Application of epigallocatechin gallate as arcanobacterium pyogenes virulence inhibitor

Abstract

The invention discloses application of epigallocatechin gallate as an arcanobacterium pyogenes virulence inhibitor, and belongs to the technical field of medicines. The invention proposes that the epigallocatechin gallate can target arcanobacterium pyogenes tatA and tatC for the first time, the toxicity of the arcanobacterium pyogenes is effectively inhibited, and the medical application of the epigallocatechin gallate is expanded. The epigallocatechin gallate, as an arcanobacterium pyogenes virulence inhibitor, has an effective inhibition effect on the formation of an arcanobacterium pyogenes biofilm. According to the invention, epigallocatechin gallate is used as an active component of the arcanobacterium pyogenes virulence inhibitor, so that the adhesion and invasion ability of arcanobacterium pyogenes to cells and the mRNA expression level of tatA and tatC genes can be effectively inhibited.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of epigallocatechin gallate as a virulence inhibitor of fusobacterium nucleatum. BACKGROUND

[0002] Fusobacterium nucleatum is a common conditional pathogenic bacterium in veterinary clinics, and can cause tracheitis, endometritis, mastitis and other infections in various economic animals such as cattle, pigs and sheep. Trueperella pyogenes , T. pyogenes The drug resistance problem is becoming more and more serious, which has caused serious threat to the breeding industry and public health safety. Under the background of "reducing antibiotics and replacing antibiotics", in order to control the diseases such as bovine mastitis caused by fusobacterium nucleatum and other pathogenic bacteria, the antibacterial virulence strategy has gradually become a research hotspot. At the same time, it is found that there are TatA and TatC systems related to the formation ability of bacterial biofilm and pathogenicity in fusobacterium nucleatum, but the regulation of virulence of fusobacterium nucleatum has not been reported.

[0003] Due to the great increase in the treatment difficulty of drug-resistant bacteria infection, the development of new anti-infective drugs is imminent. It is reported that plant natural products have good in vitro antibacterial activity against fusobacterium nucleatum, staphylococcus and streptococcus and other pathogenic bacteria, and can inhibit the expression of virulence factors related to the pathogenic process, thereby reducing the virulence of bacteria.

[0004] However, there is no report on the inhibition of fusobacterium nucleatum virulence by epigallocatechin gallate. SUMMARY

[0005] Therefore, the purpose of the present application is to provide the application of epigallocatechin gallate as a virulence inhibitor of fusobacterium nucleatum, which expands the medical application of epigallocatechin gallate. The structural formula of epigallocatechin gallate is as follows, and the chemical name is (2R, 3R)-2-(3, 4, 5-trihydroxyphenyl)-3, 4-dihydro-1 (2H)-benzopyran-3, 5, 7-triol 3-(3, 4, 5-trihydroxybenzoic acid ester).

[0006] .

[0007] In order to achieve the above purpose, the present application provides the following technical scheme: In a first aspect, the present application provides the application of epigallocatechin gallate as a virulence inhibitor of fusobacterium nucleatum.

[0008] Based on the above technical scheme, further, the epigallocatechin gallate inhibits the formation of the biofilm of the fusobacterium nucleatum.

[0009] Based on the above technical scheme, further, the epigallocatechin gallate inhibits the adhesion and invasion ability of the P. acnes to cells.

[0010] Based on the above technical scheme, further, the epigallocatechin gallate inhibits the adhesion and invasion ability of the P. acnes to cells. tatA and tatC mRNA expression level of the genes.

[0011] Based on the above technical scheme, further, the effective concentration of the epigallocatechin gallate is 19.5 μg / mL-78 μg / mL.

[0012] In a second aspect, the present application provides a pharmaceutical composition for inhibiting the virulence of P. acnes, wherein the active ingredient of the pharmaceutical composition comprises epigallocatechin gallate.

[0013] Based on the above technical scheme, further, the only active ingredient of the pharmaceutical composition is epigallocatechin gallate.

[0014] Compared with the prior art, the present application has the following beneficial effects: 1. The present application first proposes that epigallocatechin gallate can target P. acnes tatA and tatC genes, effectively inhibit the virulence of P. acnes, and expand the medical application of epigallocatechin gallate.

[0015] 2. The present application first proposes that epigallocatechin gallate as a P. acnes virulence inhibitor can effectively inhibit the formation of P. acnes biofilm.

[0016] 3. The present application first proposes that using epigallocatechin gallate as the active ingredient of a P. acnes virulence inhibitor can effectively inhibit the adhesion and invasion ability of P. acnes to cells and the mRNA expression level of tatA and tatC genes. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0018] Figure 1 To knock out the plasmid pBAV1K-T5-GFP-Δ tatA Identification results: A is the identification result of bacterial liquid PCR; B is the enzyme digestion identification diagram; C is the sequencing identification diagram; Figure 2 To knock out the plasmid pBAV1K-T5-GFP-Δ tatCIdentification results: A is PCR identification results of bacterial liquid; B is enzyme digestion identification; C is sequencing identification; Figure 3 Identification results of BMH 06-3C tatA tatA Identification results of upstream and downstream homologous arms; C is plo identification results of virulence genes of P. acnes; plo Figure 4 Identification results of BMH 06-3C tatC tatC Identification results; B is tatC Identification results of upstream and downstream homologous arms; C is plo identification results of virulence genes of P. acnes; D is sequencing results; Figure 5 Identification results of complemented strain BMH 06-3C tatA tatA Identification results; B is tatA up-down identification results; C is sequencing results; Figure 6 Identification results of complemented strain BMH 06-3C tatC tatC Identification results; B is tatC up-down identification results; C is sequencing results; Figure 7 Three-dimensional structure diagram of TatA protein of P. acnes: A is SWISS-MODEL modeling; B is phyre2 modeling; Figure 8 Three-dimensional structure diagram of TatC protein of P. acnes: A is SWISS-MODEL modeling; B is phyre2 modeling; Figure 9 Docking model diagram of EGCG and TatA protein of P. acnes; Figure 10 Docking model diagram of EGCG and TatC protein of P. acnes; Figure 11 Comparison diagram of the influence of EGCG on biofilm formation of P. acnes Tat system gene deletion strain and wild strain: “**” in the diagram indicates p<0.01, indicating that the difference between the EGCG treatment group and the control group is extremely significant; “ns” indicates p>0.05, indicating that there is no significant difference between the EGCG treatment group and the control group; Figure 12 Comparison diagram of the influence of EGCG on biofilm structure of P. acnes Tat system gene deletion strain and wild strain; ​​​​​Figure 13 The influence of EGCG on the mRNA expression of biofilm-related genes of the wild type and the Tat system gene deletion strain of F. necrophorum is shown in the following chart: A is rbsB The mRNA expression of genes is shown in the following chart: B is rbsC The mRNA expression of genes is shown in the following chart: C is luxS The mRNA expression of genes is shown in the following chart: Figure 14 The influence of EGCG on the adhesion ability of the wild type and the Tat system gene deletion strain of F. necrophorum is shown in the following chart: Figure 15 The influence of EGCG on the invasion ability of the wild type and the Tat system gene deletion strain of F. necrophorum is shown in the following chart: Figure 16 The influence of EGCG on the mRNA relative expression of the wild type and the Tat system gene deletion strain of F. necrophorum is shown in the following chart: tatA The mRNA relative expression of genes is shown in the following chart: Figure 17 The influence of EGCG on the mRNA relative expression of the wild type and the Tat system gene deletion strain of F. necrophorum is shown in the following chart. tatC The mRNA relative expression of genes is shown in the following chart. DETAILED DESCRIPTION

[0019] The application will be described in detail below with examples, but the embodiments of the application are not limited thereto. Obviously, the examples described below are only some of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0020] The application identifies potential target genes that regulate the virulence of F. necrophorum by using gene knockout technology, and screens target gene inhibitors that regulate virulence by using molecular docking technology, so as to provide a theoretical basis for the research and development of anti-F. necrophorum virulence drugs.

[0021] The application uses the following research methods: 1. Construction of F. necrophorum gene deletion strain tatA , tatC The gene deletion strain and the complementation strain of F. necrophorum TatA and TatC are constructed by using homologous recombination technology. tatA and tatC

[0022] 2. Screening of Tat system inhibitors of F. necrophorum: The three-dimensional structures of TatA and TatC proteins are predicted by using homology modeling method; and the inhibitors of TatA and TatC proteins are virtually screened by using molecular docking technology.

[0023] 3. Targeting of candidate inhibitors tatA , tatC ​Inhibition of virulence of F. necrophorum: analysis of candidate inhibitors on F. necrophorum tatA and tatC Influence of gene deletion strains and wild strains on biofilm formation, cell viability, adhesion ability and invasion ability; analysis of candidate inhibitors on F. necrophorum tatA , tatC Influence on gene mRNA expression.

[0024] The virtual screening process of F. necrophorum TatA and TatC inhibitors is as follows.

[0025] 1. Construction and evaluation of F. necrophorum Tat system protein model.

[0026] Using the online tool of Swiss-model website, the three-dimensional structure of TatA and TatC proteins was predicted by homology modeling principle. Then, after entering saves (https: / / saves.mbi.ucla.edu / ), the quality of the structures of the two Tat system proteins predicted by Swiss-model and phyre2 websites was evaluated using the procheck website, and the highest scoring model was selected for subsequent docking. The highest scoring TatA and TatC proteins were predicted for potential binding pockets using the DoGSiteScorer online tool (https: / / proteins.plus / ).

[0027] 2. Establishment of drug virtual screening library.

[0028] A total of 90 flavonoids, alkaloids, terpenes, organic acids, anthraquinones and polysaccharide compounds reported in the literature were selected, and the 3D structures of the corresponding compounds were downloaded in SDF format in PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ). After that, the energy minimization of TatA, TatC proteins and 90 kinds of ligands was carried out in the molecular docking software (MOE2019), and the all-atom docking and active pocket docking of Tat system proteins were carried out, respectively. When the binding energy of molecular docking is negative, it means that the two molecules have formed a relatively stable structure, and the negative sign indicates attraction, and the smaller the negative number, the greater the attraction. When the binding energy is less than-5 kcal / mol, it is determined to be good binding.

[0029] 3. Sensitivity of F. necrophorum to Tat system candidate inhibitors.

[0030] F. necrophorum BMH06-3 was selected as the test strain, and ATCC19411 was used as the quality control strain. The micro-broth dilution method was used to determine the antibacterial activity of the candidate compounds on the two strains.

[0031] 4. Constructing the molecular docking model of the candidate inhibitor of the Tat system of F. necrophorum.

[0032] As shown in Figures 7 to 10 MOE2019 was used to establish the docking model of the preferred candidate inhibitor and TatA and TatC proteins, and the optimal binding site of the two in space was analyzed.

[0033] The molecular docking results of some candidate inhibitors and TatA and TatC proteins are shown in Table 1.

[0034] The minimum inhibitory concentration (μg / mL) of the candidate inhibitor of the Tat system on F. necrophorum is shown in Table 2.

[0035] Table 1 .

[0036] Table 2 .

[0037] It is found that epigallocatechin gallate can target the tatA and tatC genes of F. necrophorum, effectively inhibit the virulence of F. necrophorum, and specifically inhibit the formation of biofilm of F. necrophorum, the adhesion and invasion ability of F. necrophorum to cells, and the mRNA expression level of tatA and tatC genes, thereby expanding the medical application of epigallocatechin gallate.

[0038] In the following examples, F. necrophorum is used as the research object to investigate the inhibitory effect of epigallocatechin gallate on the formation of biofilm, cell adhesion, cell invasion ability and mRNA expression level of tatA and tatC genes of F. necrophorum. Epigallocatechin gallate can be directly commercially purchased.

[0039] Example 1 Constructing tatA and tatC gene deletion strains and complementation strains of F. necrophorum.

[0040] 1. Construction of recombinant plasmid for tatA and tatC gene knockout.

[0041] PCR amplification of the flanking sequences of tatA and tatC genes with BMH06-3 genome as template; purification of the upstream and downstream homologous arm fragments of tatA and tatC genes. Double enzyme digestion of the PCR products of tatA and tatC genes and pBAV1K-T5-GFP plasmid, respectively, with the reaction condition of 37℃ for 1.5h. After the enzyme digestion, the products were subjected to 1% agarose gel electrophoresis with the condition of 80V for 45min. The purified gene fragments were ligated to pBAV1K-T5-GFP with the reaction condition of 16℃ for 16h. The recombinant plasmid was transformed and identified by PCR, enzyme digestion and sequencing.

[0042] The identification results are shown in Figure 1 and 2 , and the results show that the construction is successful.

[0043] 2. tatA , tatC Screening of the gene deletion strain.

[0044] Preparation of the F. necrophorum competent cells, and the above constructed gene knockout plasmid was electroporated into the F. necrophorum competent cells and plated on blood agar plates containing 25μg / mL kanamycin. Single colonies on the blood agar plates were picked and inoculated in BHI medium for amplification. The upstream and downstream homologous arm sequences were amplified by PCR with the bacterial liquid as template, and the single crossover strain was identified. The single crossover strain was subcultured in BHI containing 25μg / mL kanamycin for 5 generations, and then subcultured in BHI without kanamycin to induce double crossover of the recombinant plasmid and the genome. The BMH06-3Δ tatA and BMH06-3Δ tatC were screened by replica plating method.

[0045] The identification results are shown in Figure 3 and 4 , and the results show that the construction is successful.

[0046] 3. Construction of F. necrophorum tatA , tatC gene complementation strain The method is the same as that for constructing the gene knockout strain. The gene knockout recombinant plasmid was transformed into the competent cells of BMH06-3Δ tatA and BMH06-3Δ tatC , and the F. necrophorum tatA , tatC gene complementation strain was screened by replica plating method.

[0047] The identification results are shown in Figure 5 and 6 , and the results show that the construction is successful.

[0048] Example 2 Using the gene-deleted strain and wild-type strain constructed in Example 1 as subjects, the effect of epigallocatechin gallate on the biofilm formation of the gene-deleted strain and wild-type strain of Cryptobacterium pyogenes Tat system was investigated.

[0049] Take culture to OD 600nm =0.6 BMH06-3, BMH06-3Δ tatA BMH06-3CΔ tatA BMH06-3Δ tatC and BMH06-3Δ tatC Bacterial suspension (diluted 1000-fold) and candidate inhibitor EGCG (final concentration 1 / 4 MIC, i.e., 19.5 μg / mL) were added to 24-well plates, 1 mL per well, and incubated at 37ºC for 72 h, repeated 3 times. After discarding the supernatant, the plates were gently washed 3 times with 0.1M PBS (pH=7.4), and 200 μL of 0.1% crystal violet staining solution was added to each well, and staining was performed for 30 min. The plates were then rinsed 3 times with PBS (pH=7.4), reconstituted with 1000 μL of 95% ethanol, and the OD was measured. 570nm .

[0050] The MIC concentration was 78 μg / mL, at which bacterial growth was completely inhibited.

[0051] Among them, EGCG refers to epigallocatechin gallate.

[0052] The results are as follows Figure 11 As shown, the results indicate that epigallocatechin gallate has an inhibitory effect on biofilm formation in both Tat system gene-deleted and wild-type strains of Cryptococcus pyogenes.

[0053] Example 3 Using the gene-deleted strain and wild-type strain constructed in Example 1 as subjects, the effect of epigallocatechin gallate on the membrane structure of the Tat system gene-deleted strain and wild-type strain of Cryptorchidica pyogenes was investigated.

[0054] Take culture to OD 600nm =0.6 BMH06-3, BMH06-3Δ tatA BMH06-3CΔ tatA BMH06-3Δ tatC and BMH06-3Δ tatCBacterial suspension (diluted 1000-fold) and candidate inhibitor EGCG (final concentration 1 / 4 MIC) were added to laser confocal microscopy bacterial culture dishes, 1 mL of bacterial suspension per dish, and cultured for 72 h. The dishes were rinsed three times with PBS (pH=7.4), and 200 μL of LIVE / DEAD Bac Light dye was added. The dishes were incubated in the dark for 25 min. After rinsing three times with PBS (pH=7.4), the biofilm structure of *Cryptobacillus pyogenes* was observed under CLSM.

[0055] The results are as follows Figure 12 As shown, the results indicate that epigallocatechin gallate has an inhibitory effect on the biofilm structure of both Tat system gene-deleted and wild-type strains of Cryptorchidism pyogenes.

[0056] Example 4 Using the gene-deleted strain and wild-type strain constructed in Example 1 as subjects, the effect of epigallocatechin gallate on the transcription of membrane formation-related genes in the Tat system of Cryptorchidica pyogenes was investigated.

[0057] Select BMH06-3 and BMH06-3Δ tatA BMH06-3CΔ tatA BMH06-3Δ tatC and BMH06-3Δ tatC Single colonies were added to 5 mL of BHI (8% fetal bovine serum) broth and cultured with shaking until OD reached. 600nm =0.6. Afterwards, the cells were inoculated into 50 mL of NB medium (containing EGCG at a final concentration of 1 / 4 MIC) and cultured for 36 h. RNA was extracted from 15 strains treated with EGCG using liquid nitrogen grinding. gapA The gene was used as an internal reference gene, and the gene related to biofilm formation of Cryptococcus pyogenes was detected by qR-PCR. rbsB , rbsC , luxS The transcriptional level.

[0058] The results are as follows Figure 13 As shown, the results indicate that epigallocatechin gallate inhibits the transcription of biofilm formation-related genes in both Tat system deletion strains and wild-type strains of Cryptococcus pyogenes.

[0059] Example 5 Using the gene-deleted strain and wild-type strain constructed in Example 1 as subjects, the effect of epigallocatechin gallate on the adhesion ability of the gene-deleted strain and wild-type strain of Cryptorchidica pyogenes Tat system was investigated.

[0060] Cultivate to OD 600nm=0.6% Cryptococcus pyogenes was centrifuged at 3000 rpm for 5 min at 4℃, washed 3 times, and then resuspended in DMEM incomplete medium (containing 1 / 4 MIC EGCG). MAC-T cells were infected with this medium at an MOI of 10 and co-cultured at 37℃ and 5% CO2 for 2 h. After washing 3 times with PBS (pH=7.4), 200 μL of trypsin was added to each well, and digestion was performed at 37℃ for 9 min. Digestion was terminated by adding an equal volume of DMEM incomplete medium, and the medium was discarded. The cells were washed 5 times with PBS (pH=7.4). 200 μL of PBS (pH=7.4, containing 1% Triton X-100) was added along the cell wall to fully lyse the MAC-T cells. The reaction was carried out at room temperature for 10 min, and lysis was terminated by adding 800 μL of sterile PBS (pH=7.4) to each well. 100 μL of the mixed suspension was aspirated from each well and diluted 10-fold with sterile PBS (pH=7.4) to a final concentration of 10. 4 10 5 10 6 Cell suspensions were spread on MHA blood plates and incubated overnight at 37°C. After colony counting, bacterial adhesion ability was assessed. The percentage of colony count in the experimental group relative to the colony count in the positive control group (BMH06-3) was the bacterial adhesion rate.

[0061] The results are as follows Figure 14 As shown, the results indicate that epigallocatechin gallate has an inhibitory effect on the adhesion ability of both Tat system gene-deleted and wild-type strains of Cryptococcus pyogenes.

[0062] Example 6 Using the gene-deleted strain and wild-type strain constructed in Example 1 as subjects, the effect of epigallocatechin gallate on the invasive ability of the gene-deleted strain and wild-type strain of Cryptorchidica pyogenes Tat system was investigated.

[0063] Cultivate to OD 600nm=0.6% Cryptococcus pyogenes was centrifuged at 3000 rpm for 5 min at 4 °C, washed 3 times, and then resuspended in DMEM incomplete medium (containing 1 / 4 MIC EGCG). MAC-T cells were infected at an MOI of 10 and incubated at 37 °C in a 5% CO2 cell culture incubator for 2 h. After co-culturing, each well was washed 5 times with sterile PBS (pH=7.4). Residual liquid was aspirated, and 1 mL of gentamicin (final concentration 300 μg / mL) was added to each well for further co-culturing for 1 h to kill extracellular bacteria. Then, 200 μL of trypsin was added, and digestion was carried out at 37 °C for 9 min, followed by the addition of an equal volume of DMEM incomplete medium to terminate the digestion. After complete digestion of the MAC-T cells, 200 μL of PBS (pH=7.4, containing 1% Tritocin) was added to each well for adhesion. Apply nX-100 to each test well and mix thoroughly by pipetting to ensure complete cell lysis. After reacting at room temperature for 10 min, add 800 μL of sterile PBS (pH=7.4) to each well to terminate the lysis reaction. After mixing thoroughly by pipetting, take 100 μL of suspension from each test well and serially dilute the sample 10-fold using sterile PBS (pH=7.4). Perform three dilutions and continue culturing in a 37℃ (containing 5% CO2) incubator for 48 h. Count the number of colonies and use the BMH06-3 group as the positive control group to calculate the invasion rate of each group. The percentage of colonies in the test group relative to the number of colonies in the positive control group (BMH06-3) is the bacterial invasion rate.

[0064] The results are as follows Figure 15 As shown, the results indicate that epigallocatechin gallate has an inhibitory effect on the invasive ability of both Tat system gene-deleted and wild-type strains of Cryptococcus pyogenes.

[0065] Example 7 Using the gene-deleted strain and wild-type strain constructed in Example 1 as subjects, the effects of epigallocatechin gallate on the Tat system gene-deleted strain and wild-type strain of Cryptococcus pyogenes were investigated. tatA , tatC The effect of gene mRNA expression levels.

[0066] *Cryptococcus pyogenes* was treated with EGCG at a final concentration of 1 / 4 MIC for 36 h. Total RNA was extracted from *C. pyogenes* and reverse transcribed. Using *C. pyogenes* cDNA as a template and the gapA gene as an internal reference, qR-PCR was used to analyze the RNA of 18 *C. pyogenes* isolates treated with 1 / 4 MIC EGCG. tatA , tatC The relative expression levels of mRNA were determined.

[0067] The results are as follows Figure 16 and 17As shown, the results indicate that epigallocatechin gallate has an effect on both Tat system gene deletion strains and wild-type strains of Cryptococcus pyogenes. tatA , tatC Gene mRNA expression has an inhibitory effect.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of epigallocatechin gallate as a virulence inhibitor of Cryptococcus pyogenes.

2. The application according to claim 1, characterized in that, The epigallocatechin gallate inhibits the formation of the biofilm of Cryptococcus pyogenes.

3. The application according to claim 1, characterized in that, The epigallocatechin gallate inhibits the ability of *Cryptobacter pyogenes* to adhere to and invade cells.

4. The application according to claim 1, characterized in that, The epigallocatechin gallate inhibits the Cryptococcus pyogenes. tatA and tatC mRNA expression level of genes.

5. The application according to any one of claims 1 to 4, characterized in that, The effective concentration of the epigallocatechin gallate is 19.5 μg / mL-78 μg / mL.

6. A pharmaceutical composition for inhibiting the virulence of Cryptococcus pyogenes, characterized in that, The active ingredient of the pharmaceutical composition includes epigallocatechin gallate.

7. The pharmaceutical composition for inhibiting the virulence of Cryptococcus pyogenes according to claim 6, characterized in that, The sole active ingredient in the pharmaceutical composition is epigallocatechin gallate.

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