Antibacterial peptide Z34B3 and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide Z34B3 and application thereof. The amino acid sequence of the antibacterial peptide Z34B3 provided by the invention is shown as SEQ ID NO: 1, the secondary structure is an alpha helix, the molecular weight is small, and the antibacterial peptide Z34B3 can change the cell membrane permeability of various gram-negative bacteria including pasteurella multocida, escherichia coli and acinetobacter baumannii, inhibit the generation of cell membranes and play antibacterial and bactericidal roles. Moreover, the antibacterial peptide Z34B3 provided by the invention has low hemolytic activity and no cytotoxicity, and can be applied to preparation of products with bacteriostatic and bactericidal effects and preparation of drugs for preventing and / or treating bacterial infectious diseases. Furthermore, the C tail end of the antibacterial peptide Z34B3 is subjected to amidation modification, and compared with non-modification treatment, the stability can be improved.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide Z34B3 and its applications. Background Technology

[0002] Pasteurella multocida ( Pasteurella multocida Pasteurella multocida is a Gram-negative bacterium that can infect various animals such as poultry, pigs, cattle, and sheep. In cattle and sheep, infection with Pasteurella multocida usually manifests as hemorrhagic septicemia. In poultry, infection can cause fowl cholera, and in pigs, infection can cause swine pneumonia, making it one of the pathogens that endanger the livestock industry.

[0003] Antimicrobial peptides (AMPs) are a class of small-molecule polypeptides that inhibit the growth of bacteria, fungi, and viruses. They are widely distributed in organisms and are an important component of the innate immune system. Antimicrobial peptides typically possess a net positive charge and an amphiphilic α-helix structure. This structure allows them to target and bind to the negatively charged bacterial plasma membrane. Furthermore, the amphiphilic nature promotes the interaction between the antimicrobial peptide and the bacterial plasma membrane, facilitating penetration and disruption of the bacterial cell membrane, ultimately leading to bacterial death. Antimicrobial peptides are not only less likely to induce bacterial resistance but also exhibit advantages such as broad-spectrum antibacterial activity, high efficacy with minimal dosage, and immunomodulation. However, existing antimicrobial peptide development primarily targets Staphylococcus aureus, Escherichia coli, and various clinically resistant bacteria, with very little research on antimicrobial peptides targeting Pasteurella multocida. Moreover, not all small-molecule polypeptides possess antimicrobial activity, and even those with antimicrobial activity face many limitations in practical production and application. For example, instability in practical applications, along with unknown hemolytic activity and cytotoxicity, severely limit the development of antimicrobial peptides. Therefore, it is necessary to artificially design and synthesize new antimicrobial peptides to inhibit Pasteurella multocida and develop low-toxicity, highly effective antimicrobial drugs. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide Z34B3 and its application, which has good bactericidal and bacteriostatic effects on Gram-negative bacteria, including Pasteurella multocida, alters bacterial cell membrane permeability, inhibits bacterial biofilm formation, and does not have hemolytic activity or cytotoxicity, thus exhibiting good safety.

[0005] This invention provides an antimicrobial peptide Z34B3, the amino acid sequence of which is shown in SEQ ID NO:1.

[0006] Preferably, the C-terminus of the antimicrobial peptide Z34B3 is modified by amidation.

[0007] The present invention also provides the application of the antimicrobial peptide Z34B3 described in the above technical solution in one or more of the following: (1) Preparation of bactericides; (2) Preparation of bacteriostatic agents; (3) Preparation of drugs for preventing bacterial infectious diseases; (4) Preparation of drugs for treating bacterial infectious diseases; (5) Preparation of anti-biofilm drugs.

[0008] Preferably, the bactericide and / or bacteriostatic agent targets Gram-negative bacteria; The bacteria in question are Gram-negative bacteria; The anti-biofilm drug is used to inhibit the formation of biofilms by Gram-negative bacteria.

[0009] Preferably, the Gram-negative bacteria include Pasteurella multocida (Pasteurella multocida). Pasteurella multocida ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter baumannii One or more of the following.

[0010] This invention provides a product with bactericidal and / or bacteriostatic functions, wherein the active ingredient of the product includes the antimicrobial peptide Z34B3 described in the above technical solution.

[0011] Preferably, the concentration of antimicrobial peptide Z34B3 in the product is ≥0.9375μM.

[0012] Preferably, the product is a medicine or a skin care product.

[0013] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0014] Preferably, the skincare product also includes excipients acceptable in the cosmetics field.

[0015] Beneficial effects: This invention provides an antimicrobial peptide Z34B3, the amino acid sequence of which is shown in SEQ ID NO:1. The antimicrobial peptide Z34B3 provided by this invention has an α-helix secondary structure, a small molecular weight, and can alter the cell membrane permeability of various Gram-negative bacteria, including Pasteurella multocida, Escherichia coli, and Acinetobacter baumannii, and inhibit cell membrane formation, thereby exerting antibacterial and bactericidal effects. Furthermore, the antimicrobial peptide Z34B3 provided by this invention has low hemolytic activity and no cytotoxicity, and can be used in the preparation of products with antibacterial and bactericidal effects, and in the preparation of drugs for the prevention and / or treatment of bacterial infectious diseases.

[0016] Furthermore, the present invention performs amidation modification on the C-terminus of the antimicrobial peptide Z34B3, which improves its stability compared to the unmodified form. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The kinetic curve of the peptide Z34B3 against Pasteurella multocida ATCC 43137 is shown. Figure 2 The results show the cytotoxicity of peptide Z34B3 on human immortalized keratinocytes HaCat. Figure 3 The results show the cytotoxicity of peptide Z34B3 against mouse fibroblast L929 cells. Figure 4 The hemolytic activity of polypeptide Z34B3 against human erythrocytes; Figure 5 The effect of peptide Z34B3 on the outer membrane permeability of Pasteurella multocida ATCC 43137 cells; Figure 6 The inhibitory effect of polypeptide Z34B3 on the biofilm of Pasteurella multocida ATCC 43137; Where NS indicates no significant difference express P <0.005; express P <0.01; express P＜ 0.0001. Detailed Implementation

[0019] This invention provides an antimicrobial peptide Z34B3, the amino acid sequence of which is shown in SEQ ID NO:1, specifically: GLFGRLRDSLRRRIKDIFRG.

[0020] As one embodiment, the C-terminus of the antimicrobial peptide Z34B3 of the present invention is modified by amidation. Compared with unmodified treatment, the amidation modification of the C-terminus of the antimicrobial peptide Z34B3 of the present invention has the advantage of improving the stability of the antimicrobial peptide.

[0021] The present invention also provides the application of the antimicrobial peptide Z34B3 described in the above technical solution in one or more of the following: (1) preparation of bactericides; (2) preparation of bacteriostatic agents; (3) preparation of drugs for preventing bacterial infectious diseases; (4) preparation of drugs for treating bacterial infectious diseases; (5) preparation of antibiofilm drugs.

[0022] In one embodiment, the bactericide and / or bacteriostatic agent of the present invention targets Gram-negative bacteria. In one embodiment, the bacteria of the present invention are Gram-negative bacteria. In one embodiment, the anti-biofilm drug of the present invention is used to inhibit the formation of biofilms by Gram-negative bacteria.

[0023] In one embodiment, the Gram-negative bacteria of the present invention include one or more of Pasteurella multocida, Escherichia coli, and Acinetobacter baumannii. In one embodiment, the Pasteurella multocida of the present invention includes one or more of Pasteurella multocida ATCC 43137, Pasteurella multocida HB03, and Pasteurella multocida HN05. In one embodiment, the Escherichia coli of the present invention includes Escherichia coli ATCC 25922. In one embodiment, the Acinetobacter baumannii of the present invention includes Acinetobacter baumannii ATCC 19606. The Pasteurella multocida HB03 and Pasteurella multocida HN05 of the present invention are disclosed in the prior art: Peng Zhong. Isolation, Identification, and Whole Genome Resequencing of Pasteurella multocida in Porcines [D]. Huazhong Agricultural University, 2018.

[0024] This invention provides a product with bactericidal and / or bacteriostatic functions, wherein the active ingredient of the product includes the antimicrobial peptide Z34B3 described in the above technical solution.

[0025] In one embodiment, the concentration of antimicrobial peptide Z34B3 in the product of this invention is ≥0.9375 μM. In another embodiment, the product of this invention is a pharmaceutical or skincare product. In one embodiment, the pharmaceutical product of this invention further includes pharmaceutically acceptable excipients. In another embodiment, the skincare product of this invention further includes cosmetically acceptable excipients. This invention does not impose strict requirements on the type of excipients; they can be conventionally selected according to the dosage form of the pharmaceutical product.

[0026] To further illustrate the present invention, the following detailed description of an antimicrobial peptide Z34B3 and its applications, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 The polypeptide sequence described in SEQ ID NO:1 (GLFGRLRDSLRRRIKDIFRG) was synthesized artificially, and its carboxyl terminus was amidated, denoted as Z34B3. The polypeptide Z34B3 has a molecular weight of 2430.85 Da, an isoelectric point (pI) of 12.151, and a secondary structure of α-helix.

[0028] Example 2 1. Test strain Pasteurella multocida ATCC 43137, Pasteurella multocida HB03, Pasteurella multocida HN05, Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606.

[0029] 2. Determination of minimum inhibitory concentration (MIC) (1) The activated test strain was inoculated into liquid culture medium and cultured in a constant temperature shaker until the logarithmic growth phase. The bacterial concentration was measured by diluting the culture with fresh sterile liquid culture medium to 2 × 10⁻⁶. 6 (2) Take a sterile transparent 96-well plate, add 180 μL of fresh sterile liquid culture medium to the first column of wells, and add 100 µL of fresh sterile liquid culture medium to the remaining wells. Add 20 µL of the test sample solution diluted to a certain concentration and filtered through a 0.22 µm filter membrane to the first column of wells (the test samples in rows A, B, and C are polypeptide Z34B3 obtained in Example 1, and the test samples in rows D, E, and F are norfloxacin). (3) After pipetting and mixing evenly, add 100 µL to the second column of wells, and dilute sequentially. When the last well is aspirated, discard the 100 µL of sterile liquid. (4) Add 100 µL of the pre-diluted test strain solution to each well. At this time, the sample concentration in the first column is the highest (the sample concentration in the first well is 80 µM), and the concentrations in the remaining wells are half of the previous column. (5) The 96-well plate was placed in a constant temperature incubator at 37℃ and incubated statically. The average concentration of the sterile growth well and the previous well was taken as the minimum inhibitory concentration. The results are shown in Table 1.

[0030] Table 1. Results of minimum inhibitory concentration (μM) detection

[0031] 3. Minimum bactericidal concentration (MBC) determination (1) Take 50 µL of culture medium from each of the wells with the MIC concentration measured in step 2, as well as the wells with concentrations below MIC (1 / 2MIC, MIC, 2×MIC, 4×MIC). (2) Spread these culture media onto fresh blood agar plates (purchased from Huankai Biotechnology, catalog number 024070) without drugs, and incubate the plates at 37℃ for 24 h. (3) Observe the number of colonies on the plates. The lowest drug concentration that kills 99.9% of bacteria is defined as MBC, and the results are shown in Table 2.

[0032] Table 2. Minimum bactericidal concentration test results (μM)

[0033] The results, as shown in Table 1, indicate that the minimum inhibitory concentrations (MICs) of peptide Z34B3 against *Pasteurella multocida* ATCC 43137, *Pasteurella multocida* HB03, *Pasteurella multocida* HN05, *Escherichia coli* ATCC 25922, and *Acinetobacter baumannii* ATCC 19606 were 0.9375 μM, 1.8750 μM, 0.9375 μM, 1.8750 μM, and 1.8750 μM, respectively. Table 2 shows that the MICs of peptide Z34B3 against *Pasteurella multocida* ATCC 43137, *Pasteurella multocida* HB03, and *Pasteurella multocida* HN05 were 0.9375 μM, 1.8750 μM, and 1.8750 μM, respectively. The peptide Z34B3 obtained in this invention possesses both bactericidal and bacteriostatic activities and is an antimicrobial peptide.

[0034] Example 3 kinetics of the bactericidal activity of peptide Z34B3 Pasteurella multocida ATCC 43137 was used as the test strain, and the polypeptide Z34B3 obtained in Example 1 was used as the test polypeptide. The positive control was norfloxacin at the same concentration, and the negative control was physiological saline.

[0035] Use an inoculation loop to pick a single colony and inoculate it into liquid culture medium. Incubate in a shaking incubator (37℃, 220 rpm) until the logarithmic growth phase. Dilute the bacterial culture to 2×10⁻⁶ with fresh liquid culture medium. 6 CFU / mL, add peptide Z34B3 / positive control to the diluted bacterial solution to a final concentration of 1×MIC and 2×MIC. For the negative control, add the corresponding volume of sterile ultrapure water. Immediately place the bacterial solution containing the sample in a 37℃ incubator. At 0 min, 1 min, 10 min, 30 min, 60 min, and 120 min, take 10 μL of the bacterial solution and dilute it with sterile physiological saline to 1×10⁻⁶. 3 Take 50 μL of the culture medium and spread it onto a solid culture plate. Then, incubate the plate upside down at 37°C for 16 h and count the colonies. Perform three replicates for each group and calculate the mean ± SEM of the experimental results.

[0036] The results are as follows Figure 1 As shown, compared with the positive control drug norfloxacin, which cannot completely kill bacteria within 2 hours, the bactericidal kinetic curve of peptide Z34B3 can efficiently and thoroughly kill bacteria within 1 hour.

[0037] Example 4 Peptide Z34B3 cytotoxicity assay The toxicity of peptide Z34B3 from Example 1 to human immortalized keratinocytes (HaCat) and mouse fibroblasts (L929) was determined using the CCK-8 colorimetric method. The specific steps were as follows: Cell suspension was prepared, cells were counted using a counting chamber, and the suspension was diluted with DMEM cell culture medium to a final concentration of 2 × 10⁻⁶. 4 Cells / mL. The peptide Z34B3 was then diluted to different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC), with 5 replicates per group. After cell adhesion, different concentrations of Z34B3 dilutions were added. After cell interaction, 110 µL of 10% CCK8 solution (i.e., CCK8 to culture medium volume ratio of 1:10) was added to each well of a 96-well plate, and the plate was incubated for another 4 h. The absorbance at OD 450 nm was measured using a microplate reader.

[0038] The results are as follows Figure 2 and Figure 3 As shown, at 2×MIC, the survival rate of both cell types was above 80%, indicating that peptide Z34B3 has a certain degree of safety.

[0039] Example 5 Hemolysis assay of peptide Z34B3 Human red blood cells were used. The red blood cells were washed with PBS by centrifugation until the supernatant was clear and colorless. The cells were then diluted and resuspended in PBS to a 4% (v / v) red blood cell suspension. 500 μL of the red blood cell solution was transferred to a 1.5 mL centrifuge tube, and 500 μL of different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC) of the peptide Z34B3 from Example 1 were added. PBS was used as the negative control, and 1% (v / v) Triton-100 was used as the positive control. After mixing, the mixture was incubated at 37°C and 220 rpm for 1 h. The red blood cell pellet was obtained by centrifugation. The absorbance at 540 nm was measured using the supernatant, and the hemolysis rate was calculated using the formula: Hemolysis rate (%) = (Experimental group OD540 - Negative control group OD540) / (Positive control group OD540 - Negative control group OD540) × 100%.

[0040] The results are as follows Figure 4 As shown, the hemolytic activity was consistently less than 1% at 16×MIC, indicating that peptide Z34B3 has a certain degree of safety.

[0041] Example 6 Experiment on outer membrane permeability of peptide Z34B3 bacteria Colonies of Pasteurella multocida ATCC 43137 were transferred to BHI liquid medium and incubated at 37°C with shaking until the logarithmic growth phase. The culture was then washed three times with 5 mM HEPES buffer and diluted to OD600 = 0.5. The diluted bacterial solution was incubated with 1-phenylnaphthylamine (NPN) dissolved in anhydrous ethanol for 30 min to a final NPN concentration of 10 μM. Background fluorescence was recorded and subtracted using a full-wavelength microplate reader at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. In sterile black 96-well microtiter plates, peptide Z34B3 from Example 1 was added to the bacterial suspension to final concentrations of 1×MIC, 2×MIC, 4×MIC, and 8×MIC (FI). peptide Fluorescence intensity was recorded over time to detect changes in bacterial outer membrane permeability. The negative control was PBS (FI0), and the positive control was polymyxin B (0.2 mg / mL) (FI0). 100 Penetration rate (%) = (FIpeptide - FI0) / (FI 100 -FI0)×100%.

[0042] The results are as follows Figure 5 As shown, as the concentration gradually increases, the permeability of peptide Z34B3 also gradually increases, and is higher than that of the positive control. This indicates that peptide Z34B3 uses the disruption of bacterial outer membrane permeability as one of its bactericidal mechanisms, and this effect is reflected in the concentration.

[0043] Example 7 Biomembrane inhibition function of peptide Z34B3 Pasteurella multocida ATCC 43137 was cultured overnight at 37°C and 220 rpm. The overnight culture was then used for transfer. Once the bacteria reached the logarithmic growth phase, they were diluted with fresh culture medium to a concentration of 2 × 10⁻⁶. 7 CFU / mL. 100 μL of bacterial culture was added to 96-well plates containing 100 μL of antimicrobial solutions (Example 1 peptide Z34B3 and norfloxacin) at different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC) and incubated at 37°C for 24 h. The positive control (PC) consisted of bacterial culture with sterile water, and the negative control (NC) consisted of culture medium with sterile water. The plates were incubated at 37°C for 48 h. The bacterial culture was aspirated, and floating bacteria were washed away with physiological saline. After incubation for 24 h, the solution was aspirated, the bacterial membrane was dried and fixed, and treated with crystal violet staining solution for 20 min. The staining solution was aspirated, the membrane was washed and dried, and 200 µL of anhydrous ethanol was added and allowed to stand for 20 min to completely dissolve the dye. The OD value was measured at 595 nm. The percentage of remaining biofilm after treatment with the antimicrobial peptide and positive control was calculated as: (Measured value - Blank group) / (Positive group - Blank group) × 100%. The plates were treated in the same manner as described above, and the inhibitory effect on biofilm formation was calculated.

[0044] The results are as follows Figure 6 As shown, the inhibitory effect of peptide Z34B3 on biofilm gradually increases with increasing concentration, indicating that peptide Z34B3 uses the inhibition of bacterial biofilm formation as one of its bactericidal mechanisms, and this effect is concentration-dependent.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide Z34B3, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The antimicrobial peptide Z34B3 according to claim 1, characterized in that, The C-terminus of the antimicrobial peptide Z34B3 is modified by amidation.

3. The use of the antimicrobial peptide Z34B3 according to claim 1 or 2 in one or more of the following: (1) Preparation of bactericides; (2) Preparation of bacteriostatic agents; (3) Preparation of drugs for preventing bacterial infectious diseases; (4) Preparation of drugs for treating bacterial infectious diseases; (5) Preparation of anti-biofilm drugs.

4. The application according to claim 3, characterized in that, The bactericides and / or bacteriostatic agents target Gram-negative bacteria; The bacteria in question are Gram-negative bacteria; The anti-biofilm drug is used to inhibit the formation of biofilms by Gram-negative bacteria.

5. The application according to claim 4, characterized in that, The Gram-negative bacteria include Pasteurella multocida (…). Pasteurella multocida ), Escherichia coli ( Escherichia coli ) and Acinetobacter baumannii ( Acinetobacter baumannii One or more of the following.

6. A product having bactericidal and / or bacteriostatic functions, characterized in that, The active ingredient of the product includes the antimicrobial peptide Z34B3 as described in claim 1 or 2.

7. The product according to claim 6, characterized in that, The concentration of antimicrobial peptide Z34B3 in the product is ≥0.9375μM.

8. The product according to claim 6 or 7, characterized in that, The product in question is a medicine or a skincare product.

9. The product according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The product according to claim 8, characterized in that, The skincare products also include excipients acceptable in the cosmetics field.

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