Use of tuberactinomycin or an analogue thereof for the preparation of a medicine for inhibiting pseudomonas aeruginosa

Abstract

The application belongs to the technical field of microorganisms, and discloses application of tuberculo naphthoquinone or an analogue thereof in preparation of a pseudomonas aeruginosa inhibiting drug. The tuberculo naphthoquinone of the application is a natural metabolite of mycobacterium tuberculosis, has good adaptability to the human body, and can inhibit the quorum sensing system of pseudomonas aeruginosa without causing toxicity problems. Meanwhile, the analogue of the tuberculo naphthoquinone has a similar molecular structure to the naphthoquinone, and thus also has an inhibiting effect on the quorum sensing system and good adaptability to the human body. The corresponding drug prepared by using the tuberculo naphthoquinone or the analogue thereof can effectively inhibit the pseudomonas aeruginosa, and thus treat related infectious diseases, and has great clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of naphthoquinone or its analogues in the preparation of drugs that inhibit Pseudomonas aeruginosa. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa *Pseudomonas aeruginosa* is a pathogenic bacterium of significant research value: it can cause numerous acute infections (such as pneumonia and sepsis) and chronic infections (such as implant prosthesis infections, intubation infections, and chronic obstructive pulmonary disease infections) in hospitals, and it exhibits multidrug resistance. It is one of the six leading pathogens causing high morbidity and mortality rates globally. The quorum sensing (QS) system is an important component of the *Pseudomonas aeruginosa* virulence regulatory network, playing a crucial regulatory role in the expression of virulence factors such as pyocyanin and elastase, as well as bacterial biofilm formation and motility, thus being closely related to clinical bacterial infections. The QS regulatory system is an intercellular signaling system used to coordinate the behavior of different bacterial individuals in various bacterial population densities. It is synergistically regulated by four pathways: Las, Rhl, Pqs, and Iqs. The 3,4-dihydroxy-2-heptaylquinolone signal (PQS) is an important self-inducible signaling molecule in the QS regulatory system. Therefore, if quorum sensing is suppressed, bacteria will find it more difficult to colonize the host and form biofilms, and the expression of related virulence factors will also decrease accordingly.

[0003] In related technologies, the prevention and control of Pseudomonas aeruginosa infection generally targets biofilms. Biofilm inhibitors can specifically inhibit the biofilm structure of microorganisms without killing the microorganisms themselves, thus avoiding stress on the microorganisms and preventing the development of new "drug resistance." Currently, there are few inhibitors and drugs targeting quorum sensing of Pseudomonas aeruginosa, and most research on antimicrobial compounds for Pseudomonas aeruginosa involves screening through compound libraries. However, anti-Pseudomonas compounds often have the potential for drug toxicity issues that can lead to human drug sensitivity.

[0004] Both Mycobacterium tuberculosis and Pseudomonas aeruginosa are lung-colonizing bacteria that share an ecological niche, inevitably leading to some competitive or synergistic relationships. Z-100, a polysaccharide-rich extract from the Mycobacterium tuberculosis strain Qingshan B, has been shown to protect immunocompromised mice from Pseudomonas aeruginosa infection. An immunocompromised model was established by injecting mice with cyclophosphamide, followed by treatment with different doses of Z-100 for 5 days. The control group received no treatment. One day after treatment, mice were infected with Pseudomonas aeruginosa, and mortality was assessed. Compared to the untreated group, the treated group showed significantly enhanced protective ability, indicating that the secretion of Mycobacterium tuberculosis, Z-100, has an inhibitory effect on Pseudomonas aeruginosa. Based on this, a competitive relationship exists between Mycobacterium tuberculosis and Pseudomonas aeruginosa; that is, a natural product of Mycobacterium tuberculosis can inhibit some toxic behaviors of Pseudomonas aeruginosa. As a crucial component of Pseudomonas aeruginosa's toxicity, the quorum sensing system holds promise for the development of drugs by identifying molecules and analogues that inhibit the quorum sensing system of other bacteria (including Pseudomonas aeruginosa) from natural products of Mycobacterium tuberculosis. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes the application of naphthoquinone or its analogues in the preparation of drugs that inhibit the quorum sensing system of *Pseudomonas aeruginosa*. The naphthoquinone of the present invention is a natural metabolite of *Mycobacterium tuberculosis*, and the naphthoquinone analogues have similar molecular structures to naphthoquinone and both have good adaptability to the human body.

[0006] This invention also proposes the application of naphthoquinone or its analogues in the preparation of drugs that inhibit the synthesis of Pseudomonas aeruginosa virulence factors.

[0007] This invention also proposes the application of nodular naphthoquinone or its analogues in the preparation of biofilm formation inhibitory drugs.

[0008] The present invention also proposes the use of naphthoquinone or its analogues in the preparation of medicaments for treating Pseudomonas aeruginosa infection or diseases caused by said Pseudomonas aeruginosa.

[0009] According to one aspect of the present invention, the use of naphthoquinone or its analogues in the preparation of Pseudomonas aeruginosa quorum sensing system inhibitors is proposed, wherein the analogues of naphthoquinone include at least one of vitamin K1, vitamin K2 or vitamin K3;

[0010] The chemical structural formula of vitamin K1 is as follows:

[0011] ;

[0012] The chemical structural formula of vitamin K2 is as follows:

[0013] ;

[0014] The chemical structural formula of vitamin K3 is as follows:

[0015] .

[0016] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved:

[0017] Naphthoquinone is a natural metabolite of Mycobacterium tuberculosis and exhibits good adaptability to the human body. Its use in inhibiting the quorum sensing system of Pseudomonas aeruginosa does not present toxicity issues. Furthermore, some analogues of naphthoquinone, due to their similar molecular structure, also possess the same inhibitory effect on the quorum sensing system of Pseudomonas aeruginosa and show good adaptability to the human body. Drugs prepared using naphthoquinone or its analogues can effectively inhibit Pseudomonas aeruginosa, thereby treating related infectious diseases and possessing significant clinical application value.

[0018] In some embodiments of the present invention, the chemical structural formula of the nodular naphthoquinone is as follows:

[0019] .

[0020] In some preferred embodiments of the present invention, the n value of the vitamin K2 is any one of 2, 4 or 7.

[0021] The aforementioned naphthoquinone analogues are mainly synthesized in the liver, and therefore have good adaptability to the human body and do not produce drug toxicity.

[0022] In some embodiments of the present invention, the naphthoquinone or its analogues competitively bind to the receptor protein of the signaling molecule 3,4-dihydroxy-2-heptyl-quinolone (PQS) in the Pseudomonas aeruginosa quorum sensing system, thereby inhibiting the Pseudomonas aeruginosa quorum sensing system.

[0023] In some embodiments of the present invention, the receptor protein is an aromatic hydrocarbon receptor protein.

[0024] Aromatic hydrocarbon receptor protein (AhR protein) is an important and highly conserved ligand-dependent transcription factor in the human body, primarily regulating various developmental and physiological functions, including neurogenesis, tracheal and salivary duct formation, toxin metabolism, circadian rhythms, response to hypoxia, and hormone receptor function. AhR can sense environmental toxins and endogenous ligands, thereby inducing the activation of detoxification enzymes to regulate immune cell differentiation and response. AhR can directly recognize phenazine (QS) molecules secreted by Pseudomonas aeruginosa, thereby regulating the expression of detoxification enzymes, chemokines, and cytokine genes.

[0025] Both PQS molecules from *Pseudomonas aeruginosa* and naphthoquinone (Pht) molecules from *Mycobacterium tuberculosis* are ligands of human AhR regulatory proteins. Since *P. aeruginosa* and *Mycobacterium tuberculosis* share an ecological niche in the lungs, PQS and Pht compete with each other. Pht molecules can inhibit the expression of PQS molecules in *P. aeruginosa*, thereby suppressing the quorum sensing system of *P. aeruginosa*. Analogs of naphthoquinone, due to their similar molecular structure to naphthoquinone, can also exert the same inhibitory effect on the quorum sensing system of *P. aeruginosa*.

[0026] In some embodiments of the present invention, the naphthoquinone or its analogues have an inhibitory effect on the Pseudomonas aeruginosa quorum sensing system in the concentration range of 500 to 750 nM.

[0027] In some preferred embodiments of the present invention, the signaling molecule 3,4-dihydroxy-2-heptyl-quinolone has a promoting effect on the Pseudomonas aeruginosa quorum sensing system at a concentration of less than 2000 nM; the naphthoquinone or its analogue competitively binds to the receptor protein of the signaling molecule 3,4-dihydroxy-2-heptyl-quinolone in a concentration range of 500-750 nM, thereby inhibiting the Pseudomonas aeruginosa quorum sensing system.

[0028] In some preferred embodiments of the present invention, the concentration of the signaling molecule 3,4-dihydroxy-2-heptyl-quinolone at 750–1000 nM has a significant promoting effect on the quorum sensing system of Pseudomonas aeruginosa.

[0029] In some preferred embodiments of the present invention, the naphthoquinone or its analogues exhibit the most significant inhibitory effect on the quorum sensing system of Pseudomonas aeruginosa at a concentration of 500 nM.

[0030] According to a second aspect of the present invention, the use of the said naphthoquinone or its analogues in the preparation of a drug for inhibiting the synthesis of Pseudomonas aeruginosa virulence factors is proposed, wherein the analogues of said naphthoquinone include at least one of vitamin K1, vitamin K2 or vitamin K3;

[0031] The chemical structural formula of vitamin K1 is as follows:

[0032] ;

[0033] The chemical structural formula of vitamin K2 is as follows:

[0034] ;

[0035] The chemical structural formula of vitamin K3 is as follows:

[0036] .

[0037] In some embodiments of the present invention, the chemical structural formula of the nodular naphthoquinone is as follows:

[0038] .

[0039] In some embodiments of the present invention, the virulence factor includes at least one of pyocyanin or rhamnolipin.

[0040] Pseudomonas aeruginosa (PCN) and rhamnolipid (RL) are important virulence factors secreted by Pseudomonas aeruginosa, which can induce apoptosis and other pathological manifestations.

[0041] According to a third aspect of the present invention, the use of the said naphthoquinone or an analogue thereof in the preparation of a drug to inhibit the formation of Pseudomonas aeruginosa biofilm is proposed, wherein the chemical structural formula of the naphthoquinone is as follows:

[0042] ;

[0043] The analogues of the tuberculonaphthoquinone include at least one of vitamin K1, vitamin K2, or vitamin K3;

[0044] The chemical structural formula of vitamin K1 is as follows:

[0045] ;

[0046] The chemical structural formula of vitamin K2 is as follows:

[0047] ;

[0048] The chemical structural formula of vitamin K3 is as follows:

[0049] .

[0050] The biofilm is an important ecological manifestation of the Pseudomonas aeruginosa community and can induce resistance to multiple antibiotics.

[0051] According to a fourth aspect of the invention, the use of naphthoquinone or an analogue thereof in the preparation of a medicament for treating Pseudomonas aeruginosa infection or diseases caused by said Pseudomonas aeruginosa is provided, wherein the analogue of naphthoquinone includes at least one of vitamin K1, vitamin K2 or vitamin K3;

[0052] The chemical structural formula of vitamin K1 is as follows:

[0053] ;

[0054] The chemical structural formula of vitamin K2 is as follows:

[0055] ;

[0056] The chemical structural formula of vitamin K3 is as follows:

[0057] .

[0058] In some embodiments of the present invention, the infection includes at least one of an acute infection or a chronic infection.

[0059] In some embodiments of the present invention, the disease includes at least one of pneumonia, sepsis, disease caused by implanted prosthesis infection, intubation infection, or chronic obstructive pulmonary disease infection. Attached Figure Description

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0061] Figure 1 This is a schematic diagram of the chemical structures of 3,4-dihydroxy-2-heptyl-quinolone and nodular naphthoquinone in Example 1 of the present invention;

[0062] Figure 2 This is a growth curve diagram of the six groups of Pseudomonas aeruginosa cultured in Example 1 of the present invention;

[0063] Figure 3 This is a graph showing the effect of adding different concentrations of 3,4-dihydroxy-2-heptaylquinone and naphthoquinone on the expression of the green fluorescent protein gene in Example 2 of the present invention; where A represents the effect of adding 3,4-dihydroxy-2-heptaylquinone on the expression of the green fluorescent protein gene. pqsA The effect on gene expression, where B represents the effect of adding naphthoquinone on gene expression. pqsA The effects on gene expression;

[0064] Figure 4 for Figure 3 Enlarged views of parts a and b in the middle;

[0065] Figure 5 This is a schematic diagram illustrating the determination of pyocyanin production in five groups of Pseudomonas aeruginosa in Example 3 of the present invention.

[0066] Figure 6 This is a schematic diagram illustrating the determination of rhamnolipid production in five groups of Pseudomonas aeruginosa in Example 3 of the present invention.

[0067] Figure 7 This is a schematic diagram illustrating the determination of biofilm formation in 5 groups of Pseudomonas aeruginosa in Example 4 of the present invention;

[0068] Figure 8 This is a schematic diagram of the chemical structures of vitamins K1, K2, and K3 in Example 5 of the present invention;

[0069] Figure 9 The growth curves of five groups of Pseudomonas aeruginosa cultured in Example 5 of the present invention are shown.

[0070] Figure 10 This is a graph showing the effect of adding different concentrations of PQS on the expression of the green fluorescent protein gene in Example 6 of the present invention.

[0071] Figure 11 for Figure 10 A magnified view of part a in the middle;

[0072] Figure 12 This is a graph showing the effect of adding different concentrations of vitamin K1 on the expression of the green fluorescent protein gene in Example 6 of the present invention.

[0073] Figure 13 This is a graph showing the effect of adding different concentrations of vitamin K2 on the expression of the green fluorescent protein gene in Example 6 of the present invention.

[0074] Figure 14 This is a graph showing the effect of adding different concentrations of vitamin K3 on the expression of the green fluorescent protein gene in Example 6 of the present invention. Detailed Implementation

[0075] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0076] In the description of this invention, unless otherwise explicitly defined, terms such as cultivation and extraction should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0077] In the description of this invention, references to terms such as "one embodiment," "some embodiments," etc., indicate that a specific feature, method, or material described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, methods, and materials described may be combined in any suitable manner in one or more embodiments.

[0078] Unless otherwise specified, the experimental methods used in the examples are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0079] Example 1

[0080] This embodiment describes the culture of *Pseudomonas aeruginosa* under different treatments. The specific process is as follows:

[0081] Six groups were set up: blank control group, WT (Wild Type) group, WT+PQS group, and △ pqsC Group, △ pqsC+Pht Groups and △ pqsR Groups, each with 6 repetitions. The specific settings for each group are as follows:

[0082] Blank control group: 200µL of ABTGC medium (200µL of medium B (0.1% magnesium chloride, 0.1% calcium chloride, 0.1% ferric chloride) plus 10% of medium A (0.2% glucose and 0.2% casein amino acids)) was added.

[0083] WT group: Add 200µL of Pseudomonas aeruginosa PAO1 bacterial suspension (from ATCC strain bank, strain number ATCC15692, and the Pseudomonas aeruginosa PAO1 bacterial suspension mentioned below is from the same source).

[0084] WT+PQS group: Add 200µL of Pseudomonas aeruginosa PAO1 bacterial culture, and then add 2000nM of PQS (3,4-dihydroxy-2-heptyl-quinolone).

[0085] △ pqsC Group: Add 200µL of Pseudomonas aeruginosa PAO1△ pqsC The bacterial solution, specifically the one in which Pseudomonas aeruginosa was knocked out. pqsC Gene;

[0086] △ pqsC+Pht Group: Add 200µL of Pseudomonas aeruginosa PAO1△ pqsC Add 2000 nM Pht (nucleotide naphthoquinone) to the bacterial culture.

[0087] △ pqsR Group: Add 200µL of Pseudomonas aeruginosa PAO1△ pqsR The bacterial solution, specifically the one in which Pseudomonas aeruginosa was knocked out. pqsR Gene.

[0088] The chemical structural formulas of PQS and Pht are as follows: Figure 1 As shown, the two molecules have similar structures.

[0089] Plant the seedlings in the culture plate as described above, place the culture plate in a microplate reader, and measure the absorbance (OD) every hour at 37°C. 600 The measurement lasted for 24 hours, and the results are as follows: Figure 2 As shown.

[0090] Figure 2The results showed that the growth rate and corresponding trend of P. aeruginosa in P. aeruginosa bacterial culture with the addition of PQS or Pht were nearly identical to those of P. aeruginosa alone, indicating that the addition of PQS or Pht did not have a significant impact on the growth of P. aeruginosa PAO1. At the same time, the gene knockout group could be compared with the experimental group with the inhibition of the quorum sensing system to a certain extent, which also proved that the absence of PQS had no effect on the growth of P. aeruginosa PAO1.

[0091] Example 2

[0092] This example demonstrates the effects of different concentrations of PQS or Pht on Pseudomonas aeruginosa. pqsA The analysis of the effects on gene expression is conducted as follows:

[0093] pqsA The gene is an important gene in the quorum sensing system of Pseudomonas aeruginosa. Here, we characterize it by detecting the expression of green fluorescent protein (gfp). pqsA Gene expression status.

[0094] Extraction of Pseudomonas aeruginosa strain PAO1 PpqsA-gfp plasmid PUCP22- pqsA-gfp (Constructed on PUCP22 plasmid) pqsA - gfp The gene was then introduced into PAO1 competent cells and cultured to obtain plasmid PUCP22- pqsA- gfp ), import the plasmid △pqsC competent cells (Pseudomonas aeruginosa PAO1 cell knockout) pqsC (Originally obtained from gene sequencing) Pseudomonas aeruginosa PAO1 was constructed. pqsC-pqsA-gfp strains, among which gfp The gene encoding green fluorescent protein is used as a reporter gene for characterization. pqsA Gene expression status.

[0095] Then, 200 μL of Pseudomonas aeruginosa PAO1 was added to the culture plate. pqsC-pqsA-gfp Different concentrations of PQS molecules (0, 125, 250, 375, 500, 750, 1000, 1500, 2000 nM) were added to the bacterial suspension as a positive control, because it is known that the addition of PQS can restore the expression of pqsA in the Pseudomonas aeruginosa quorum sensing system. 200 μL of Pseudomonas aeruginosa PAO1 bacterial suspension was added as a negative control; another group was prepared using Pseudomonas aeruginosa PAO1 Δ... pqsC-pqsA-gfpDifferent concentrations of Pht molecules (0, 125, 250, 375, 500, 750, 1000, 1500, 2000 nM) were added to the bacterial culture. The culture plate was placed in a microplate reader, and the absorbance (OD) was measured every 1 hour at 37°C. 600 The measurement lasted for 24 hours, and the results are as follows: Figure 3 and Figure 4 As shown.

[0096] Figure 3 The display shows that A represents the effect after adding PQS. pqsA The effect on gene expression is shown in the figure, where the concentrations of PQS from top to bottom are 0, 125, 250, 500, 750, 1000, 1500, and 2000 nM; B represents the effect of Pht addition on gene expression. pqsA The effect on gene expression, where the concentration of Pht in the legend is from top to bottom as 0, 125, 250, 500, 750, 1000, 1500, 2000 nM. Figure 4 for Figure 3 Enlarged views of parts a and b are shown, with the concentrations of added PQS and Pht marked. At concentrations less than 2000 nM, PQS has the following effect on... pqsA Gene expression was promoted in all cases, with the effect being more pronounced at concentrations between 750 and 1000 nM. Specifically, PQS promoted the quorum sensing system of *Pseudomonas aeruginosa* at concentrations below 2000 nM, with a more significant effect at concentrations between 750 and 1000 nM. In Figure B, after the addition of Pht, the effect increased with increasing Pht concentration. pqsA Gene expression is suppressed, with significant inhibition occurring at concentrations between 500 and 750 nM, and the most significant inhibition at 500 nM. This indicates that Pht concentrations of 500–750 nM have a more pronounced inhibitory effect on the quorum sensing system of *Pseudomonas aeruginosa*, suggesting a competitive relationship between Pht and PQS. Increased Pht concentration competitively inhibits the function of PQS, thereby suppressing its activity. pqsA Gene expression further suppresses the quorum sensing system.

[0097] Example 3

[0098] This embodiment analyzes the effect of Pht on the synthesis of virulence factors in Pseudomonas aeruginosa. The specific process is as follows:

[0099] 1. Determination of Pyrosisin (PCN) Yield

[0100] Pseudomonas aeruginosa is an important virulence factor secreted by Pseudomonas aeruginosa, which can induce various pathological manifestations such as apoptosis and immune escape. Measuring its levels can determine the effect of Pht on inhibiting Pseudomonas aeruginosa. Five groups were set up: WT group, WT+Pht group, Δ... lasI △ rhlI Group, △ pqsC Groups and △ pqsC +PQS groups, with 3 replicates per group. Specific settings for each group:

[0101] WT group: 200µL of Pseudomonas aeruginosa PAO1 bacterial suspension was added;

[0102] WT+Pht group: Add 200µL of Pseudomonas aeruginosa PAO1 bacterial solution, and then add 750nM Pht;

[0103] △ lasI △ rhlI Group: Add 200µL of Pseudomonas aeruginosa PAO1△ lasI △ rhlI The bacterial solution, specifically the one in which Pseudomonas aeruginosa was knocked out. lasI and rhlI Gene;

[0104] △ pqsC Group: Add 200µL of Pseudomonas aeruginosa PAO1△ pqsC The bacterial solution, specifically the one in which Pseudomonas aeruginosa was knocked out. pqsC Gene;

[0105] △ pqsC+PQS Group: Add 200µL of Pseudomonas aeruginosa PAO1△ pqsC Add 750 nM PQS to the bacterial culture.

[0106] Among them, △ lasI △ rhlI The group served as the negative control group. lasI and rhlI Both genes are very important in the Pseudomonas aeruginosa quorum sensing system. After these two genes are knocked out, the two most important QS molecules, 3-o-C12-L-HSL and C4-L-HSL, which regulate the quorum sensing system, cannot be produced, and the QS system cannot function at all.

[0107] Five groups of *Pseudomonas aeruginosa* were cultured in King's medium (20 g / L peptone, 1.5 g / L potassium hydrogen phosphate, 1.5 g / L magnesium sulfate) enriched with pyocyanin. The supernatant was collected, chloroform and hydrochloric acid were added, and the absorbance at 520 nm was measured. The absorbance was then divided by 17.072 to convert the concentration (mg / mL). The results are as follows: Figure 5 As shown.

[0108] Figure 5 The results showed that the concentration of pyocyanin was highest in the WT group, △ lasI △ rhlI The group had the lowest concentration of pyocyanin, △ pqsC+PQS Group ratio △ pqsC The high concentration of pyocyanin in the WT+Pht group served as a positive control, while the decreased concentration of pyocyanin in the WT+Pht group indicated that Pht inhibited the production of pyocyanin. The WT group, WT+Pht group, and Δ lasI △ rhlI Group, △ pqsC Groups and △ pqsC The concentrations of pyocyanin in the +PQS group were 1.85, 0.97, 0.67, 0.72, and 1.47 mg / mL, respectively.

[0109] 2. Determination of rhamnolipid yield

[0110] Rhamnolipid is another important virulence factor secreted by *Pseudomonas aeruginosa*. Measuring its levels can reveal the effect of Pht on inhibiting rhamnolipid. Five groups were set up: WT group, WT+Pht group, and Δ group. lasI △ rhlI Group, △ pqsC Groups and △ pqsC +PQS group, 3 replicates per group, see settings in the Pseudomonas aeruginosa production assay for specific settings.

[0111] First, the bacterial supernatant was extracted with ethyl acetate, evaporated overnight, and the organic phase was collected. Then, it was extracted with melanin dissolved in sulfuric acid, reacted at 80°C for 10 min, and the absorbance was measured at 421 nm. The results are as follows: Figure 6 As shown.

[0112] Figure 6 The results showed that the concentration of rhamnolipin was highest in the WT group, △ lasI △ rhlI The concentration of rhamnolipin was lowest in the group, △ pqsC+PQS Group ratio △ pqsC The high concentration of rhamnolipin in the WT+Pht group served as a positive control, while the decreased concentration of rhamnolipin in the WT+Pht group indicated that Pht inhibited the production of rhamnolipin. The WT group, WT+Pht group, and Δ lasI △ rhlI Group, △ pqsC Groups and △ pqsC The concentrations of rhamnolipin in the +PQS group were 1.77, 1.51, 0.25, 0.96, and 1.38 mg / mL, respectively.

[0113] Example 4

[0114] This embodiment analyzes the effect of Pht on the biofilm yield of Pseudomonas aeruginosa. The specific process is as follows:

[0115] Biofilm is an important ecological manifestation of the self-formed community of *Pseudomonas aeruginosa*, and can induce resistance to multiple antibiotics. Measuring its indices can reveal the effect of Pht on biofilm inhibition. Five groups were set up: WT group, WT+Pht group, Δ... lasI △ rhlI Group, △ pqsC Groups and △ pqsC +PQS group, 3 replicates per group, see the settings in the Pseudomonas aeruginosa production determination in Example 3 for specific settings.

[0116] After inoculation, the culture plate was placed in a 37℃ CO2 incubator for 72 hours. The bacterial culture in the wells was aspirated, the plate was washed twice with pure water, and after drying, 125 μL of 0.1% crystal violet staining solution was added to each well. The plate was incubated at room temperature for 10 minutes, and the solution was transferred to a new 96-well plate. The absorbance at 550 nm was measured. The results are as follows: Figure 7 As shown.

[0117] Figure 7 The results showed that the WT group formed the most biofilm, △ lasI △ rhlI Fewer biofilms formed in the group, △ pqsC+PQS Group ratio △ pqsC The group that formed more biofilm served as a positive control, while the group that formed less biofilm showed reduced biofilm formation, indicating that Pht inhibits biofilm formation; WT group, WT+Pht group, △ lasI △ rhlI Group, △ pqsC Groups and △ pqsC The biofilm formation rates in the +PQS group were 100%, 78%, 48%, 42%, and 92%, respectively.

[0118] Example 5

[0119] This embodiment describes the culture of *Pseudomonas aeruginosa* under different treatments. The specific process is as follows:

[0120] Five groups were set up: a blank control group, a WT group, a WT + vitamin K1 group, a WT + vitamin K2 group, and a WT + vitamin K3 group, with six replicates in each group. The specific settings for each group were as follows:

[0121] Blank control group: 200 µL of ABTGC medium was added;

[0122] WT group: 200µL of Pseudomonas aeruginosa PAO1 bacterial suspension was added;

[0123] WT + Vitamin K1 group: Add 200µL of Pseudomonas aeruginosa PAO1 bacterial solution, and then add 2000nM of Vitamin K1.

[0124] WT + Vitamin K2 group: Add 200µL of Pseudomonas aeruginosa PAO1 bacterial solution, and then add 2000nM of Vitamin K2.

[0125] WT + Vitamin K3 group: Add 200µL of Pseudomonas aeruginosa PAO1 bacterial solution, and then add 2000nM of Vitamin K3.

[0126] The chemical structural formulas of vitamins K1, K2, and K3 are as follows: Figure 8 As shown, the structures of the three molecules are similar to those of nodular naphthoquinone.

[0127] Plant the seedlings in the culture plate as described above, place the culture plate in a microplate reader, and measure the absorbance (OD) every hour at 37°C. 600 The measurement lasted for 24 hours, and the results are as follows: Figure 9 As shown.

[0128] Figure 9 The results showed that the growth rate and corresponding trend of Pseudomonas aeruginosa with the addition of vitamins K1, K2 and K3 to the bacterial culture were almost consistent with those of Pseudomonas aeruginosa alone, indicating that the addition of vitamins K1, K2 and K3 did not have a very significant effect on the growth of Pseudomonas aeruginosa PAO1.

[0129] Example 6

[0130] This example demonstrates the effects of different concentrations of vitamin K1, K2, and K3 on Pseudomonas aeruginosa. pqsA The analysis of the effects on gene expression followed the same procedure as in Example 2:

[0131] By comparing the compound library of PubChem, analogues of Pht were identified as vitamins K1, K2, and K3.

[0132] Extraction of Pseudomonas aeruginosa strain PAO1 PpqsA-gfp plasmid PUCP22- pqsA-gfp The plasmid was introduced △pqsC Constructing Pseudomonas aeruginosa PAO1 in competent cells △ pqsC-pqsA-gfp strains, among which gfp The gene encoding green fluorescent protein is used as a reporter gene for characterization. pqsA Gene expression status.

[0133] Then, 200 μL of Pseudomonas aeruginosa PAO1 was added to the culture plate. pqsC-pqsA-gfpThe bacterial suspension was then supplemented with different concentrations of PQS molecules (0, 125, 250, 375, 500, 750, 1000, 1500, 2000 nM) as a positive control, and divided into 3 groups. A negative control was prepared by adding 200 μL of *Pseudomonas aeruginosa* PAO1 bacterial suspension. Additionally, the *Pseudomonas aeruginosa* PAO1 Δ... pqsC-pqsA-gfp Different concentrations of vitamin K1, K2, and K3 molecules were added to the bacterial culture (concentrations of vitamin K1, K2, and K3 were set to 0, 125, 250, 375, 500, 750, 1000, 1500, and 2000 nM, respectively). The culture plates were placed in a microplate reader, and the absorbance (OD) was measured every 1 hour at 37°C. 600 The measurement lasted for 24 hours, and the results are as follows: Figure 10 , 11 As shown in 12, 13 and 14.

[0134] Figure 10 To add PQS to pqsA The effect on gene expression, where the concentrations of PQS in the legend are 0, 125, 250, 500, 750, 1000, 1500, and 2000 nM from top to bottom; Figure 11 for Figure 10 A magnified view of part a, showing the concentration of added PQS; Figure 12 To improve the effect of adding vitamin K1 pqsA The effect on gene expression, where the concentrations of vitamin K1 in the figure from top to bottom are 0, 125, 250, 500, 750, 1000, 1500, and 2000 nM; Figure 13 To improve the effect of adding vitamin K2 pqsA The effect on gene expression, where the concentrations of vitamin K2 in the figure from top to bottom are 0, 125, 250, 500, 750, 1000, 1500, and 2000 nM; Figure 14 To improve the effect of adding vitamin K3 pqsA The effect on gene expression, where the concentrations of vitamin K3 in the figure from top to bottom are 0, 125, 250, 500, 750, 1000, 1500, and 2000 nM. Figure 10 and Figure 11 In the case of PQS at concentrations less than 2000 nM, PQS has the effect of... pqsA Gene expression was promoted by all of them, with the promotion effect being more pronounced at concentrations in the range of 750–1000 nM. Specifically, PQS promoted the quorum sensing system of *Pseudomonas aeruginosa* at concentrations below 2000 nM, and the promotion effect was more pronounced at concentrations of 750–1000 nM. The addition of vitamins K1, K2, or K3 further enhanced the effect. Figure 12 , Figure 13 , Figure 14The results show that as the concentration of vitamin K1, K2, or K3 increases, pqsA Gene expression is suppressed, with the most significant inhibitory effect observed at a concentration of 500 nM. This indicates that a concentration of 500 nM of vitamin K1, K2, or K3 most significantly inhibits the quorum sensing system of *Pseudomonas aeruginosa*, suggesting a competitive relationship between vitamin K1, K2, and K3 and PQS. Increased concentrations of vitamin K1, K2, and K3 competitively inhibit the function of PQS, thereby suppressing its activity. pqsA Gene expression further inhibits the quorum sensing system. This is similar to previous findings on Pht; overall, the naphthoquinone analogs vitamins K1, K2, and K3 have the function of inhibiting the quorum sensing system of Pseudomonas aeruginosa.

[0135] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of naphthoquinone analogues in the preparation of drugs to inhibit Pseudomonas aeruginosa, characterized in that, The tuberculoquinone analogue is at least one of vitamin K2 or vitamin K3; The chemical structural formula of vitamin K2 is as follows: ； The chemical structural formula of vitamin K3 is as follows: 。

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