Application of 3-phenyl-1-propylamine in inhibiting growth and pathogenicity of bacillus cereus by regulating quorum sensing system of bacillus cereus

Abstract

The invention discloses an application of 3-phenyl-1-propylamine in inhibiting growth and pathogenicity of bacillus cereus by regulating and controlling a quorum sensing system of the bacillus cereus. 3-phenyl-1-propylamine is used, and compared with traditional chemical antibacterial agents such as antibiotics, the 3-phenyl-1-propylamine is safer to the environment and human bodies, and drug residues and risks of generation of drug-resistant strains are reduced. Research results show that the green natural product can play a role at low concentration (1 / 8 MIC), so that the use cost of the green natural product is low, and the green natural product can not only inhibit pathogenicity, but also effectively control proliferation of bacillus cereus and reduce the risk of food pollution and food-borne disease infection through targeted regulation of expression of a plurality of key genes (such as Spo0A, CodY, rpoN and the like), so that the green natural product has good application prospects. Important application prospects are realized in the field of food safety.

Description

Technical Field

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

[0002] Bacillus cereus is a Gram-positive bacterium of the genus Bacillus, which is facultatively aerobic and widely distributed in nature. Pathogenic Bacillus cereus is commonly found in foods such as rice and flour products, and can produce a variety of toxins, threatening food safety. It is a very important foodborne pathogen and conditional pathogen, and it ranks at the forefront in terms of the number of food poisoning incidents and the total number of poisoning cases. Therefore, it is very necessary to study the process of the formation of Bacillus cereus hazards in food, and it is of great significance to inhibit it.

[0003] The proliferation and pathogenicity of Bacillus cereus are regulated by signaling pathways, such as quorum sensing systems (QS) and pleiotropic transcriptional regulators. The production of degradation enzymes, virulence factors, and biofilm formation of many bacteria rely on QS. The QS pathway can effectively control bacterial growth and virulence, but traditional methods lack targeting and are difficult to achieve precise regulation. Existing prevention and control measures such as chemical antimicrobial agents and high-temperature sterilization have problems such as drug residues, increased risk of drug resistance, and affected food taste. Therefore, a safer and more effective green antibacterial method is urgently needed.

[0004] Green natural products have attracted much attention due to their safety and environmental friendliness, but how to accurately regulate the quorum sensing signal pathway of pathogenic bacteria still needs in-depth research. As a natural product, 3-phenyl-1-propylamine has good biocompatibility and can target and regulate the signal pathway of Bacillus cereus at low concentrations, thereby inhibiting the quorum sensing signal of Bacillus cereus and the expression of virulence genes, reducing pathogenicity and not easily causing bacterial resistance. These natural products can regulate gene expression such as the PlcR-PapR quorum sensing system and the Spo0A transcriptional regulator to achieve an antibacterial effect, have high safety and low-cost advantages, and are increasingly valued today when antibiotics are rampant. As a drug-resistant bacterium, the development of quorum sensing signal inhibitors for Bacillus cereus is of great significance, which is of great help to the development of new anti-Bacillus cereus drugs and even the hazard control of Bacillus cereus. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and 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 invention is achieved through the following technical solutions:

[0007] 3-phenyl-1-propylamine is used to inhibit the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus. At low concentrations, 3-phenyl-1-propylamine can inhibit the growth and pathogenicity of various Bacillus cereus.

[0008] Application of 3-phenyl-1-propylamine in the preparation of antibacterial agent for Bacillus cereus.

[0009] Application of 3-phenyl-1-propylamine in the preparation of preparations for inhibiting the pathogenicity of Bacillus cereus.

[0010] Application of 3-phenyl-1-propylamine in preparing a preparation for inhibiting biofilm formation of Bacillus cereus.

[0011] Application of 3-phenyl-1-propylamine in the preparation of a preparation for inhibiting the quorum sensing system of Bacillus cereus.

[0012] The inhibition of the quorum sensing system of Bacillus cereus is to inhibit the expression level of the genes related to the quorum sensing system.

[0013] The quorum sensing system-related genes include at least one of plcR, papR and LuxS.

[0014] A method for utilizing 3-phenyl-1-propylamine to regulate the quorum sensing system of Bacillus cereus to inhibit its growth and pathogenicity comprises the following steps:

[0015] 3-phenyl-1-propylamine is added to the system to inhibit the growth and pathogenicity of Bacillus cereus.

[0016] The added amount of the 3-phenyl-1-propylamine is 0.08-5 mg / mL, preferably 0.3125 mg / mL.

[0017] The Bacillus cereus includes 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.

[0018] The inhibition of the pathogenicity of Bacillus cereus is to inhibit the expression level of virulence genes.

[0019] The virulence gene comprises at least one of hblB, cytK, nheB and clo.

[0020] Compared with the prior art, the present invention has the following advantages and effects:

[0021] The present invention uses 3-phenyl-1-propylamine, 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 the green natural product can play a role at a low concentration (1 / 8MIC), which makes its use cost low. The present invention can not only inhibit pathogenicity, but also effectively control the proliferation of Bacillus cereus, reduce food contamination and infection of foodborne diseases, and has important application prospects in the field of food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the growth curve of free Bacillus cereus at 1 / 8MIC.

[0023] Figure 2 Changes in quorum sensing gene expression at 1 / 8 MIC 3-phenyl-1-propylamine.

[0024] Figure 3 Changes in toxicity gene expression at 1 / 8 MIC 3-phenyl-1-propylamine.

[0025] Figure 4 Changes in gene expression of pleiotropic transcriptional regulators at 1 / 8 MIC 3-phenyl-1-propylamine.

[0026] Figure 5 Changes in protease gene expression at 1 / 8 MIC 3-phenyl-1-propylamine.

[0027] Figure 6 The expression changes of other key genes under 1 / 8MIC 3-phenyl-1-propylamine. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0029] If the specific experimental conditions are not specified in the following embodiments, the conventional experimental conditions or the experimental conditions recommended by the reagent company are usually followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0030] Example 1 Determination of the Minimum Inhibitory Concentration (MIC) of 3-phenyl-1-propylamine against Bacillus cereus

[0031] (1) Glycerol-frozen Bacillus cereus ATCC 14579, Bacillus cereus IAM 12605, Bacillus cereus CCM 2010, and two strains of Bacillus cereus 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, cultured at 37°C for 24 h, and then single colonies were picked and inoculated into 2 mL of TSB liquid medium, and cultured overnight at 37°C and 200 r / min for 12 h.

[0032] (2) Weigh 10 mg of 3-phenyl-1-propylamine and dissolve it in 1 mL of DMSO. Filter and sterilize to obtain a 10 mg / mL 3-phenyl-1-propylamine solution.

[0033] (3) Dilute the bacterial suspension of five strains of Bacillus cereus cultured overnight to an initial concentration of 10 5 CFU / mL. In a sterile 96-well plate, the final concentrations of the green natural product solution in wells 1 to 7 were set to 5, 2.5, 1.25, 0.625, 0.313, 0.106, and 0.053 mg / mL, respectively, and 5 parallels and a blank control were set accordingly. After culturing at 37°C, 200 rpm for 24 hours, the OD 600nm .

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

[0035] Table 1 OD after adding 3-phenyl-1-propylamine and culturing for 24h 600nm

[0036]

[0037]

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

[0039] The five overnight cultures of Bacillus cereus were diluted in culture tubes to an initial concentration of 10 5 CFU / mL, referring to the conclusion of Example 1, add 1 / 8MIC (0.3125mg / mL) of 3-phenyl-1-propylamine solution, culture on a shaker at 37°C, 200rpm, sample 200μL of bacterial solution in a 96-well plate every 2h, measure its absorbance at 600nm, and set the bacterial solution without adding drug solution as a control.

[0040] The results are as follows Figure 1 As shown, 3-phenyl-1-propylamine at a concentration of 1 / 8 MIC had no effect on the growth of Bacillus cereus, and this concentration can be used to study the inhibitors of quorum sensing signals of Bacillus cereus.

[0041] Example 3 Effect of 1 / 8 MIC concentration of 3-phenyl-1-propylamine on gene expression of free Bacillus cereus

[0042] 3.1 Detection of genes

[0043] The present invention selects multiple key genes for detection to explore the effect of 1 / 8MIC concentration of 3-phenyl-1-propylamine on the gene expression of free-state Bacillus cereus, so as to evaluate its potential for reducing toxicity. First, select the quorum sensing system genes plcR, papR and LuxS, which regulate the formation of biofilm and the synthesis of toxins, directly affecting the pathogenicity of bacteria. In addition, the detection of virulence genes hblB, cytK, nheB and clo is crucial, and the pathogenic factors encoded by these genes are directly related to the toxicity performance of bacteria. Protease genes nprB, colA, clpP and sfp participate in the maturation of quorum sensing signal molecules and the degradation of extracellular matrix, which is of great significance for understanding the regulation of biofilm formation and pathogenicity of bacteria. Pleiotropic transcriptional regulator genes codY and spo0A regulate multiple biological processes, involving the motility and biofilm formation of Bacillus cereus, revealing the adaptation mechanism of bacteria to changes in the external environment. Finally, the detection of other key genes, tlpA, sigH, and rpoN, will help to fully understand the role of Bacillus cereus in growth, metabolism, and toxin production. Through the systematic study of the expression of these genes, we can analyze the effect of 3-phenyl-1-propylamine on Bacillus cereus and verify whether it has the effect of reducing virulence, providing important theoretical basis and data support for the development of new antibacterial strategies.

[0044] 3.2 Primer design

[0045] Bacillus cereus 16srRNA was selected as the internal reference gene, quorum sensing system genes plcR, papR and LuxS; virulence genes hblB, cytK, nheB and clo; protease genes nprB, colA, clpP and sfp; pleiotropic transcription regulator genes codY and spo0A; other genes tlpA, sigH and rpoN were selected as detection genes, and RT-qPCR primers were designed using Primer Premier software. The primers were synthesized by Guangzhou Aiji 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 Establishment of real-time fluorescence quantitative PCR system

[0050] (1) Total RNA extraction from Bacillus cereus

[0051] The five Bacillus cereus cultures were diluted to an initial concentration of 10 5 CFU / mL, 3-phenyl-1-propylamine solution with a concentration of 1 / 8 MIC was added. Samples were taken at 4h, 8h, 12h, and 24h at 37°C and 200rpm, and RNA was extracted using the Trizol method.

[0052] (2) Reverse transcription

[0053] gDNA was removed using RNase free DNase I kit (Thermol), and RNA was reverse transcribed into cDNA using a reverse transcription kit (Biosharp).

[0054] (3) Real-time fluorescence quantitative PCR

[0055] cDNA was diluted 10 times, and the primer concentration was diluted to 10μM for use. Real-time fluorescence quantitative PCR reaction system: 10μL 2×Mastermix, 2μL cDNA template (after dilution), 0.4μL each of upstream and downstream (F / R) primers (10μM), and finally 20μL was supplemented with RNase free water. Amplification program: 94℃ pre-denaturation for 3min; 94℃ denaturation for 15s, 55℃ annealing for 15s, 72℃ extension for 20s, these three steps were cycled 39 times.

[0056] (4) Statistics

[0057] The relative quantitative 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 the error. The group cultured for 4 hours was used as the reference, and after normalization with the internal reference gene, 2 –ΔΔCt The value of (Livak method) represents the fold change of the expression level of the target gene.

[0058] 3.4 Experimental Results

[0059] Figure 1The growth curve of free Bacillus cereus at 1 / 8MIC is shown in the figure. As shown in the figure, 1 / 8MIC hordenine inhibited the growth of Bacillus cereus to a certain extent. For example, the bacterial concentration of ATCC 14579 was lower than that of the control group when it was cultured for 0-14h, CMCC 2010 was cultured for 7-11h, and IAM 12605 was cultured for 6-14h.

[0060] Figure 2 It shows that when the expression of the PlcR-PapR quorum sensing system of Bacillus cereus is slightly weak under 1 / 8MIC of 3-phenyl-1-propylamine, the expression of the system can be completely inhibited. Under the action of 3-phenyl-1-propylamine, except for strain L1, the plcR of other Bacillus cereus was completely inhibited within 24h or before 24h. The same is true for the LuxS gene. It proves that 3-phenyl-1-propylamine can target and inhibit the plcR, PlcR-PapR, LuxS signaling pathways and the proliferation of Bacillus cereus to a certain extent.

[0061] Figure 3 The results showed that the NheB gene was completely inhibited after 8 hours of culture under the action of 3-phenyl-1-propylamine, and strains IAM12605 and CCM 2010 even had no expression after 4 hours of culture. The NheB gene is an important gene encoding the non-hemolytic enterotoxin of Bacillus cereus. The decrease in its expression indicates that 3-phenyl-1-propylamine can effectively reduce the Nhe toxicity of Bacillus cereus, and to some extent reduce the harm caused by the proliferation of Bacillus cereus.

[0062] Figure 4 The results showed that under the action of 3-phenyl-1-propylamine, the expression of Spo0A gene in strains IAM 12605 and CMCC 2010 was completely inhibited after 24 hours of culture. 3-phenyl-1-propylamine significantly promoted the expression of pleiotropic transcriptional regulator gene Spo0A in most cases studied, and it could promote the sporulation and biofilm formation of Bacillus cereus, making the bacteria more inclined to protect themselves rather than invade the host when the survival pressure is high.

[0063] Figure 5 The results showed that the effects of 3-phenyl-1-propylamine on the expression of clpP, nprB and sfp genes were quite different, indicating that 3-phenyl-1-propylamine had no significant effect on the expression of protease genes.

[0064] Figure 6 The results showed that the expression of sigH gene was mostly up-regulated under the action of 3-phenyl-1-propylamine. The promotion of sigH gene expression by 3-phenyl-1-propylamine can make the bacteria enter a dormant state under unfavorable conditions.

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

[0066] The above experimental results show that the green natural product 3-phenyl-1-propylamine selected by the present invention has a wide range of application prospects, and its effect on the expression of signal pathway genes is not only applicable to Bacillus cereus, but can also be extended to other strains and even the food field. It is of great value for the growth detection of Bacillus cereus and in-depth understanding of the proliferation mechanism of Bacillus cereus, which will hopefully improve food safety management and provide useful reference and reference for microbiology research and other fields of application.

[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

Application of 1.3-phenyl-1-propylamine in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system. Application of 2.3-phenyl-1-propylamine in the preparation of Bacillus cereus antibacterial agent.

3. Application of 3-phenyl-1-propylamine in the preparation of preparations for inhibiting the pathogenicity of Bacillus cereus.

4. Application of 3-phenyl-1-propylamine in the preparation of preparations for inhibiting biofilm formation of Bacillus cereus.

5. Application of 3-phenyl-1-propylamine in the preparation of preparations for inhibiting the quorum sensing system of Bacillus cereus.

6. The use according to claim 5, characterized in that: The inhibition of the quorum sensing system of Bacillus cereus is to inhibit the expression level of genes related to the quorum sensing system; The quorum sensing system-related genes include at least one of plcR, papR and LuxS.

7. A method for inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus using 3-phenyl-1-propylamine, characterized in that The steps include: 3-phenyl-1-propylamine is added to the system to inhibit the growth and pathogenicity of Bacillus cereus.

8. The method according to claim 7, characterized in that: The added amount of the 3-phenyl-1-propylamine is 0.08-5 mg / mL.

9. The use according to any one of claims 1 to 6 or the method according to any one of claims 7 to 8, characterized in that: The Bacillus cereus includes 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.

10. The use according to any one of claims 1 to 3 or the method according to any one of claims 7 to 8, characterized in that: The inhibition of pathogenicity is to inhibit the expression level of virulence genes; The virulence gene comprises at least one of hblB, cytK, nheB and clo.

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