CN113633651B

CN113633651B - Application of genistin in preparation of Listeria monocytogenes sortase inhibitor

Info

Publication number: CN113633651B
Application number: CN202111048012.8A
Authority: CN
Inventors: 王建锋; 刘敏达; 张思琪; 周永林; 沈雪; 王琳; 冯海华; 邓旭明
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2023-03-21
Anticipated expiration: 2041-09-08
Also published as: CN113633651A

Abstract

The invention relates to application of genistin in preparing a Listeria monocytogenes sortase inhibitor, which is verified that the genistin can effectively inhibit SrtA catalytic activity through an in vitro test SrtA peptidase activity inhibition test, a biofilm formation inhibition test and a bacterial internalization analysis test, so that the toxicity of Listeria monocytogenes is inhibited; in vivo experiments are carried out to establish a mouse Listeria monocytogenes systemic infection model, which proves that the genistin has good treatment effect on the Listeria monocytogenes infection.

Description

Application of genistin in preparation of Listeria monocytogenes sortase inhibitor

Technical Field

The invention relates to an application of genistin in preparing a medicine for inhibiting virulence factor sortase A and resisting Listeria monocytogenes infection, belonging to the technical field of medical pharmacy.

Background

Listeria monocytogenes (Listeria monocytogenes) is a gram-positive, facultative, food-borne pathogen, the causative agent of Listeria disease. The listeriosis is mainly characterized by gastroenteritis, meningitis, abortion and the like of human and animals, and the listeriosis monocytogenes has strong resistance to extreme environments (such as low pH value, high-concentration salt or low temperature) so that the listeriosis is more easily infected with food source, thereby causing great economic loss to the livestock breeding industry. Sortase a (SrtA) is a cysteine transpeptidase widely present in listeria monocytogenes and other gram-positive pathogenic bacteria, and is one of the important virulence factors of listeria monocytogenes. The catalytic activity of SrtA is involved in the process of anchoring many surface proteins to the cell wall, which are strongly associated with virulence such as bacterial adhesion and biofilm formation. Therefore, the key role of SrtA in the pathogenicity of listeria monocytogenes makes it an important potential target for the development of anti-virulent drugs against listeria monocytogenes infection.

The traditional antibiotics can achieve the purpose of sterilization or bacteriostasis by interfering with key links in the growth process of bacteria, and various drug-resistant bacteria continuously appear under the pressure of the treatment strategy. In order to avoid drug resistance of bacteria or recover sensitivity of drug-resistant bacteria to antibiotics, research on an anti-virulence strategy is particularly important, and anti-virulence drugs promote the elimination of pathogenic bacteria by a host natural immune system mainly by inhibiting the expression or function of virulence factors of the pathogenic bacteria. Genistin, an important isoflavone compound, exists in various edible plants such as soybean, kudzu root, etc. Genistin has become more important as a food additive and dietary supplement, and has various biological properties such as antioxidant activity, protecting cells from oxidative damage caused by free radicals generated by oxidation in normal metabolism, and reducing osteoporosis, anticancer, antioxidant, cardioprotective, anti-apoptotic, neuroprotective, hepatoprotective, and antimicrobial activities. At present, the report that the genistin resists the listeria monocytogenes infection by inhibiting the catalytic activity of a listeria monocytogenes sortase is not seen at home and abroad.

Disclosure of Invention

The genistin CAS of the invention has the accession number of 529-59-9 and the molecular formula of C ₂₁ H ₂₀ O ₁₀ Molecular weight is 432.38.

The chemical structural formula of the genistin is as follows:

the research takes Listeria monocytogenes SrtA as a target spot, a SrtA inhibitor is obtained by screening from a natural compound and is used as genistin, and the inhibitor can resist the infection of Listeria monocytogenes by reducing the catalytic activity of SrtA. The research further researches the inhibition effect of the genistin on the virulence of Listeria monocytogenes, and shows that the genistin can effectively inhibit the catalytic activity of SrtA and has a certain inhibition effect on the virulence of Listeria monocytogenes through an SrtA peptidase activity inhibition test, a biofilm formation inhibition test and a bacterial internalization analysis test; in vivo animal infection tests show that genistin has good treatment effect on listeria monocytogenes infection.

Drawings

FIG. 1: genistin inhibits the activity of Listeria monocytogenes sortase enzyme;

FIG. 2: genistin inhibits the formation of Listeria monocytogenes biofilm;

FIG. 3: genistin inhibits the internalization of listeria monocytogenes;

FIG. 4 is a schematic view of: therapeutic effect of genistin on listeria monocytogenes infected mouse model.

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the invention in any way, and any modifications or changes that can be easily made by a person skilled in the art without departing from the technical solution of the invention will fall within the scope of the claims of the invention.

1. Influence of genistin on Listeria monocytogenes sortase catalytic activity

The inhibition effect of genistin on the catalytic activity of listeria monocytogenes SrtA is detected by Fluorescence Resonance Energy Transfer (FRET). The purified SrtA protein and different concentrations of genistin (0, 2, 4 and 8 mu g/mL) are incubated in a black 96-well plate at 37 ℃ for 30 minutes, a fluorescent peptide substrate (Dabcyl-QALPNTGEE-Edans) is added into a reaction system and incubated for 1h in a dark place, the fluorescence intensity of each sample is measured by a microplate reader at an excitation wavelength of 350nm and an emission wavelength of 520nm respectively, and the result is shown in figure 1.

And (4) conclusion: the study detects the inhibitory effect of genistin on the activity of SrtA enzyme by a fluorescence resonance energy transfer method. The result shows that the genistin can inhibit the enzymatic activity of SrtA in a dose-dependent manner, wherein the inhibition rate of 8 mu g/mL of the genistin can reach more than 50%.

2. Effect of genistin on Listeria monocytogenes biofilm formation

In the research, a crystal violet dyeing method is used for dyeing the biofilm formed by the listeria monocytogenes, and the influence of different concentrations of genistin on the formation of the listeria monocytogenes biofilm is analyzed according to the crystal violet dyeing degree. Overnight culturing wild Listeria monocytogenes strain BUG1600 and SrtA knockout strain BUG1777, and culturing the bacterial suspension (1 × 10) in middle logarithmic growth phase ⁸ CFU/mL) was expanded with 1mL of fresh sterile TSB medium at 1. After incubation, the medium was aspirated and the plates were gently washed 3 times with 1mL sterile PBS. The well plates were then air dried and stained with 400 μ L of 0.1% crystal violet for 1 hour. Excess stain was aspirated and the plate was washed 3 times with sterile distilled water. After drying the 24-well plate, 500. Mu.L of 33% glacial acetic acid was added per well. OD measurement Using microplate reader _570nm Absorbance at wavelength, results are averaged over three replicates. The results are shown in FIG. 2.

And (4) conclusion: the absorbance results of the crystal violet staining indicate that the genistin concentration-dependently inhibits the formation of listeria monocytogenes biofilm.

3. Effect of genistin on internalization of Caco-2 cells by Listeria monocytogenes

The research detects the influence of different concentrations of genistin on the internalization of the listeria monocytogenes through a listeria monocytogenes internalization Caco-2 cell test. Caco-2 cells were cultured in DMEM medium containing 10% bovine serum without antibiotics at 2X 10 per well ⁵ Cell density was seeded in 24-well plates at 37 ℃ and 5% CO ₂ Cultured overnight under the conditions. Caco-2 cells were infected with overnight cultured Listeria monocytogenes (BUG 1600 and BUG 1777) at an MOI of 50 and co-cultured with various concentrations of genistin (0,4, 16 and 64 μ g/mL). After 3h incubation, the medium was replaced with fresh medium containing 50. Mu.g/mL gentamicin for 30min and washed 3 times with sterile PBS. Adding sterile water to lyse the cells, diluting and counting the number of inoculations. The results are shown in FIG. 3. And (4) conclusion: the influence of genistin on the internalization of the listeria monocytogenes is preliminarily evaluated through a listeria monocytogenes internalization analysis test, and the result shows that the internalization Caco-2 cell degree of the listeria monocytogenes is in a descending trend along with the increase of the genistin concentration within 3h without the action of the genistin concentration.

4. Therapeutic effect of genistin on Listeria monocytogenes mouse infection model

Listeria monocytogenes (BUG 1600) was grown overnight, expanded in fresh TSB (1 _600nm = 0.8). After washing twice with sterile PBS, the bacteria were resuspended in sterile PBS and the bacterial suspension concentration was adjusted to 4X 10 ⁷ CFUs/mL. 20 BALB/c mice (female, about 20 g) were divided into an infected group and a treated group, 10 mice in each group were intraperitoneally injected with 100. Mu.L of bacterial suspension, the treated group was infected with 2 hours and then subcutaneously injected with genistin in 25mg/kg DMSO, and the administration was performed once every 8 hours, and the infected group was simultaneously subcutaneously injected with an equal volume of DMSO without drug. Mice livers were harvested 48h after infection, weighed, lysed in PBS with 2% Triton X-100, diluted, plated on TSB agar plates, incubated at 37 ℃ and counted. The results are shown in FIG. 4.

And (4) conclusion: intraperitoneal injection of 4X 10 in mice ⁷ CFUs/mL Listeria monocytogenes establishes a systemic infection model, and the result shows that the bacterial colonization amount of the liver of a mice in a genistin treatment group is obviously lower than that of the liver of the mice in an infected group, which indicates that the genistin has a good treatment effect on Listeria monocytogenes infection.

Claims

1. The application of the genistin in preparing the medicine for inhibiting the formation of the Listeria monocytogenes biofilm is characterized in that the genistin has an inhibiting effect on the activity of sortase A.