Application of 3-methylindole in preparing medicine for inhibiting salmonella typhimurium

Abstract

The invention discloses an application of 3-methylindole in preparation of drugs for inhibiting salmonella typhimurium, which inhibits the expression of QS related genes and virulence genes and inhibits the formation of biofilms through the combination of 3-methylindole and LsrB protein, thereby reducing the pathogenicity of salmonella typhimurium. And the 3-methylindole can inhibit invasion and adhesion of salmonella typhimurium to cells, so that the 3-methylindole can be applied to preparation of the salmonella typhimurium inhibiting medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of 3-methylindole in preparing a drug for inhibiting Salmonella typhimurium. Background Art

[0002] Salmonella enterica serovar Typhimurium (S.typhimurium) belongs to the non-typhoidal Salmonella class. The infections caused by it account for a large proportion of non-typhoidal Salmonella infections. It is a major zoonotic pathogen, and its infection incidence ranks first among Salmonella infections. S.typhimurium is infected through the oral cavity. After entering the human body, it can invade, adhere to and bypass the digestive tract (gastric acid, etc.). There are many virulence markers and determinants that play a vital role in its infection mechanism. These factors include flagella, capsule, plasmids, adhesion system and type III secretion system encoded on SPI-1 and SPI-2 in Salmonella pathogenicity islands (SPIs). Antibiotic therapy has always been the common treatment for S.typhimurium, but the abuse of antibiotics has led to the emergence of drug resistance in S.typhimurium, posing a great threat to food safety and public health. In order to address the occurrence of drug resistance, new treatments have been developed, such as phage therapy, probiotic therapy, and quorum sensing system inhibitors.

[0003] Quorum sensing (QS) is a cellular communication mechanism between cells. The QS mechanism is involved in multiple cellular processes, especially bacterial virulence, such as biofilm formation, motility, adhesion, and invasion. In addition, QS also plays a key role in regulating pathogen resistance to antibiotics. It can regulate the formation of biofilms, efflux pumps, membrane proteins, and two-component systems, thereby mediating bacterial resistance to antibiotics.

[0004] 3-Methylindole, also known as skatole, is an aromatic heterocyclic compound. It has a strong musky smell at high concentrations, and a floral odor at low concentrations. It is used as a fragrance and fixative in many perfumes and can also be used as an aromatic compound. However, its inhibition of Salmonella typhimurium has not been reported. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide application of 3-methylindole in preparing a drug for inhibiting Salmonella typhimurium.

[0006] The technical solution of the present invention is summarized as follows:

[0007] Application of 3-methylindole in the preparation of drugs for inhibiting Salmonella typhimurium.

[0008] Advantages of the present invention:

[0009] The present invention inhibits the expression of QS-related genes and virulence genes and inhibits the formation of biofilm by combining 3-methylindole with LsrB protein (PDB ID: 1TJY), thereby reducing the pathogenicity of Salmonella typhimurium. Moreover, 3-methylindole can inhibit the invasion and adhesion of Salmonella typhimurium to cells, so 3-methylindole can be used as a drug to inhibit Salmonella typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 These are the results of the surface plasmon resonance experiment (SPR) of 3-methylindole and LsrB protein.

[0011] Figure 2 The figure shows the effect of different concentrations of 3-methylindole on the growth curve of LT2 strain.

[0012] Figure 3 The effect of 3-methylindole on the biofilm formation of LT2 strain obtained by crystal violet staining. Figure A shows the staining results of 96-well plate, ****: p<0.0001; Figure B shows the results of optical microscope observation after adding 3-methylindole.

[0013] Figure 4 Effects of 0.5 mM 3-methylindole on QS-related genes and virulence genes of LT2.

[0014] Figure 5 The effect of different concentrations of 3-methylindole on cell viability, ns: p>0.05, no significant difference.

[0015] Figure 6 The effect of 3-methylindole on the invasion and adhesion of LT2 cells, where A is the effect of 3-methylindole on cell adhesion, and B is the effect of 3-methylindole on cell invasion. , *: p<0.05; ***: p<0.001; ****: p<0.0001. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0017] The 3-methylindole (CAS No: 83-34-1) involved in the following examples was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., product number: BD11685, dissolved in dimethyl sulfoxide (DMSO) and configured into a 400mM stock solution.

[0018] The strain used was the LT2 strain of Salmonella typhimurium, purchased from Beinachuanglian Biotechnology Co., Ltd. in China Mall (https: / / www.bncc.com / pro / p2 / 8 / p_316132.html), referred to as LT2.

[0019] The culture conditions were as follows: culture in LB medium at 37°C and 220 rpm; the cells used were human normal intestinal epithelial cells NCM460 (commercially available), cultured in high-glucose DMEM medium (commercially available) containing 1% double antibody (100 U / mL penicillin, 0.1 mg / mL streptomycin) and 10% fetal bovine serum, i.e., DMEM complete medium, at 37°C and 5% CO 2 The strains and cell types used are only examples and should not be used to limit the scope of application of this application.

[0020] Example 1: Surface plasmon resonance (SPR) experiment results of 3-methylindole and LsrB protein.

[0021] Experimental method: The purified LsrB protein (PDB ID: 1TJY) was fixed on a CM5 chip (commercial product) by amino coupling, and an SPR experiment was performed after the coupling was completed. The LsrB protein was used as the stationary phase, and different concentrations of 3-methylindole (solvent: DMSO) were used as the mobile phase to flow through the chip at a speed of 30 μL / min. When 3-methylindole binds to the LsrB protein, it will generate pressure on the chip, thereby obtaining a response value. In the saturated state before stopping the injection, the KD value (equilibrium dissociation constant) between the two is calculated. This value is often used to represent its binding affinity. The smaller the KD value, the greater the affinity.

[0022] Experimental results: Figure 1 As shown in the figure, the SPR results of 3-methylindole and LsrB protein were obtained, and the KD value was 1.487×10 -8 , that is, it has a strong binding force with LsrB protein in vitro, proving that 3-methylindole can bind to LsrB protein.

[0023] Example 2: Effect of 3-methylindole on the growth curve of LT2.

[0024] Experimental methods:

[0025] (1) Minimum inhibitory concentration (MIC) of 3-methylindole against LT2

[0026] The MIC of 3-methylindole to LT2 was determined by the doubling dilution method. The highest concentration of 3-methylindole solution was 40mM, which was diluted to 0.315mM with DMSO and co-cultured with LT2 at 37°C for 24h. The lowest concentration without bacterial growth was the MIC of 3-methylindole. Three parallels were set for each 3-methylindole.

[0027] (2) The overnight LT2 bacterial suspension was inoculated into LB medium at a 1% inoculation ratio. The concentrations of 3-methylindole in each sample were MIC, 4 / 5MIC, 3 / 5MIC, 2 / 5MIC, and 1 / 5MIC, respectively. No 3-methylindole was added to the positive control. Three replicates were set for each concentration. The culture was carried out at 37°C and 220 rpm in a shaker for 24 hours. The OD was recorded every 2 hours using a spectrophotometer. 600nm value.

[0028] Experimental results: Figure 2 The figure shows the effect of different concentrations of 3-methylindole on the growth curve of LT2 strain. The experimental results show that the MIC of 3-methylindole to LT2 is 2.5mM. The growth curve shows that except for 2.5mM, other concentrations of 3-methylindole solution have no effect on the growth curve of LT2. Therefore, it is concluded that when 3-methylindole is at a concentration of 0.5mM, it will inhibit its pathogenicity but will not make Salmonella typhimurium resistant.

[0029] Example 3: Effect of 3-methylindole on the biofilm of LT2 strain.

[0030] Experimental methods:

[0031] Crystal violet staining was used to determine the effect of 3-methylindole on LT2 biofilm. In this study, biofilm was studied using two methods: 96-well plate observation and optical microscope observation.

[0032] 96-well plate observation method: Pick a single LT2 colony and culture it overnight in a shaker at 37°C and 220rpm. Dilute the overnight cultured bacterial solution 100 times with LB medium, add different volumes of 3-methylindole solution to a final concentration of 2mM, 1.5mM, 1mM, and 0.5mM, mix well, and add to a 96-well plate. Incubate the 96-well plate in a 37°C incubator for 24 hours, then discard the supernatant, wash three times with PBS, and dry in an oven. Add 2% crystal violet solution to the sample wells, stain for 30 minutes in a dark place, discard the supernatant, wash three times with PBS, and dry. Finally, add an equal volume of 95% ethanol, mix well by pipetting, and use an enzyme reader to measure the OD 595nm The absorbance at was detected.

[0033] Optical microscope observation method: Dilute the overnight activated LT2 100 times with LB medium, place the sterilized cell slide in a 12-well plate, add bacterial solution and 3-methylindole solution to a final concentration of 2mM, and incubate at 37°C for 24 hours. After the culture is completed, take out the cell slide and clean the remaining impurities on the cell slide with PBS. Add a 0.1% crystal violet solution to the cell slide and stain it for 30 minutes in the dark. Take out the stained cell slide, rinse it with PBS and dry it. Use an optical microscope (10x100 times) to observe the distribution of cell membranes on the cell slide.

[0034] Experimental results: Figure 3 The results of the 96-well plate stained with crystal violet showed that 2, 1.5, 1, and 0.5 mM 3-methylindole inhibited the biofilm formation of LT2. Since it significantly inhibited the formation of biofilm at 0.5 mM, with an inhibitory effect of 20.75%, 0.5 mM 3-methylindole was observed under a microscope. Figure 3 B. Results show that the addition of 0.5 mM 3-methylindole reduced the aggregation of LT2 biofilm, effectively inhibited the formation of biofilm, and reduced the pathogenicity of LT2.

[0035] Example 4: Effects of 3-methylindole on QS-related genes and virulence genes of LT2.

[0036] Experimental method: The LT2 strain activated overnight was inoculated into LB medium at a ratio of 1%, and 3-methylindole was added at a final concentration of 0.5mM and cultured to the logarithmic phase. RNA was extracted according to Tiangen's bacterial total RNA extraction kit, and then reverse transcribed using SparkJade's reverse transcription kit, SPARKscript II RT Plus Kit, according to its instructions, and the concentration was measured using Nanodrop. qRT-PCR was performed on LightCycler 480Ⅱ, with a final reaction volume of 10μL, and the system and reaction procedures were performed according to SparkJade's 2×SYBR Green qPCR Mix instructions.

[0037] Experimental results: Figure 4The results showed that 0.5 mM 3-methylindole could inhibit the QS-related genes and virulence genes of LT2. The QS-related genes explored by qRT-PCR were: lsr operon-related genes lsrB (Gene ID: 94541) and lsrC (Gene ID: 946105) and AI-2 synthetic protein luxS (Gene ID: 1254340); related virulence genes were: flagellar synthesis-related genes flhC (Gene ID: 1253445) and fliA (Gene ID: 1253477), fimD (Gene ID: 1252066), sptP (Gene ID: 1254401), sopB (Gene ID: 1252609), sopE (Gene ID: 1253374) and invH (Gene ID: 1254423), which were related to bacterial adhesion and invasion. The results showed that 3-methylindole inhibited LT2 QS-related genes, such as lsrB and lsrC, by 3.6 and 4.1 times, respectively. qRT-PCR results also showed that 3-methylindole inhibited the flagellar synthesis gene flhC and the bacterial motility gene fimD by 3.3 and 3.6 times, respectively. In addition, 3-methylindole also inhibited the gene sopB related to LT2 invasion and adhesion, with an inhibitory effect of 9.1 times.

[0038] Example 5: Effect of 3-methylindole on cell activity.

[0039] Experimental method: NCM460 cells were cultured with DMEM complete medium at 2×10 4 The cells were seeded at a density of 100 / well in a 96-well plate and placed in a 37°C, 5% CO 2 Incubate in a humidified environment until cells adhere. Treat cells with different concentrations of 3-methylindole, and then analyze cell viability according to the instructions of Cell Counting Kit-8 (CCK-8) purchased from TargetMOI. After 24 hours of treatment, add 10 μL of CCK-8 to each well and incubate for 2 hours. All experiments were repeated three times. The wells without cells were used as blank groups, and the OD 450nm The absorbance at was detected.

[0040] Experimental results: The effects of different concentrations of 3-methylindole on cell viability are shown in Figure 5 As shown, the experiment found that the 0.5 mM concentration of 3-methylindole used in the previous experiment had no effect on cell viability, so when conducting invasion and adhesion experiments, the 3-methylindole concentration of 0.5 mM was still selected.

[0041] Example 6: Effects of 3-methylindole on LT2 adhesion and invasion.

[0042] Experimental method: NCM460 cells were seeded in 12-well plates with DMEM complete medium at a density of 5×10 5 200 μL of activated LT2 was added to 20 ml of LB medium, and the cells were cultured at 37°C for 3 h. Then, NCM460 cells were infected at a multiplicity of infection of 20:1. A control group without 3-methylindole and an experimental group containing 3-methylindole at a final concentration of 0.5 mM and 0.25 mM were set up at 37°C and 5% CO. 2 The cells were cultured for 2 h under the same conditions. After the culture was completed, the supernatant was discarded, and the cells were washed with PBS three times, and then lysed with 0.1% Triton X-100 at room temperature for 20 min. The suspension was diluted step by step, and 100 μL of each dilution was inoculated into LB solid medium. After culturing at 37°C for 16 h, the bacteria on the plate were counted to obtain the total number of bacteria infecting the cells. The number of adherent bacteria = the total number of bacteria infecting the cells - the number of bacteria invading the cells.

[0043] In the invasion experiment, the infection step is the same as the adhesion step. After the infection, the cells were washed with PBS, and then a DMEM solution containing 100 μg / ml gentamicin was added and incubated for 50 minutes to kill extracellular bacteria. Then, the cells were washed three times with PBS, and then washed and lysed with 0.1% Triton X-100. The suspension was diluted step by step, and 100 μL of each dilution was inoculated into LB solid medium. After culturing at 37°C for 16 hours, the plate bacteria were counted to obtain the total number of bacteria invading the cells.

[0044] Experimental results: The results of 3-methylindole on LT2 adhesion cells are as follows Figure 6 As shown in A, 0.25mM 3-methylindole has an inhibitory effect of 30.79%, while 0.5mM 3-methylindole has an inhibitory effect of 48.28% on cell adhesion. Figure 6 As shown in Figure B, 0.25mM 3-methylindole has an inhibitory effect of 27.29% on cell invasion, and 0.5mM 3-methylindole has an inhibitory effect of 63.04%. In summary, 3-methylindole will not be toxic to cells at a concentration of 0.5mM, but will inhibit the invasion and adhesion of Salmonella typhimurium to cells, thereby inhibiting its pathogenicity, which proves that 3-methylindole can be used as a drug to inhibit Salmonella typhimurium.

[0045] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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.

[0046] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

Application of 1.3-methylindole in the preparation of drugs for inhibiting Salmonella typhimurium.

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