Vibrio parahaemolyticus quorum sensing inhibiting peptide and application thereof

Abstract

The application discloses a Vibrio parahaemolyticus quorum sensing inhibiting peptide and application thereof, and belongs to the field of bioactive peptides. The inhibiting peptide comprises the following amino acid sequence: Ser-Phe; the application further discloses application of the quorum sensing inhibiting peptide in inhibiting Vibrio parahaemolyticus or preparing antibacterial drugs for inhibiting Vibrio parahaemolyticus. The application adopts a molecular docking technology to screen an active peptide having an inhibiting effect on a Vibrio parahaemolyticus LuxS / AI-2 quorum sensing system, ADMET property prediction is carried out on a peptide segment with better binding force, and finally, a bioactive peptide SF stably combined with LuxP, LuxQ and LuxS is obtained; the dipeptide has good in-vitro inhibiting activity on the Vibrio parahaemolyticus LuxS / AI-2 quorum sensing, and has no cytotoxicity. The dipeptide obtained by the application can be used for guiding development of a new type of antibacterial preparation applied to food, medicine and agriculture and other fields, and has great significance for human health and agricultural development.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides, and in particular to a quorum sensing inhibitory peptide of Vibrio parahaemolyticus and its application. Background Technology

[0002] Vibrio parahaemolyticus is a halophilic pathogen commonly found in seafood such as fish, shellfish, shrimp, and crab, as well as pickled foods. It can produce virulence factors such as adhesion factors, hemolysin, urease, and outer membrane proteins. It can also form mature biofilms on the surfaces of processing equipment, enhancing the bacteria's resistance to adverse external environments and thus causing food safety issues. These problems can be controlled using physical and chemical methods, such as high temperature and pressure, the use of bactericides, and antibiotics. However, these methods are costly, inefficient, and prone to causing bacterial resistance. Research shows that quorum sensing in Vibrio parahaemolyticus can regulate the expression of virulence factors and biofilm formation; therefore, quorum sensing inhibitors have become one method for controlling the pathogenicity of Vibrio parahaemolyticus.

[0003] Quorum sensing inhibitors (QSIs) target the bacterial quorum sensing system without interfering with normal microbial life activities, controlling the production of biofilms and virulence factors, thereby reducing the pathogenicity of pathogens. They offer the advantages of being less likely to induce bacterial resistance while effectively preventing bacterial diseases. Traditional methods for finding QSIs are often random, inefficient, costly, and require extensive experimentation. With the continuous development of science and technology, research combining high-performance computer-aided design of QSIs is increasing. Utilizing online data not only reduces screening intensity, manual screening, and the development cycle, but also improves the success rate of screening. Applying computer technology to simulate the enzymatic hydrolysis of myosin in Litopenaeus vannamei and predicting the properties of currently studied bioactive peptides based on online databases, after multiple rounds of virtual screening, highly efficient bioactive peptides that bind to target molecules can be finally screened, gradually becoming a hot topic in the field of bioactive peptide research. Currently, there are relatively few reported quorum sensing inhibitory peptides, requiring further research and investigation. Summary of the Invention

[0004] The purpose of this invention is to provide a quorum sensing inhibitory peptide for Vibrio parahaemolyticus and its application, in order to solve the problems existing in the prior art. This invention screens and obtains a bioactive peptide SF that stably binds to the Vibrio parahaemolyticus receptor proteins LuxP, LuxQ, and LuxS. This dipeptide has good in vitro inhibitory activity against the LuxS / AI-2 quorum sensing of Vibrio parahaemolyticus and is non-cytotoxic. The dipeptide obtained by this invention can be used to guide the development of novel antimicrobial agents and applied in the fields of food, medicine, and agriculture, which is of great significance to human health and agricultural development.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a quorum sensing inhibitory peptide of Vibrio parahaemolyticus, comprising the following amino acid sequence: Ser-Phe.

[0007] The present invention also provides the application of the quorum sensing inhibitory peptide in the inhibition of Vibrio parahaemolyticus for non-diagnostic purposes.

[0008] The present invention also provides the application of the quorum sensing inhibitory peptide described above in the preparation of an antibacterial drug for inhibiting Vibrio parahaemolyticus.

[0009] The present invention also provides the application of the quorum sensing inhibitory peptide in the preparation of food antibacterial agents.

[0010] Furthermore, the antibacterial agent is used in food, medicine, and agriculture to inhibit Vibrio parahaemolyticus infection.

[0011] The present invention also provides an antibacterial agent for inhibiting Vibrio parahaemolyticus, comprising the aforementioned quorum sensing inhibitory peptide.

[0012] The present invention discloses the following technical effects:

[0013] This invention utilizes computer technology to simulate the enzymatic hydrolysis of shrimp myosin, and employs molecular docking technology to screen for bioactive peptides that inhibit the LuxS / AI-2 quorum sensing system of Vibrio parahaemolyticus. Bioinformatics technology is then used to predict the ADMET properties of peptides with good binding affinity, ultimately yielding a bioactive peptide SF that stably binds to LuxP, LuxQ, and LuxS. The dipeptide SF was artificially synthesized, and its in vitro inhibitory activity against the LuxS / AI-2 quorum sensing of Vibrio parahaemolyticus and its cytotoxicity were verified. Results showed that the dipeptide SF exhibited good in vitro inhibitory activity against the LuxS / AI-2 quorum sensing of Vibrio parahaemolyticus and was non-cytotoxic. The dipeptide obtained using this invention can guide the development of novel antimicrobial agents for application in food, medicine, and agriculture, which is of great significance to human health and agricultural development.

[0014] In addition, this invention uses computer-aided design technology to virtually screen Vibrio parahaemolyticus quorum sensing inhibitory peptides, which reduces the number of compounds actually tested in bioassays while saving time and cost in purifying and identifying peptide sequences, thus increasing the success rate of screening; at the same time, this invention provides a new option for foodborne Vibrio parahaemolyticus quorum sensing inhibitory peptides. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The results show the molecular docking of Vibrio parahaemolyticus receptor protein with peptide SF; ac shows the three-dimensional structures of Vibrio parahaemolyticus QS receptor proteins LuxP, LuxQ, and synthetic protein LuxS, respectively; df shows the 2D schematic diagram of the docking of peptide SF with Vibrio parahaemolyticus QS receptor proteins LuxP, LuxQ, and synthetic protein LuxS, respectively.

[0017] Figure 2 The inhibitory activity of peptide SF against AI-2 in Vibrio parahaemolyticus; a: Trend of AI-2 signaling molecules in Vibrio parahaemolyticus with added SF; b: Inhibitory effect of peptide SF on AI-2 activity of Vibrio parahaemolyticus 21617 at sub-inhibitory concentrations;

[0018] Figure 3 The effect of peptide SF on the survival rate of mouse macrophages after treatment with RAW cells. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The quorum sensing inhibitory peptide disclosed in this invention is derived from myosin of Litopenaeus vannamei, with the amino acid sequence SF (Ser-Phe). The quorum sensing inhibitory peptide of Vibrio parahaemolyticus protected by this invention comprises any active peptide with the core sequence of the dipeptide SF, subject to corresponding adjustments or modifications.

[0025] Example 1

[0026] This invention utilizes an online database for multiple rounds of screening to obtain Vibrio parahaemolyticus AI-2 inhibitory peptides, comprising the following steps:

[0027] (1) Search for and analyze the myosin sequence of Litopenaeus vannamei (accession number NCBI: ROT75475.1) in the NCBI database.

[0028] (2) Myosin was virtually hydrolyzed using pepsin (pH 1.3) and trypsin via ExPASyPeptideCutter (http: / / web.expasy.org / peptide_cutter). Activity was then scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), and bioactive hydrolysates were selected (see Table 1). Using Discovery Studio 2017 R2 software, five bioactive peptides were molecularly docked with the Vibrio parahaemolyticus AI-2 receptor proteins LuxP, LuxQ, and the synthetic protein LuxS (the docking results are shown in Table 2 and...). Figure 1 The dipeptides SF and DW, which had higher docking scores and better binding affinity, were selected for ADMET property prediction (results are shown in Table 3). Then, the dipeptide SF, which showed better prediction results, was artificially synthesized (by Shanghai Qiangyao Biotechnology Co., Ltd.) and subjected to in vitro AI-2 inhibitory activity and cytotoxicity tests.

[0029] Table 1. Enzyme mimics with potential biological activity

[0030]

[0031]

[0032] Table 2 Scoring of docking results between five peptide segments and Vibrio parahaemolyticus receptor proteins

[0033]

[0034] Table 3 ADMET Prediction Results

[0035]

[0036] (3) The in vitro AI-2 inhibitory activity of the active peptide was verified by the Vibrio harzianum bioluminescence method.

[0037] Activated Vibrio parahaemolyticus 21617 was inoculated into 2216E liquid medium, and then active peptide SF (concentrations of 0.2, 0.6, 0.8, and 1 mg / mL) was added, with the control group not containing SF. The medium was incubated at 37℃ with shaking for 16 h, centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter to obtain cell-free Vibrio parahaemolyticus supernatant. The Vibrio harveyi supernatant was collected as a positive control, while AB medium and 2216E medium served as negative and media controls. Activated Vibrio harveyi BB170 (fluorescent indicator bacteria) was inoculated at 1% into AB medium and incubated overnight with shaking at 30℃. The Vibrio harveyi BB170 culture was then diluted 1:1000 with fresh AB medium. Vibrio parahaemolyticus supernatant, V. harvestyi BB170 supernatant, AB medium, and 2216E medium were added to diluted V. harvestyi BB170 culture medium at a volume ratio of 1:100 and incubated at 30°C for 6 hours. Every hour, 200 μL was transferred to a black 96-well microplate, and the fluorescence intensity was measured using a microplate reader in bioluminescent mode.

[0038] The results are as follows Figure 2 As shown, Figure 2 The value 'a' indicates the trend of AI-2 signal molecule production in Vibrio parahaemolyticus with or without SF. It can be seen that at the initial point, the AI-2 signal molecule has not yet been recognized by the Vibrio harveyi indicator bacteria; therefore, the fluorescence produced at this time is generated by Vibrio harveyi. Over the next 3 hours, the concentration of the signal molecule produced by Vibrio parahaemolyticus does not reach the minimum value that the indicator bacteria can recognize, so the fluorescence intensity gradually decreases, reaching its lowest point at 3 hours. Afterward, the signal molecule is recognized by the indicator bacteria, and the fluorescence intensity gradually increases. Figure 2 In the figure, b represents the inhibition result of peptide SF on the AI-2 activity of VP21617 at a sub-inhibitory concentration. Figure 2It was found that the inhibition rates of peptide SF at concentrations of 0.2-1 mg / mL on the AI-2 activity of Vibrio parahaemolyticus were 16.53%, 34.41%, 50.16%, and 55.64%, respectively. Therefore, peptide SF can interfere with the QS system of Vibrio parahaemolyticus by inhibiting its AI-2 activity.

[0039] (4) Verify the cytotoxicity of the active peptide.

[0040] Mouse macrophage RAW toxicity assay:

[0041] Cell culture: Resuscitated mouse macrophages (RAW) were digested with 1 mL of 0.25% trypsin-EDTA and passaged every two days. After the cells grew well and adhered to the culture vessel at 80%-90%, toxicity tests were performed.

[0042] Healthy mouse macrophages (RAW cells) were selected and digested with trypsin to prepare a cell suspension. The suspension was then seeded into 96-well plates at a density of 200 μL per well, maintaining a density of 2.5 × 10⁻⁶ cells / well. 4 Cells were cultured in triplicate, with each group containing approximately 80% cell coverage in each well. The cells were incubated at 37°C with 5% CO2 for 12 hours. After cell attachment, the culture medium was discarded, and 200 μL of peptide SF at different concentrations was added to each well. Incubation continued for 24 hours. Four hours before the end of the culture period, the liquid was removed, and 100 μL of LTT solution was added to each well. The cells were incubated at 37°C for another 4 hours, at which point the culture was terminated. The solution was carefully removed from the wells, and 150 μL of DMSO solution was added. The cells were shaken at low speed for 15 minutes until the crystals were completely dissolved. The absorbance at 490 nm was measured for each well, and the inhibitory effect of peptide SF on mouse macrophage RAW cells was calculated.

[0043] The results are as follows Figure 3 As shown, peptide SF had no effect on the survival rate of mouse macrophages RAW cells after treatment. Therefore, peptide SF is not toxic.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a quorum sensing inhibitory peptide of Vibrio parahaemolyticus in the inhibition of Vibrio parahaemolyticus for non-disease diagnostic and therapeutic purposes, characterized in that, The amino acid sequence of the quorum sensing inhibitory peptide of Vibrio parahaemolyticus is Ser-Phe.

2. The application of a quorum sensing inhibitory peptide of Vibrio parahaemolyticus in the preparation of antibacterial drugs that inhibit Vibrio parahaemolyticus, characterized in that, The amino acid sequence of the quorum sensing inhibitory peptide of Vibrio parahaemolyticus is Ser-Phe.

3. The application of a quorum sensing inhibitory peptide of Vibrio parahaemolyticus in the preparation of antibacterial agents, characterized in that, The amino acid sequence of the quorum sensing inhibitory peptide of Vibrio parahaemolyticus is Ser-Phe; the antibacterial agent is an antibacterial agent of Vibrio parahaemolyticus.

4. The application according to claim 3, characterized in that, The antibacterial agent is used in food, medicine, and agriculture to inhibit Vibrio parahaemolyticus infection.

5. A bacteriostatic agent for inhibiting Vibrio parahaemolyticus, characterized in that, It contains a quorum sensing inhibitory peptide of Vibrio parahaemolyticus, wherein the amino acid sequence of the quorum sensing inhibitory peptide of Vibrio parahaemolyticus is Ser-Phe.

Images