Application of antibacterial peptide targeting vibrio parahaemolyticus outer membrane protein LptD

Abstract

The invention provides application of an antibacterial peptide targeting a vibrio parahaemolyticus outer membrane protein LptD, and belongs to the technical field of antibacterial peptides. The amino acid sequence of the antibacterial peptide targeting the vibrio parahaemolyticus outer membrane protein LptD provided by the invention is as shown in SEQ ID NO. 1. The antibacterial peptide provided by the invention can specifically inhibit vibrio parahaemolyticus, and the integrity of an outer membrane is destroyed by combining a beta-taco structural domain of LptD protein, so that cellular contents are leaked. In a ready-to-eat salmon model, the load of artificially inoculated vibrio parahaemolyticus can be reduced by 1.51 log CFU / mL by soaking 64 micrograms / mL of the antibacterial peptide disclosed by the invention for 4 hours, and meanwhile, the regeneration of a biofilm in a storage period is remarkably inhibited. Safety experiments prove that the antibacterial peptide provided by the invention has the advantage of high safety.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to the application of an antimicrobial peptide that targets the outer membrane protein LptD of Vibrio parahaemolyticus. Background Technology

[0002] Vibrio parahaemolyticus is a major pathogen causing foodborne infections, spreading through contaminated seafood (such as salmon) and leading to acute gastroenteritis and even sepsis. In recent years, the prevalence of multidrug-resistant strains (such as ATCC 17802 and ATCC 33847) has significantly increased the difficulty of clinical treatment, and the efficacy of traditional antibiotics (such as β-lactams) is limited due to the spread of resistance genes (such as blaNDM-1). LptD protein is a key channel protein in the assembly of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, mediating LPS transmembrane transport through its β-barrel domain. Disruption of its function can lead to loss of outer membrane integrity and bacterial death. However, existing antibiotics are limited by molecular size or binding sites, making it difficult to directly target LptD, and the outer membrane barrier further reduces drug penetration efficiency.

[0003] Antimicrobial peptides (AMPs) are a class of naturally occurring bioactive molecules widely distributed in insects, plants, animals, and microorganisms. They exert broad-spectrum antibacterial effects by targeting microbial membrane structures, exhibiting advantages such as low resistance induction, multiple mechanisms of action (e.g., membrane penetration, biofilm inhibition), and low cytotoxicity. Due to their membrane-targeting properties, antimicrobial peptides have become a hot research topic in anti-drug-resistant bacteria research. However, most AMPs have high minimum inhibitory concentrations (MICs) against Vibrio parahaemolyticus and exhibit hemolytic toxicity. For example, although polymyxin E can bind to LPS, its nephrotoxicity limits its application. Therefore, developing highly effective and low-toxicity AMPs targeting LptD has significant clinical implications. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an antimicrobial peptide that targets the outer membrane protein LptD of Vibrio parahaemolyticus, which has the advantages of high efficiency in killing bacteria, inhibiting bacteria and low toxicity.

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

[0006] This invention provides an antimicrobial peptide that targets the outer membrane protein LptD of Vibrio parahaemolyticus, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides the use of the above-mentioned antimicrobial peptide in the preparation of products for the prevention and / or treatment of Vibrio parahaemolyticus infection.

[0008] The present invention also provides the application of the above-mentioned antimicrobial peptide in the preparation of bacteriostatic agents or bactericides, wherein the bacteria in the bacteriostatic agents or bactericides include Vibrio parahaemolyticus.

[0009] Preferably, the concentration of the antimicrobial peptide in the bacteriostatic agent or bactericide is 64 μg / mL or higher.

[0010] This invention also provides the application of the above-mentioned antimicrobial peptides in preventing and controlling Vibrio parahaemolyticus contamination in food.

[0011] Preferably, the food includes seafood.

[0012] Preferably, the seafood includes salmon.

[0013] The present invention also provides the application of the above-mentioned antimicrobial peptide in the removal of Vibrio parahaemolyticus biofilm.

[0014] The beneficial effects of this invention are:

[0015] The targeted antimicrobial peptide against Vibrio parahaemolyticus provided by this invention demonstrates precise control advantages in the field of food sterilization. The antimicrobial peptide provided by this invention can specifically inhibit Vibrio parahaemolyticus (MIC = 64 μg / mL), which disrupts the outer membrane integrity by binding to the β-taco domain of the LptD protein, leading to leakage of cell contents. In a ready-to-eat salmon model, immersion treatment with 64 μg / mL of the antimicrobial peptide of this invention for 4 hours reduced the viral load of artificially inoculated Vibrio parahaemolyticus by 1.51 log CFU / mL, while significantly inhibiting biofilm regeneration during storage. Safety experiments confirmed that the antimicrobial peptide provided by this invention has no effect on the survival rate of HT29 cells (>95%) at food application concentrations (50 μg / mL to 1000 μg / mL), providing an efficient and safe solution for targeted control of Vibrio parahaemolyticus contamination in seafood. Attached Figure Description

[0016] Figure 1 The results show the scavenging and preventive effects of the antimicrobial peptide VPneco of this invention on the biofilm of Vibrio parahaemolyticus, where A represents the scavenging result and B represents the preventive result, and ns represents p≥0.05, * represents p<0.05, ** represents p<0.01, and **** represents p<0.0001.

[0017] Figure 2 The scanning electron microscope results show the disruptive effect of the antimicrobial peptide VPneco of this invention on the outer membrane of Vibrio parahaemolyticus.

[0018] Figure 3 The results show the effect of the antimicrobial peptide VPneco of the present invention on the membrane permeability of Vibrio parahaemolyticus, where A represents the outer membrane result and B represents the inner membrane result, and **** indicates p < 0.0001;

[0019] Figure 4This invention describes the bactericidal effect of the antimicrobial peptide VPneco on Vibrio parahaemolyticus in salmon, where ns indicates p≥0.05 and **** indicates p<0.0001. Detailed Implementation

[0020] This invention provides an antimicrobial peptide that targets the outer membrane protein LptD of Vibrio parahaemolyticus, wherein the amino acid sequence of the antimicrobial peptide is ASKKPVPIIYCNRRTRRGKCQRM (SEQ ID NO.1).

[0021] In this invention, the antimicrobial peptide with the amino acid sequence shown in SEQ ID NO.1 is named VPneco. Based on the NMR structure of thanatin (PDB ID: 5XO4), this invention employs a phenotypic screening combined with a targeted focusing strategy. While maintaining the β-hairpin conformation (Cys11-Cys18 disulfide bonds), the N-terminal glycine (G) is replaced with alanine (A) to enhance hydrophobicity and promote interaction with bacterial outer membrane phospholipids. Furthermore, a C-terminal arginine (R) cluster is introduced: an R16-R17 dual is inserted into the C-terminal ring (GKCQRM) to promote competitive substitution with divalent cations in the bacterial outer membrane, disrupting membrane stability. This ultimately yields the antimicrobial peptide VPneco of this invention, achieving significant technical effects of low toxicity and high efficiency.

[0022] The present invention also provides the application of the above-mentioned antimicrobial peptides in any of the following: (1) preparing products for the prevention and / or treatment of Vibrio parahaemolyticus infection; (2) preparing antibacterial agents or bactericides; (3) preventing and controlling Vibrio parahaemolyticus contamination in food; (4) removing Vibrio parahaemolyticus biofilm.

[0023] In this invention, the bacteria in the antibacterial agent or bactericide preferably include Vibrio parahaemolyticus. The concentration of the antimicrobial peptide in the antibacterial agent or bactericide is preferably 64 μg / mL or higher. In this invention, the food preferably includes seafood, and the seafood preferably includes salmon. The antimicrobial peptide of this invention has an inhibition rate of 21.24% against Vibrio parahaemolyticus biofilm; the biofilm formation rate of Vibrio parahaemolyticus is significantly reduced after treatment with the antimicrobial peptide of this invention, demonstrating a good preventive effect against Vibrio parahaemolyticus biofilm. The antimicrobial peptide provided by this invention has a significant effect on the cell membrane of Vibrio parahaemolyticus, effectively increasing its outer membrane permeability, while also penetrating the outer membrane and causing damage to the inner membrane structure, increasing inner membrane permeability and exacerbating cell damage. The antimicrobial peptide provided by this invention is non-cytotoxic and has high safety.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, the following embodiments are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1

[0028] (1) The antimicrobial peptide VPneco with the amino acid sequence ASKKPVPIIYCNRRTRRGKCQRM (SEQ ID NO.1) was mixed with sterile double-distilled water to prepare a VPneco stock solution with a concentration of 1000 μg / mL. The VPneco stock solution was dissolved in sterile double-distilled water and filtered through a 0.22 μm filter membrane. Subsequently, a concentration gradient (128 μg / mL to 0.125 μg / mL) was constructed in a 96-well plate using a two-fold serial dilution method and stored at -20℃ for later use.

[0029] (2) After activating Vibrio parahaemolyticus with TSB broth containing 3% NaCl, streak culture was performed on TCBS agar plates. Single colonies were picked and inoculated into TSB broth containing 3% NaCl. The mixture was then cultured at 37°C with shaking for 10-12 hours to obtain the inoculum solution for later use.

[0030] (3) Centrifuge the inoculum from step (2) and adjust the bacterial concentration to 9 log CFU / mL (equivalent to 10). 9 The bacterial culture was diluted to 6 log CFU / mL with 0.85% NaCl. 20 μL of the diluted bacterial culture was added to each well of a well-dissolved 96-well plate (obtained in step (1)). The OD was then measured. 600nm After no changes were observed, the 96-well plate was placed in a 37°C incubator. After 20 hours, the 96-well plate was removed, and the OD was measured using a microplate reader. 600nm By recording the experimental results, the minimum inhibitory concentration (MIC) value can be obtained.

[0031] The micro-broth dilution method showed that the MIC of the antimicrobial peptide VPneco of the present invention against Vibrio parahaemolyticus was 64 μg / mL, indicating that VPneco has good antimicrobial activity against Vibrio parahaemolyticus.

[0032] Example 2

[0033] The antimicrobial peptide VPneco of this invention exhibits scavenging and preventative effects against Vibrio parahaemolyticus biofilms.

[0034] Centrifuge the inoculum obtained in step (2) of Example 1, adjust the concentration to 9 log CFU / mL, place it in a 96-well plate, and incubate at 25°C for 24 h. Wash the cultured biofilm three times with 150 μL 1×PBS, then add 100 μL 1 / 2 MIC (32 μg / mL) VPneco. Use 1×PBS as a control and 100 μL 1 / 2 MIC (Thanatin MIC is 128 μg / mL, 1 / 2 MIC is 64 μg / mL) Thanatin as a positive control. After treatment at 37°C for 2 h, aspirate the inoculum, add 150 μL 0.1% (w / v) crystal violet and stain for 10 min. Wash three times with 1×PBS, then dissolve in 150 μL 95% (v / v) ethanol. Finally, transfer the dissolved liquid to a new 96-well plate and measure the OD using a microplate reader. 595 value.

[0035] Crystal violet staining assays showed that VPneco cleared 21.24% of Vibrio parahaemolyticus biofilm, while thanatin cleared 14.99% (see [link to assay]). Figure 1 In A), the biofilm density of Vibrio parahaemolyticus was significantly reduced after VPneco treatment.

[0036] The bacterial culture was diluted to 6 log CFU / mL using 0.85% NaCl. 95 μL of the diluted solution was then placed in a 96-well plate, and 1 / 2 MIC (32 μg / mL) of VPneco was added. The plate was incubated at 25°C for 24 h. 1×PBS was used as a control, and 100 μL of 1 / 2 MIC (64 μg / mL) of Thanatin was used as a positive control. The cultured biofilm was washed three times with 150 μL of 1×PBS, followed by staining with 150 μL of 0.1% (w / v) crystal violet for 10 min.

[0037] Crystal violet staining experiments showed that VPneco had a significant preventive effect against Vibrio parahaemolyticus biofilm formation. The biofilm formation in groups supplemented with the antimicrobial peptide VPneco of this invention was significantly reduced, reaching 39.40% (see...). Figure 1 In the B group, the biofilm thickness of Vibrio parahaemolyticus was significantly reduced after VPneco treatment.

[0038] Example 3

[0039] Effects of VPneco on Vibrio parahaemolyticus cell morphology

[0040] Centrifuge the inoculum obtained in step (2) of Example 1, adjust the concentration to 9 log CFU / mL, dilute the bacterial solution to 6 log CFU / mL with 0.85% NaCl, add 1 / 2 MIC (32 μg / mL) of VPneco, using the control group without VPneco. Incubate at 37°C on a shaker for 2 h, wash with 1 mL of 1×PBS buffer, add 1 mL of 2.5% glutaraldehyde, fix for 30 min, then wash three times with 1 mL of 1×PBS buffer, then dehydrate with 1 mL of 30%, 50%, 70%, 90%, and 100% ethanol for 10 min each time, and finally dehydrate with 100% ethanol for 10 min. Dry the treated coverslips, treat with gold-palladium coating, and observe under a scanning electron microscope (SEM).

[0041] The results are as follows Figure 2 As shown, the surface of Vibrio parahaemolyticus cells treated with VPneco exhibits significant depressions and wrinkles, and the cell membrane ruptures, leading to leakage of contents, indicating that the integrity of its outer membrane has been compromised.

[0042] Example 4

[0043] Effect of VPneco treatment on Vibrio parahaemolyticus membrane permeability

[0044] 1-N-Phenylenaphthylamine (NPN) staining: The prepared Vibrio parahaemolyticus bacterial solution (the inoculum obtained in step (2) of Example 1) was washed twice with 5 mM HEPES. The bacterial solution concentration was adjusted to 9 log CFU / mL. The adjusted bacterial solution and VPneco were added to a centrifuge tube at a ratio of 1:1. 1×PBS was used as a control. The solution was incubated at 37°C for 2 h. The bacterial solution was then collected, washed with 5 mM HEPES and resuspended. Then, 10 μM NPN solution was added and the solution was incubated at 25°C in the dark for 30 min. The excitation wavelength and emission wavelength were set to 350 nm and 420 nm, respectively, on the microplate reader to detect the fluorescence intensity.

[0045] NPN is a hydrophobic fluorescent dye that is strongly repelled by intact bacterial outer membranes. Only when the bacterial outer membrane is disrupted can the dye gradually penetrate the cell, increasing its fluorescence intensity. The results are as follows... Figure 3 As shown in A, compared with the control group, VPneco treatment can significantly enhance fluorescence intensity, thus achieving the effect of disrupting the cell outer membrane.

[0046] Propidium iodide (PI) is a non-membrane-permeable fluorescent dye that cannot pass through the cell membrane of living cells. It can only stain nucleic acids in cells when the cell membrane is damaged. PI dye was used to perform an inner membrane permeability experiment: The prepared Vibrio parahaemolyticus bacterial solution (the inoculum obtained in step (2) of Example 1) was washed twice with 5 mM HEPES. The bacterial solution concentration was adjusted to 9 log CFU / mL. The adjusted bacterial solution and VPneco were added to a centrifuge tube at a 1:1 ratio. 1×PBS was used as a control. The solution was incubated at 37°C for 2 h. The bacterial solution was then collected, washed with 5 mM HEPES and resuspended. Then, 10 μM PI solution was added and incubated at 37°C in the dark for 20 min. The excitation wavelength and emission wavelength were set to 535 nm and 615 nm, respectively, on the microplate reader to detect the fluorescence intensity.

[0047] The results are as follows Figure 3 As shown in B, compared with the negative control group, VPneco treatment significantly enhanced the fluorescence intensity, indicating that VPneco can penetrate the outer membrane of Vibrio parahaemolyticus and cause damage to the inner membrane structure, thereby increasing the permeability of the inner membrane and aggravating cell damage.

[0048] Example 5

[0049] Cytotoxicity assay

[0050] The toxic effects of different concentrations of VPneco on mouse monocyte / macrophage RAW264.7 and human colon cancer cell line HT29 were detected using the CCK8 assay. The specific experimental procedures are as follows: The test samples were dissolved in complete culture medium to concentrations of 50, 100, 200, 400, and 1000 μg / mL, respectively, and sterilized by 0.22 μm filtration. RAW264.7 and HT29 cell suspensions, resuspended in complete culture medium (89% DMEM + 10% FBS + 1% P / S) after two or more passages of adequately adapted cells, were seeded into 96-well plates at a rate of 0.1 mL per well. An equal volume of cells was seeded in the control group (no sample group), and an equal volume of culture medium was seeded in the blank group (no cells and sample group). PBS was added to the edge wells, and the cells were cultured overnight. The original culture medium was removed, and the corresponding concentrations of VPneco (50, 100, 200, 400, and 1000 μg / mL) were added, and the cells were cultured for another 24 h. Remove the original culture medium and add 100 μL of CCK8 working solution (90% DMEM + 10% CCK8 detection solution). Use cell-free wells as blanks. After incubation for an appropriate time, measure the absorbance at 450 nm using a microplate reader. Based on the absorbance data, calculate the cell viability for each group and the differences between groups using the following formula:

[0051]

[0052] The results showed that VPneco at all concentrations did not inhibit the viability of HT29 and RAW254.7 cells.

[0053] Example 6

[0054] Determination of antibacterial activity of Vibrio parahaemolyticus in salmon artificially inoculated with Vibrio parahaemolyticus

[0055] Vibrio parahaemolyticus was resuspended in 0.85% NaCl and diluted to 8 log CFU / mL for later use. Salmon, susceptible to Vibrio parahaemolyticus contamination, was selected as the actual sample. The salmon was washed three times with deionized water. In a laminar flow hood, the sample was cut into 2×2×1 cm pieces using a sterile microtome and placed in a sterile petri dish. Both sides of the petri dish were sterilized with UV light for 30 min. The sample was then completely immersed in the prepared bacterial suspension. After the bacterial suspension was fully incorporated into the sample, the sample was transferred to a sterile homogenizing bag and soaked in 64 μg / mL VPneco solution. An equal volume of 1×PBS was added to the control group. The samples were treated at room temperature (~10℃) for 0 h, 0.25 h, 0.5 h, 1 h, 2 h, and 4 h. Subsequently, the samples were homogenized using a homogenizer, serially diluted with 0.85% NaCl, and plate counts were performed.

[0056] The results are as follows Figure 4 As shown, compared with the control group, after 1 hour of treatment with VPneco, the number of Vibrio parahaemolyticus in salmon decreased from 6.11 log CFU / mL to 5.54 log CFU / mL; after 4 hours of treatment, the number of Vibrio parahaemolyticus in salmon decreased to 4.76 log CFU / mL, while the number of Vibrio parahaemolyticus in the control group was 6.27 log CFU / mL. This indicates that the antimicrobial peptide VPneco provided by this invention can effectively inhibit the growth of Vibrio parahaemolyticus in salmon.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide targeting the outer membrane protein LptD of Vibrio parahaemolyticus, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide of claim 1 in the preparation of products for the prevention and / or treatment of Vibrio parahaemolyticus infection.

3. The application of the antimicrobial peptide according to claim 1 in the preparation of antibacterial agents or bactericides, characterized in that, Bacteria in bacteriostatic agents or bactericides include Vibrio parahaemolyticus.

4. The application according to claim 3, characterized in that, The concentration of the antimicrobial peptide in the bacteriostatic agent or bactericide is 64 μg / mL or higher.

5. The application of the antimicrobial peptide according to claim 1 in preventing and controlling Vibrio parahaemolyticus contamination in food.

6. The application according to claim 5, characterized in that, The food products mentioned include seafood.

7. The application according to claim 6, characterized in that, The seafood includes salmon.

8. The use of the antimicrobial peptide of claim 1 in clearing biofilms of Vibrio parahaemolyticus.

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