3-phenyl-1-propanamine for use in the inhibition of the growth and pathogenicity of bacillus cereus by modulating its quorum sensing system

Abstract

The application discloses application of 3-phenyl-1-propanamine in inhibiting growth and pathogenicity of Bacillus cereus by regulating a quorum sensing system of the Bacillus cereus. Compared with traditional chemical antibacterial agents such as antibiotics, the 3-phenyl-1-propanamine is safer for the environment and human bodies, and reduces the risk of drug residue and drug-resistant strain generation. Research results show that the green natural product can play a role at a low concentration (1 / 8 MIC), so that the use cost is low. The application can target and regulate the expression of multiple key genes (such as Spo0A, CodY, rpoN and the like), so as to not only inhibit pathogenicity, but also effectively control the proliferation of the Bacillus cereus, reduce the risk of food contamination and foodborne disease infection, and has important application prospects in the field of food safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of 3-phenyl-1-propylamine in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus. BACKGROUND

[0002] Bacillus cereus is a gram-positive bacterium of the genus Bacillus, facultative anaerobic, widely distributed in nature. Pathogenic Bacillus cereus is often found in food such as rice products, which can produce a variety of toxins and threaten food safety. It is a very important foodborne pathogen and opportunistic pathogen, ranking first in the number of food poisoning events and the total number of poisoning. Therefore, it is necessary to study the process of the harm of Bacillus cereus in food, and it is of great significance to inhibit it.

[0003] The proliferation and pathogenicity of Bacillus cereus are regulated by signal pathways such as quorum sensing system (QS) and pleiotropic transcriptional regulators. The production of many bacterial degradation enzymes, virulence factors and biofilm formation processes depend on QS, which can effectively control bacterial growth and virulence through the QS pathway. However, traditional methods lack targeting and are difficult to achieve precise regulation. Existing prevention and control measures such as chemical antibacterial agents and high-temperature sterilization have problems such as drug residues, increasing the risk of drug resistance, and affecting the taste of food, so there is an urgent need for a safer and more effective green antibacterial means.

[0004] Green natural products are attracting attention due to their safety and environmental friendliness, but how to precisely regulate the quorum sensing signal pathway of pathogenic bacteria still needs further research. As a natural product, 3-phenyl-1-propylamine has good biocompatibility and can target regulate the signal pathway of Bacillus cereus at low concentrations, thereby inhibiting the quorum sensing signal and virulence gene expression of Bacillus cereus, reducing pathogenicity and not easily causing bacterial drug resistance. These natural products can regulate gene expression targeting PlcR-PapR quorum sensing system, Spo0A transcriptional regulator, etc., to achieve antibacterial effect, have high safety and low cost advantage, and are more and more valued in today's antibiotic epidemic. As an easy-to-drug-resistant bacterium, the development of Bacillus cereus quorum sensing signal inhibitors has great significance, and the development of new anti-Bacillus cereus drugs and even the control of Bacillus cereus hazards are of great help. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide an application of 3-phenyl-1-propylamine in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] 3-phenyl-1-propanamine in the preparation of a Bacillus cereus bacteriostatic agent.

[0008] 3-phenyl-1-propanamine in the preparation of a Bacillus cereus bacteriostatic agent.

[0009] 3-phenyl-1-propanamine in the preparation of a Bacillus cereus bacteriostatic agent.

[0010] 3-phenyl-1-propanamine in the preparation of a Bacillus cereus bacteriostatic agent.

[0011] 3-phenyl-1-propanamine in the preparation of a Bacillus cereus bacteriostatic agent.

[0012] The inhibition of the quorum sensing system of the Bacillus cereus includes inhibiting the expression level of a quorum sensing system related gene.

[0013] The quorum sensing system related gene includes at least one of the following: plcR, papR and LuxS.

[0014] A method for inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of the Bacillus cereus by using 3-phenyl-1-propanamine, comprising the following steps:

[0015] Adding 3-phenyl-1-propanamine in the system to inhibit the growth and pathogenicity of the Bacillus cereus.

[0016] The adding amount of the 3-phenyl-1-propanamine is 0.08-5 mg / mL; preferably 0.3125 mg / mL.

[0017] The Bacillus cereus includes at least one of the following: Bacillus cereus ATCC 14579, Bacillus cereus IAM 12605, Bacillus cereus CCM 2010, Bacillus cereus Bcer A9 and Bacillus cereus Bcer L1.

[0018] The inhibition of the pathogenicity of the Bacillus cereus includes inhibiting the expression level of a virulence gene.

[0019] The virulence gene includes at least one of the following: hblB, cytK, nheB and clo.

[0020] The present application has the following advantages and effects relative to the prior art:

[0021] The application uses 3-phenyl-1-propanamine, which is safer for the environment and human body than traditional chemical antibacterial agents such as antibiotics, and reduces the risk of drug residues and drug-resistant strains. And the research results show that green natural products with low concentration (1 / 8 MIC) can play a role, which makes the use cost low, and the application can regulate the expression of multiple key genes (such as Spo0A, CodY, rpoN, etc.) through targeting, not only can inhibit pathogenicity, but also can effectively control the proliferation of Bacillus cereus, reduce the risk of food contamination and foodborne disease infection, and has important application prospect in the field of food safety. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is a growth curve diagram of Bacillus cereus in a free state under 1 / 8 MIC.

[0023] Figure 2 Figure 2 is the expression change of quorum sensing genes under 1 / 8 MIC 3-phenyl-1-propanamine.

[0024] Figure 3 Figure 3 is the expression change of virulence genes under 1 / 8 MIC 3-phenyl-1-propanamine.

[0025] Figure 4 Figure 4 is the expression change of pleiotropic transcriptional regulator genes under 1 / 8 MIC 3-phenyl-1-propanamine.

[0026] Figure 5 Figure 5 is the expression change of protease genes under 1 / 8 MIC 3-phenyl-1-propanamine.

[0027] Figure 6 Figure 6 is the expression change of other key genes under 1 / 8 MIC 3-phenyl-1-propanamine. DETAILED DESCRIPTION

[0028] The application will be further described in detail below in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0029] In the following examples, if the specific test conditions are not specified, the general test conditions or the test conditions recommended by the reagent company are usually used. If not specified, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0030] Example 1 Determination of minimum inhibitory concentration (MIC) of 3-phenyl-1-propanamine on Bacillus cereus

[0031] (1) Glycerol stocks of B. cereus ATCC 14579, B. cereus IAM 12605, B. cereus CCM 2010, and two B. cereus strains preserved in our laboratory, numbered Bcer A9 (abbreviated as A9) and Bcer L1 (abbreviated as L1) (purchased from Guangzhou Peptide Valley Co., Ltd.), were streaked onto TSA solid medium and incubated at 37°C for 24 h. Single colonies were picked and inoculated into 2 mL of TSB liquid medium, which was incubated at 37°C with shaking at 200 r / min for 12 h.

[0032] (2) 10 mg of 3-phenyl-1-propylamine was dissolved in 1 mL of DMSO, filtered to remove bacteria, and prepared into a 10 mg / mL 3-phenyl-1-propylamine solution.

[0033] (3) The overnight culture of the five B. cereus strains was diluted to an initial concentration of 10 5 CFU / mL. In sterile 96-well plates, the final concentrations of green natural product solutions in the first to seventh wells were 5, 2.5, 1.25, 0.625, 0.313, 0.106, and 0.053 mg / mL, respectively, and five parallels were set up accordingly, with a blank control. After incubation at 37°C with shaking at 200 rpm for 24 h, the OD 600nm was measured.

[0034] The results are shown in Table 1. When the concentration of 3-phenyl-1-propylamine solution was 2.5 mg / mL, the OD 600nm was consistent with that of the blank culture medium, indicating that this concentration inhibited the growth of B. cereus. Therefore, the MIC of 3-phenyl-1-propylamine for B. cereus was 2.5 mg / mL.

[0035] Table 1 OD of 24 h culture with 3-phenyl-1-propylamine 600nm

[0036]

[0037]

[0038] Example 2 Determination of the growth curve of free B. cereus

[0039] The overnight culture of the five B. cereus strains was diluted to an initial concentration of 10 5 CFU / mL in a culture tube, and 1 / 8 MIC (0.3125 mg / mL) of 3-phenyl-1-propylamine solution was added according to the results of Example 1. The culture was incubated at 37°C with shaking at 200 rpm, and 200 μL of bacterial solution was sampled every 2 h in a 96-well plate to measure the absorbance at 600 nm. The bacterial solution without the addition of the drug was set as a control.

[0040] Results as shown in Figure 1 Figure 1, 3-phenyl-1-propylamine at a concentration of 1 / 8 MIC had no effect on the growth of B. cereus, and this concentration can be used for the study of B. cereus quorum sensing signal inhibitors.

[0041] Example 3 Effect of 3-phenyl-1-propylamine at a concentration of 1 / 8 MIC on the expression of free-state B. cereus genes

[0042] 3.1 Detection genes

[0043] The present application selects multiple key genes for detection to explore the effect of 3-phenyl-1-propylamine at a concentration of 1 / 8 MIC on the expression of free-state B. cereus genes, thereby evaluating its potential to reduce virulence. First, the quorum sensing system genes plcR, papR and LuxS are selected, which regulate biofilm formation and toxin synthesis, directly affecting the pathogenicity of bacteria. In addition, the detection of virulence genes hblB, cytK, nheB and clo is crucial, as these genes encode pathogenic factors directly related to the virulence of bacteria. Protease genes nprB, colA, clpP and sfp are involved in the maturation of quorum sensing signal molecules and the degradation of extracellular matrix, which is important for understanding the regulation of bacterial biofilm formation and pathogenic ability. The multiple effector transcriptional regulator genes codY and spo0A regulate multiple biological processes, involving B. cereus motility and biofilm formation, revealing the adaptive mechanisms of bacteria to external environmental changes. Finally, the detection of other key genes tlpA, sigH and rpoN will help to fully understand the role of B. cereus in growth, metabolism and toxin production. Through systematic study of the expression of these genes, we can analyze the effect of 3-phenyl-1-propylamine on B. cereus and verify whether it has the function of reducing virulence, providing important theoretical basis and data support for the development of new antibacterial strategies.

[0044] 3.2 Primer design

[0045] B. cereus 16s rRNA is selected as the internal reference gene, and the quorum sensing system genes plcR, papR and LuxS; virulence genes hblB, cytK, nheB and clo; protease genes nprB, colA, clpP and sfp; multiple effector transcriptional regulator genes codY and spo0A; other genes tlpA, sigH and rpoN are detected genes, and RT-qPCR primers are designed using Primer Premier software. The primers are synthesized by Guangzhou Aik Biotechnology Co., Ltd., and the primer sequences are shown in Table 2.

[0046] Table 2 Primer sequences for RT-qPCR

[0047]

[0048]

[0049] 3.3 Real-time PCR system establishment

[0050] (1) Extraction of total RNA from Bacillus cereus

[0051] The overnight culture of five Bacillus cereus strains was diluted to an initial concentration of 10 5 CFU / mL in a culture tube using TSB medium, and then 1 / 8 MIC 3-phenyl-1-propanamine solution was added. At 37°C, 200 rpm, samples were taken at 4h, 8h, 12h, and 24h, and RNA was extracted using the Trizol method.

[0052] (2) Reverse transcription

[0053] gDNA was removed using the Rnase free DNase I kit (Thermol). RNA was reverse transcribed into cDNA using the reverse transcription kit (Biosharp).

[0054] (3) Real-time PCR

[0055] The cDNA was diluted 10-fold, and the primer concentration was diluted to 10 mM for standby. The real-time PCR reaction system was as follows: 10 mL of 2x Mastermix, 2 mL of cDNA template (after dilution), 0.4 mL of upstream and downstream (F / R) primers (10 mM) each, and finally 20 mL of RNase free water. The amplification program was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 15 s, 55°C annealing for 15 s, and 72°C extension for 20 s, for 39 cycles.

[0056] (4) Data statistics

[0057] The relative quantification method was used to determine the changes in the expression of the target detection gene of Bacillus cereus over time. Three replicates were set for each sample to reduce errors. The 4h culture group was used as a reference, and the expression of the target gene was normalized using the internal reference gene, and the value of 2 –ΔΔCt (Livak method) was used to represent the fold change in the expression of the target gene.

[0058] 3.4 Experimental results

[0059] Figure 1Figure 1 shows the growth curve of B. cereus under 1 / 8 MIC of malt. As shown in the figure, 1 / 8 MIC of malt inhibited the growth of B. cereus to some extent. For example, the concentration of ATCC 14579 was less than the control group after 0-14h of culture, the concentration of CMCC 2010 was less than the control group after 7-11h of culture, and the concentration of IAM 12605 was less than the control group after 6-14h of culture.

[0060] Figure 2 Figure 4 shows that 3-phenyl-1-propylamine at 1 / 8 MIC completely inhibited the expression of PlcR-PapR quorum sensing system when the expression of PlcR-PapR was slightly weak. Under the action of 3-phenyl-1-propylamine, the plcR of B. cereus was completely inhibited at 24h or before 24h, except for strain L1. The same was true for the LuxS gene. This proved that 3-phenyl-1-propylamine could target and inhibit the plcR, PlcR-PapR, LuxS signaling pathway, and the multiplication of B. cereus to some extent.

[0061] Figure 3 Figure 5 shows that under the action of 3-phenyl-1-propylamine, the NheB gene was completely inhibited after 8h of culture, and strains IAM 12605 and CCM 2010 had no expression even after 4h of culture. The NheB gene is an important gene that encodes the non-hemolytic enterotoxin of B. cereus. The decrease in its expression indicates that 3-phenyl-1-propylamine can effectively reduce the Nhe toxicity of B. cereus, and to some extent, reduce the harm caused by the multiplication of B. cereus.

[0062] Figure 4 Figure 6 shows that under the action of 3-phenyl-1-propylamine, the expression of the Spo0A gene of strains IAM 12605 and CMCC 2010 was completely inhibited after 24h of culture. 3-phenyl-1-propylamine had a significant promoting effect on the expression of the pleiotropic transcriptional regulator gene Spo0A in most cases studied, which could promote the sporulation and biofilm formation of B. cereus, and make the bacteria more inclined to protect themselves rather than invade the host under greater survival pressure.

[0063] Figure 5 Figure 7 shows that the effects of 3-phenyl-1-propylamine on the expression of clpP, nprB, and sfp genes were quite different. This indicates that 3-phenyl-1-propylamine had no significant effect on the expression of protease genes.

[0064] Figure 6 Figure 8 shows that under the action of 3-phenyl-1-propylamine, the expression of the sigH gene was mostly up-regulated. The promotion of 3-phenyl-1-propylamine on the expression of the sigH gene could make the bacteria enter a dormant state under unfavorable conditions.

[0065] In summary, the B. cereus under 1 / 8 MIC of 3-phenyl-1-propylamine can completely inhibit the expression of the PlcR-PapR quorum sensing system when the expression of the system is slightly weak. The green natural product can basically completely inhibit the expression of the nheB gene after a period of time, effectively reduce the Nhe toxicity of B. cereus, and indirectly inhibit the proliferation. The promotion of 3-phenyl-1-propylamine to the expression of the pleiotropic transcriptional regulator gene Spo0A can also slow down the growth rate of B. cereus. The promotion of 3-phenyl-1-propylamine to the expression of the sigH gene can inhibit the colonization and pathogenic potential of B. cereus.

[0066] The above experimental results show that the selected green natural product 3-phenyl-1-propylamine of the present application has a wide application prospect, and its influence on the expression of the signal pathway gene is not only suitable for B. cereus, but also can be extended to other strains and even the food field. It has important value for the growth detection and in-depth understanding of the proliferation mechanism of B. cereus, and will be expected to improve food safety management, and provide beneficial reference and reference for microbiology research and other fields of application.

[0067] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. Use of 3-phenyl-1-propanamine in the preparation of a Bacillus cereus bacteriostatic agent.

2. Use of 3-phenyl-1-propanamine in the preparation of a preparation for inhibiting the pathogenicity of Bacillus cereus.

3. The use according to claim 1 or 2, wherein the 3-phenyl-1-propanamine is used to inhibit the growth or pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus.

4. The use according to claim 1 or 2, wherein the 3-phenyl-1-propanamine is used to inhibit the growth or pathogenicity of Bacillus cereus by inhibiting the formation of a biofilm of Bacillus cereus.

5. The use according to claim 3, wherein the quorum sensing system of Bacillus cereus is inhibited by regulating the expression level of a gene related to the quorum sensing system of Bacillus cereus. plcR papR LuxS The quorum-sensing system-related gene includes at least one of 6. The use according to claim 1 or 2, wherein the Bacillus cereus comprises at least one of Bacillus cereus ATCC 14579, Bacillus cereus IAM 12605, Bacillus cereus CCM 2010, Bacillus cereus Bcer A9, and Bacillus cereus Bcer L1. , 7. The use according to claim 2, wherein the pathogenicity of Bacillus cereus is inhibited by regulating the expression level of a virulence gene of Bacillus cereus. and hblB . cytK nheB clo ​ The virulence genes include at least one of ​ , ​ , ​ and ​ .

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