimental results: The results are as follows Figure 3 4-Imidazolecarboxaldehyde at the experimental concentration reduced the production of QS system-regulated virulence factors, including proteolytic enzymes, pyocyanin, and rhamnolipids.

[0044] Example 4: Effect of 4-imidazolecarboxaldehyde on biofilm.

[0045] Experimental method: Pseudomonas aeruginosa PAO1 monoclonal strain was selected and placed in 5 mL of fresh liquid LB, cultured at 37°C, 200 rpm for 12 h. The overnight culture was adjusted to OD 600is 0.5. 20 μL of bacterial solution was added to each well of a 12-well plate, wherein each well contained 2 mL of fresh liquid LB and LB liquid culture medium with 4-imidazole formaldehyde concentrations of 160 μg / mL and 80 μg / mL, respectively. The control group was a liquid LB culture medium without 4-imidazole formaldehyde. After the 12-well plate was incubated at 37°C for 24 hours, the bacterial solution was discarded and each well was washed with PBS to remove the residue attached to the well. After cleaning, 3 mL of 1% crystal violet solution was added to each well of the 12-well plate to stain the biofilm for 15 minutes. The crystal violet solution was discarded and the staining of the biofilm was observed after rinsing with PBS. The biofilm was rinsed with 1 mL of 95% alcohol and the OD was measured. 595 , to determine the biofilm production.

[0046] Experimental results: The results are as follows Figure 4 4-Imidazolecarboxaldehyde showed an inhibitory effect on the formation of biofilm.

[0047] Example 5: Effects of 4-imidazolecarboxaldehyde on the motility (swarming, swimming, and gliding) of Pseudomonas aeruginosa.

[0048] Preparation of swimming medium: Dissolve 1 g of peptone, 3 g of NaCl and 0.3 g of agar in water to 100 mL.

[0049] Preparation of cell culture medium: Dissolve 1 g peptone, 3 g NaCl, 0.5 g agar and 0.5 g glucose in water to 100 mL.

[0050] Preparation of culture medium: Dissolve 1 g tryptone, 0.5 g yeast extract, 3 g NaCl and 0.3 g agar in water to 100 mL.

[0051] Experimental method: Pick a single colony of Pseudomonas aeruginosa PAO1 in 5 mL of fresh liquid LB and culture it at 37°C and 200 rpm for 12 hours. Adjust the overnight culture to an OD 600 The concentration of 4-imidazole formaldehyde was 0.5. 2 μL of bacterial solution was spotted on the center of the surface of swimming medium and clustering medium containing 160 μg / mL and 80 μg / mL of 4-imidazole formaldehyde, respectively. The control group was liquid LB medium without 4-imidazole formaldehyde. For rubbing motility, use a pipette tip to puncture the center of the rubbing medium containing 160 μg / mL and 80 μg / mL of 4-imidazole formaldehyde, and inject 2 μL of bacterial solution into the puncture hole in the center of the rubbing medium. The control group was liquid LB medium with a 4-imidazole formaldehyde concentration of 0 μg / mL. After the plate was allowed to stand for 30 minutes, it was transferred to a 37°C incubator for 24 hours to observe the movement of the bacteria.

[0052] Experimental results: The results are as follows Figure 54-Imidazole carboxaldehyde reduced the swimming, swarming and motility of Pseudomonas aeruginosa.

[0053] Example 6: The effect of 4-imidazolecarboxaldehyde on the pathogenicity of Pseudomonas aeruginosa was detected using the cabbage model and the Drosophila model.

[0054] 1. Cabbage model: Pick a monoclonal colony of Pseudomonas aeruginosa PAO1 in 5 mL of fresh liquid LB medium and culture it at 37 ° C and 200 r / min to the logarithmic phase for activation. The activated bacterial solution was transferred to liquid LB with a 4-imidazole formaldehyde concentration of 160 μg / mL and 80 μg / mL at an inoculum volume fraction of 1%. The control group was a liquid LB medium without 4-imidazole formaldehyde. It was cultured at 37 ° C and 200 r / min for 12 hours and centrifuged at 8000 r / min for 5 minutes to obtain the bacteria. The bacteria were resuspended with 10 mM sterile MgSO4 to an OD 600 Cut fresh cabbage tubers to an appropriate size and disinfect the stem surface with 1% H₂O₂. Place the disinfected cabbage in the center of a glass plate containing filter paper soaked in a 10 mM MgSO₄ solution. Inject 10 μL of the resuspended bacterial solution into the stems and incubate the plates at 30°C for 4 days. Observe and record the decay of the cabbage.

[0055] 2. Drosophila model: Pick a single clone of Pseudomonas aeruginosa PAO1 in 5mL fresh liquid LB, culture at 37℃, 200r / min to the logarithmic phase for activation. The activated bacterial solution was transferred to liquid LB with 4-imidazole formaldehyde concentrations of 160μg / mL and 80μg / mL, respectively. The positive control group was LB liquid culture medium without 4-imidazole formaldehyde, cultured at 37℃, 200r / min for 12h, and the bacterial solution was centrifuged at 8000r / min for 5min to obtain the bacteria. The bacteria were resuspended in 5% sucrose solution to OD 600 The concentration of 5% sucrose was 2.0. A 5% sucrose medium was prepared in a glass culture bottle. After sterilization and air drying, the surface of the sucrose medium was covered with sterile filter paper. Finally, 200 μL of the resuspended bacterial solution was injected onto the filter paper surface. Ten starved and cryo-anesthetized male fruit flies were added to each glass bottle and cultured at 25°C for 10 days. The survival of the fruit flies was recorded. A blank control group was injected with a 1% DMSO solution alone, while a negative control group was injected with a 160 μg / mL 4-imidazole formaldehyde solution alone.

[0056] Experimental results: The experimental results of the cabbage model are as follows Figure 6As shown in the results, 4-imidazole carboxaldehyde reduced the rot area of ​​cabbage, and the number of bacteria in the cabbage was not significantly affected two days after inoculation with the drug and Pseudomonas aeruginosa PAO1, indicating that the inhibition of 4-imidazole carboxaldehyde on PAO1 is by affecting virulence rather than bacterial growth. Figure 7 and Figure 8 As shown, 4-imidazole carboxaldehyde increased the survival rate of fruit flies. Compared with the blank control and positive control groups, the negative control group showed no effect on the survival of fruit flies when using 4-imidazole carboxaldehyde alone. At the same time, the effect of 4-imidazole carboxaldehyde alone on the survival of fruit flies was not significantly different from that of DMSO alone. This shows that 4-imidazole carboxaldehyde has an anti-pathogenic effect on Pseudomonas aeruginosa and does not show obvious biological toxicity in animals.

[0057] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0058] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0059] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

Application of 1.4-imidazolecarboxaldehyde in the preparation of Pseudomonas aeruginosa infection inhibitors.

2. The use according to claim 1, characterized in that The Pseudomonas aeruginosa infection inhibitor inhibits infection by inhibiting the quorum sensing ability of Pseudomonas aeruginosa.

3. The use according to claim 2, characterized in that The inhibiting of the quorum sensing ability of Pseudomonas aeruginosa includes inhibiting the production of virulence factors and inhibiting the movement ability of Pseudomonas aeruginosa.

4. The use according to claim 3, characterized in that The virulence factors include pyocyanin, proteolytic enzymes and rhamnolipids.

5. The use according to claim 3, characterized in that The athletic ability includes swimming ability, clump movement ability and crawling ability.

6. A Pseudomonas aeruginosa infection inhibitor, characterized in that The active ingredient of the Pseudomonas aeruginosa infection inhibitor is the 4-imidazole carboxaldehyde described in claim 1.

7. The Pseudomonas aeruginosa infection inhibitor according to claim 6, characterized in that The concentration of 4-imidazole carboxaldehyde in the Pseudomonas aeruginosa infection inhibitor is 80 μg / mL to 160 μg / mL.

8. The Pseudomonas aeruginosa infection inhibitor according to claim 6, characterized in that The dosage form of the Pseudomonas aeruginosa infection inhibitor is any one of emulsifiable concentrate, suspension, microemulsion, suspoemulsion, aqueous emulsion and dispersible oil suspension.

Images