Application of hordenine in inhibiting growth and pathogenicity of bacillus cereus by regulating quorum sensing system of bacillus cereus

Abstract

The invention discloses an application of hordenine in inhibiting growth and pathogenicity of bacillus cereus by regulating and controlling a quorum sensing system of the bacillus cereus. Compared with traditional chemical antibacterial agents, such as antibiotics, the hordenine which is a green natural product is used, so that the hordenine 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 hordenine can play a role at low concentration (1 / 8 MIC), so that the hordenine is low in use cost, the expression of a plurality of key genes (such as Spo0A, CodY, rpoN and the like) can be regulated in a targeted manner, the pathogenicity can be inhibited, the proliferation of bacillus cereus can be effectively controlled, the risk of food pollution and food-borne disease infection is reduced, and the hordenine has a good application prospect. 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 hordenine 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, hordenine 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. Natural products can regulate gene expression such as the PlcR-PapR quorum sensing system and the Spo0A transcriptional regulator to achieve an antibacterial effect. They 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 hordenine 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] The application of hordenine in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus. At low concentrations, hordenine can inhibit the growth and pathogenicity of various Bacillus cereus.

[0008] Application of hordenine in the preparation of bacillus cereus antibacterial agent.

[0009] Application of hordenine in the preparation of preparations for inhibiting the pathogenicity of Bacillus cereus.

[0010] Application of hordenine in the preparation of preparations for inhibiting biofilm formation of Bacillus cereus.

[0011] Application of hordenine in the preparation of preparations 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 hordenine to regulate the quorum sensing system of Bacillus cereus to inhibit its growth and pathogenicity comprises the following steps:

[0015] Adding hordenine into the system can inhibit the growth and pathogenicity of Bacillus cereus.

[0016] The added amount of hordenine 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 the green natural product hordenine, which is safer for the environment and human body than traditional chemical antibacterial agents, such as antibiotics, etc., and reduces the risk of drug residues and drug-resistant strains. And the research results show that hordenine 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 the risk of infection of foodborne diseases, and has important application prospects in the field of food safety by targeting and regulating the expression of multiple key genes (such as Spo0A, CodY, rpoN, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 The expression changes of quorum sensing genes under 1 / 8 MIC hordenine.

[0024] Figure 3 The changes in virulence gene expression under 1 / 8MIC hordenine.

[0025] Figure 4 The expression changes of pleiotropic transcriptional regulator genes under 1 / 8MIC hordenine.

[0026] Figure 5 The expression changes of protease genes under 1 / 8 MIC hordenine.

[0027] Figure 6 The expression changes of other key genes under 1 / 8MIC hordenine. 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 no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally 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 hordenine against Bacillus cereus

[0031] (1) Glycerol-frozen Bacillus cereus ATCC 14579, Bacillus cereus IAM 12605, Bacillus cereus CCM 2010, Bcer A9 (purchased from Guangzhou Peptide Valley Co., Ltd.) and Bcer L1 (purchased from Guangzhou Peptide Valley Co., Ltd.) were streaked onto TSA solid medium, cultured at 37°C for 24 h, and then a single colony was 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 hordenine and dissolve it in 1 mL of DMSO. Filter and sterilize to prepare a 10 mg / mL hordenine solution.

[0033] (3) Dilute the bacterial suspension of five strains of Bacillus cereus cultured overnight to an initial concentration of 10 5 CFU / mL. Add 180 μL of diluted bacterial solution to each well from the first to the seventh well of each of the first five columns of a sterile 96-well plate, add culture medium without bacterial solution to the eighth well as a blank control, and then add 20 μL of hordenine solution of different concentrations diluted by a dilution factor of 2 to the bacterial solution, so that the final concentrations of the green natural product solution in the first to seventh wells are 5, 2.5, 1.25, 0.625, 0.313, 0.106, and 0.053 mg / mL, respectively. Incubate on a shaker at 37°C, 200 rpm for 24 hours, and then use a full-wavelength microplate reader to measure OD 600nm .

[0034] The results are shown in Table 1. MIC (minimuminhibitory concentration) is an important indicator for measuring the antibacterial activity of drugs. It refers to the lowest concentration of drugs that can inhibit the growth and reproduction of pathogens in the culture medium after 18-24 hours of in vitro bacterial culture. In this experiment, when the concentration of hordenine 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 hordenine against Bacillus cereus was 2.5 mg / mL.

[0035] Table 1 OD600nm after adding hordenine for 24h

[0036]

[0037] Example 2 Determination of the growth curve of free Bacillus cereus

[0038] The five overnight cultures of Bacillus cereus were diluted in culture tubes to an initial concentration of 10 5CFU / mL, refer to the conclusion of Example 1, add 1 / 8MIC, i.e. 0.3125mg / mL of green natural product hordenine solution, culture on a shaker at 37°C, 200rpm, sample 200μL of bacterial solution every 2h in a 96-well plate, and measure its OD 600nm The absorbance value at 1 was set, and the bacterial solution without drug solution was set as the control.

[0039] The results are as follows Figure 1 As shown, 1 / 8 MIC concentration of hordenine has 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.

[0040] Example 3 Effect of 1 / 8 MIC concentration of hordenine on gene expression of free Bacillus cereus

[0041] 3.1 Selection of detection genes

[0042] (1) The quorum sensing system is a mechanism for information exchange between cells. When the secreted signal molecules (auto-inducers, AIs) of microorganisms accumulate to a certain concentration during their growth, the QS system is activated, regulating the expression of specific genes so that cells can adapt and survive through group behavior. PlcR-PapR is a quorum sensing system unique to the Bacillus cereus group. Among them, the activity of PlcR requires the activation of the signal molecule encoded by the papR gene directly downstream of the plcR gene. After the papR gene is processed and transported, two PapR molecules bind to two PlcR molecules to form a tetramer, which binds to the PlcR box at a specific site on the DNA, and finally initiates the transcription of the PlcR regulator. The PlcR regulator regulates the transcription of hundreds of genes, including the formation of biofilms, the synthesis of toxins and proteases, etc. The LuxS / AI-2 system is confirmed to exist in Bacillus cereus. The signal molecule AI-2 is synthesized by the luxS gene and involves multiple enzymatic reactions. The initial metabolic substrate undergoes methylation, hydrolysis, and cleavage reactions to generate precursors, which then spontaneously cyclize through their own chemical properties to form the active chemical form of AI-2, thereby participating in the bacterial quorum sensing process. In Bacillus cereus, the LuxS / AI-2 quorum sensing system participates in the formation of biofilms and may also be related to the synthesis of vomitoxin in vomiting strains. Therefore, the Bacillus cereus quorum sensing system genes plcR, papR, and LuxS were selected as detection genes.

[0043] (2) Common diarrheal enterotoxins in Bacillus cereus include hemolytic enterotoxin (Hbl), non-hemolytic enterotoxin (Nhe) and cytotoxin K (CytK), all of which are secreted through the Sec pathway. The virulence genes related to Hbl are hblA, hblB, hblC and hblD, of which hblA, hblC and hblD encode the three protein subunits Hbl B, L2 and L1 that constitute the Hbl complex, respectively. The protein Hbl B' encoded by hblB has been shown to balance the number of Hbl B-L1 complex and the corresponding free subunits, which is a way for Bacillus cereus to regulate its own toxicity. The genes encoding the three subunits of Nhe are nheA, nheB and nheC, which encode the protein subunits with the corresponding names. Similarly, toxins such as CytK and Clo are encoded by genes such as cytK and clo, respectively. Therefore, the virulence genes hblB, cytK, nheB and clo of Bacillus cereus were selected as detection genes.

[0044] (3) The NprB protease encoded by the NprB gene of Bacillus cereus is an important protease involved in catalyzing the maturation of the quorum sensing signal molecule PapR. The ColA gene encodes a protease that can cut the three-dimensional structure of triple-helical collagen and promote bacterial pathogenicity and the shedding of biofilms by hydrolyzing the extracellular matrix. The ClpP protease encoded by the ClpP gene maintains intracellular protein stability and regulates cell growth by controlling the amount of intracellular protein. It has been proven to be related to biofilm formation, spore production and motility. The protein encoded by the Sfp gene is a typical subtilisin family protease, which is mainly related to the synthesis of surfactants in Bacillus. Therefore, the Bacillus cereus protease genes nprB, colA, clpP and sfp were selected as detection genes.

[0045] (4) Pleiotropic transcriptional regulator genes are involved in regulating a large number of genes and affect various biological behaviors. CodY is not only involved in the expression of motility and virulence genes of Bacillus cereus, but also affects the formation of biofilms by inhibiting the production of an unknown protease. Spo0A is the main regulatory factor for spore production in Bacillus cereus. There are as many as hundreds of genes affected by spo0A, which in turn affects the transcription of more genes. Therefore, spo0A is related to various biological behaviors of Bacillus cereus, such as surface movement and biofilm formation. Therefore, the pleiotropic transcriptional regulator genes codY and spo0A were selected as detection genes.

[0046] (5) There are many other key genes in Bacillus cereus, which have their own functions and roles and jointly regulate the biological behavior of Bacillus cereus. For example, tlpA, which encodes the methyl-accepting chemotaxis transducer protein, sigH, the spore production regulatory factor, and RpoN, which are related to growth metabolism, motility, biofilm formation, and toxin production. They may play a very critical role in Bacillus cereus, so they are also selected as detection genes.

[0047] 3.2 Primer design

[0048] Bacillus cereus 16srRNA was selected as the internal reference gene, and several key genes of Bacillus cereus were selected as detection genes, including: 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. PCR primers were designed according to the selected genes, and the specific primer design process was as follows:

[0049] (1) Search the entire genome sequence of Bacillus cereus on GenBank, and continue to search and obtain the gene sequence of the target detection target;

[0050] (2) Directly search for the target gene sequence on GenBank and use Clustaw software for alignment analysis. By performing multiple alignments between different sequences, the most conservative regional sequence is obtained.

[0051] (3) Further use Primer Premier and other software to analyze the obtained regional sequences, and at the same time, combine the sequencing platform and method used for the sequence to perform a comprehensive fault tolerance analysis. For example, the Sanger sequencing method has low credibility of the first 20-30 and last 20-30 base sequences, which can be eliminated by testing both ends; for example, PacBio sequencing has base mispairing, which can be proofread by Illumina or Sanger sequencing; comprehensive analysis is used to further accurately judge the sequence fault tolerance, obtain the most conservative regional sequence of the target detection gene, and use it to design primers.

[0052] The primers were synthesized by Guangzhou Aiji Biotechnology Co., Ltd. and the primer sequences are shown in Table 2.

[0053] Table 2 Primer sequences for RT-qPCR

[0054]

[0055]

[0056] 3.3 Establishment of real-time fluorescence quantitative PCR system

[0057] (1) Total RNA extraction from Bacillus cereus

[0058] The five Bacillus cereus cultures were diluted to an initial concentration of 10 5 CFU / mL, 1 / 8 MIC hordenine solution was added. Samples were taken at 4h, 8h, 12h, and 24h at 37°C and 200rpm, and RNA was extracted using the Trizol method.

[0059] (2) Reverse transcription

[0060] gDNA was removed using an RNase free DNase I kit (Thermol), and reverse transcription of RNA into cDNA was performed using a reverse transcription kit (Biosharp).

[0061] (3) Real-time fluorescence quantitative PCR

[0062] 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 40 times.

[0063] (4) Data Statistics

[0064] 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.

[0065] 3.4 Results and Discussion

[0066] Figure 1 The 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 10-14h, CMCC 2010 for 6-12h, IAM 12605 for 8-14h, L1 for 8-16h, and A9 for 6-14h.

[0067] Figure 2 It was shown that 1 / 8MIC of hordenine in Bacillus cereus could completely inhibit the expression of the PlcR-PapR quorum sensing system when the system was slightly weakly expressed. Under the action of hordenine, the plcR gene of strains ATCC 14579, A9 and L1 was completely inhibited when the strains were cultured for 24 hours, and PapR was inhibited when ATCC 14579 was cultured for 24 hours. The LuxS gene was completely inhibited when the strain ATCC 14579 was cultured for 24 hours, the strain IAM 12605 was cultured for 8 hours, and the strain CCM 2010 was cultured for 4 hours and 8 hours. It was proved that hordenine can target and inhibit the plcR, PlcR-PapR, LuxS signaling pathways and the proliferation of Bacillus cereus to a certain extent.

[0068] Figure 3 It was shown that under the action of hordenine, except for strain IAM 12605 which expressed after 12 hours of culture, the expression of nheB gene of the other four strains was completely inhibited after 8 hours of culture, and some even stopped expressing after 4 hours. Therefore, it is believed that hordenine has a very obvious inhibitory effect on the nheB virulence gene of Bacillus cereus, and can effectively reduce the Nhe toxicity of Bacillus cereus, which can be considered to reduce the harm caused by the proliferation of Bacillus cereus to some extent.

[0069] Figure 4 The results showed that under the action of hordenine, the expression of Spo0A gene was basically regulated at each time point. Except for strain L1, the expression of CodY gene was inhibited in the other four bacteria after 24 hours of culture. In most cases of the study, hordenine played a significant role in promoting the expression of the pleiotropic transcriptional regulator gene Spo0A, which can promote the spore production 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. The inhibition of CodY gene expression by hordenine can also slow down the growth rate of bacteria.

[0070] Figure 5 The results showed that the effects of hordenine on the expression of clpP, nprB and sfp genes were quite different. This indicated that hordenine had no significant effect on the expression of protease genes. The expression of protease genes of strain CCM 2010 was significantly inhibited by hordenine, but the growth of strain CCM 2010 was still unaffected in this case, indicating that these proteases were not necessary for the growth of Bacillus cereus.

[0071] Figure 6 The results showed that the expression of other key genes was completely suppressed at multiple time points under the action of hordenine, and the rpoN gene was completely suppressed. The inhibitory effect of hordenine on the expression of the rpoN gene may reduce the nitrogen metabolism efficiency of Bacillus cereus, thereby inhibiting its colonization and pathogenic potential.

[0072] In summary, 1 / 8MIC of hordenine in Bacillus cereus can completely inhibit the expression of the PlcR-PapR quorum sensing system when the system is slightly weakly expressed. After a period of time, hordenine can basically completely inhibit the expression of the nheB gene, which can 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 hordenine and the inhibition of the expression of the CodY gene by hordenine can also slow down the growth rate of Bacillus cereus. The inhibition of rpoN gene expression by hordenine can inhibit the colonization and pathogenic potential of Bacillus cereus.

[0073] The green natural product hordenine selected by the present invention has a wide range of application prospects. Its influence 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 is expected to improve food safety management and provide useful reference and reference for microbiology research and other fields of application.

[0074] 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

1. Application of hordenine in inhibiting the growth and pathogenicity of Bacillus cereus by regulating its quorum sensing system.

2. Application of hordenine in the preparation of Bacillus cereus antibacterial agent.

3. Application of hordenine in the preparation of preparations for inhibiting the pathogenicity of Bacillus cereus.

4. Application of hordenine in the preparation of preparations for inhibiting biofilm formation of Bacillus cereus.

5. Application of hordenine 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 using hordenine to regulate the quorum sensing system of Bacillus cereus to inhibit its growth and pathogenicity, characterized in that The steps include: Adding hordenine into the system can inhibit the growth and pathogenicity of Bacillus cereus.

8. The method according to claim 7, characterized in that: The added amount of the hordenine 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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