Antibacterial peptide MAP34B-2 and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide MAP34B-2 and application thereof. The amino acid sequence of the antibacterial peptide MAP34B-2 provided by the invention is as shown in SEQ ID NO: 1, and the C terminal of the antibacterial peptide MAP34B-2 can be subjected to amidation modification. The antibacterial peptide MAP34B-2 provided by the invention is small in molecular weight, can change the permeability of cell membranes of escherichia coli and acinetobacter baumannii and inhibit the generation of the cell membranes, and plays a role in inhibiting and killing bacteria; moreover, the antibacterial peptide MAP34B-2 provided by the invention is low in hemolytic activity and free of cytotoxicity, can be applied to preparation of products with antibacterial and bactericidal effects, and can be developed into antibacterial peptide antibacterial bactericides and medicines for preventing and / or treating bacterial infectious diseases.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide MAP34B-2 and its applications. Background Technology

[0002] Escherichia coli ( Escherichia coli Bacterium tumefaciens (BLT) is a Gram-negative, flagellated, motile facultative anaerobic bacterium. It is also an opportunistic pathogen, primarily transmitted through human-to-human and animal-to-human contact, fecal-oral transmission, and contaminated water and food, causing peritonitis, appendicitis, and pneumonia. The advent of antibiotics has improved the prognosis of patients with bacterial infections and reduced mortality from bacterial infections. However, with the overuse of antibiotics, coupled with bacterial evolution and selection, bacteria have gradually developed antibiotic resistance. To address the increasingly serious problem of bacterial resistance, the development of new antibiotics or alternative drugs is necessary.

[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 not only do not readily induce bacterial resistance but also exhibit broad-spectrum antibacterial activity, high efficacy with minimal dosage, and immunomodulatory effects. However, not all small-molecule polypeptides possess antimicrobial activity, and even those with antimicrobial activity face numerous limitations in practical production and application. For example, instability in practical applications, unknown hemolytic activity, and cytotoxicity severely restrict the development of antimicrobial peptides. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide MAP34B-2 and its application, which has good bactericidal and bacteriostatic effects on Gram-negative bacteria, including Escherichia coli and Acinetobacter baumannii, thereby damaging bacterial growth and cell membrane synthesis.

[0005] This invention provides an antimicrobial peptide MAP34B-2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0006] Preferably, the C-terminus of the antimicrobial peptide MAP34B-2 is modified by amidation.

[0007] This invention provides the application of the antimicrobial peptide MAP34B-2 described above in the preparation of bactericides and / or bacteriostatic agents.

[0008] Preferably, the bactericide and / or bacteriostatic agent is targeted at Gram-negative bacteria.

[0009] Preferably, the Gram-negative bacteria include Escherichia coli (Escherichia coli). Escherichia coli ) and / or Acinetobacter baumannii ( Acinetobacter baumnnii ).

[0010] This invention provides the use of the antimicrobial peptide MAP34B-2 described above in the preparation of medicaments for the prevention and / or treatment of bacterial infectious diseases.

[0011] Preferably, the bacteria include Gram-negative bacteria.

[0012] Preferably, the Gram-negative bacteria include Escherichia coli and / or Acinetobacter baumannii.

[0013] This invention provides a bactericidal and / or bacteriostatic product, the active ingredient of which includes the antimicrobial peptide MAP34B-2 described in the above-mentioned scheme.

[0014] Preferably, the active ingredient further includes other antibacterial and bactericidal components; the other antibacterial and bactericidal components include antibiotics.

[0015] Beneficial effects: The antimicrobial peptide MAP34B-2 provided by this invention has a small molecular weight, which can alter the permeability of the cell membranes of *Escherichia coli* and *Acinetobacter baumannii* and inhibit cell membrane formation, thereby exerting antibacterial and bactericidal effects. Furthermore, the antimicrobial peptide MAP34B-2 provided by this invention has low hemolytic activity and no cytotoxicity, and can be applied to the preparation of products with antibacterial and bactericidal effects, developing it into an antimicrobial peptide antimicrobial and bactericidal agent, and preparing drugs for the prevention and / or treatment of bacterial infectious diseases. Attached Figure Description

[0016] 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.

[0017] Figure 1 The results of cytotoxicity assay of antimicrobial peptide MAP34B-2 on human immortalized keratinocytes Hacat are presented. Figure 2 The results show the cytotoxicity of the antimicrobial peptide MAP34B-2 against mouse fibroblast L929 cells. Figure 3 The antimicrobial peptide MAP34B-2 exhibits hemolytic activity against human erythrocytes; Figure 4 The effect of the antimicrobial peptide MAP34B-2 on the outer membrane permeability of Escherichia coli cells; Figure 5 The effect of the antimicrobial peptide MAP34B-2 on the outer membrane permeability of Acinetobacter baumannii; Figure 6 The effect of antimicrobial peptide MAP34B-2 on the inner membrane permeability of Escherichia coli cells; in, express P <0.001; P represents ＜ 0.0001. Detailed Implementation

[0018] This invention provides an antimicrobial peptide MAP34B-2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0019] In one embodiment, the C-terminus of the antimicrobial peptide MAP34B-2 of the present invention is modified by amidation. Compared with unmodified treatment, the amidation modification of the C-terminus of the antimicrobial peptide MAP34B-2 of the present invention has the advantage of improving the stability of the antimicrobial peptide.

[0020] This invention provides the application of the antimicrobial peptide MAP34B-2 described above in the preparation of bactericides and / or bacteriostatic agents.

[0021] In one embodiment, the bactericides and / or bacteriostatic agents of the present invention target Gram-negative bacteria. In another embodiment, the Gram-negative bacteria of the present invention include *Escherichia coli* and / or *Acinetobacter baumannii*.

[0022] This invention provides the use of the antimicrobial peptide MAP34B-2 described above in the preparation of medicaments for the prevention and / or treatment of bacterial infectious diseases.

[0023] In one embodiment, the bacteria described in this invention include Gram-negative bacteria. In one embodiment, the Gram-negative bacteria described in this invention include *Escherichia coli* and / or *Acinetobacter baumannii*. In one embodiment, the *Escherichia coli* described in this invention is *Escherichia coli* ATCC 25922. In one embodiment, the *Acinetobacter baumannii* described in this invention is *Acinetobacter baumannii* ATCC 19606.

[0024] This invention provides a bactericidal and / or bacteriostatic product, the active ingredient of which includes the antimicrobial peptide MAP34B-2 described in the above-mentioned scheme.

[0025] In one embodiment, the active ingredient of the present invention further includes other antibacterial and bactericidal components; the other antibacterial and bactericidal components include antibiotics. In one embodiment, the antibiotic of the present invention includes ampicillin.

[0026] In one embodiment, the product of this invention includes pharmaceuticals or skincare products. In another embodiment, the product of this invention also includes excipients. This invention does not impose strict requirements on the type of excipients; they can be conventionally selected according to the type of product. When the product is a pharmaceutical, the excipients are pharmaceutically acceptable; when the product is a skincare product, the excipients are cosmetically acceptable.

[0027] To further illustrate the present invention, the following detailed description of an antimicrobial peptide MAP34B-2 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.

[0028] Example 1 The polypeptide sequence described in SEQ ID NO:1 (RWGWRWKRWFR) was synthesized artificially and its carboxyl terminus was amidated, designated as MAP34B-2. The molecular weight was 1719.01 Da, the isoelectric point (pI) was 12.48, and the secondary structure was an α-helix.

[0029] Example 2 Minimum inhibitory concentration (MIC) determination 1. Using Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 as the test strains, the activated strains were inoculated into LB liquid medium and cultured in a constant temperature shaker until the logarithmic growth phase.

[0030] 2. To determine the bacterial concentration, dilute the culture medium to 2 × 10⁻⁶ with fresh, sterile liquid culture medium. 6 CFU / mL available for use.

[0031] 3. Take a sterile transparent 96-well plate, add 180 μL of fresh sterile liquid culture medium to the first row 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 row of wells (the test sample in rows A, B, and C is the polypeptide MAP34B-2 obtained in Example 1, and the test sample in rows D, E, and F is ampicillin).

[0032] 4. After mixing thoroughly with a pipette, add 100 µL to the second row of wells and continue diluting until the last well is reached. Discard the 100 µL of sterile liquid that has been drawn.

[0033] 5. Add 100 µL of pre-diluted bacterial solution to each well. At this point, the sample concentration in the first column is the highest (the concentration of the first well is 100 µg / mL), and the concentrations of the others are half of the previous column.

[0034] 6. The 96-well plate was placed in a 37℃ incubator for static incubation. The average concentration of the sterile growth well and the previous well was taken as the minimum inhibitory concentration (MIC). The results are shown in Table 1. The MIC of peptide MAP34B-2 against Escherichia coli ATCC 25922 was 18.75 μg / mL, and the MIC against Acinetobacter baumannii ATCC 19606 was >100.00 μg / mL.

[0035] Table 1. MIC (μg / mL) of the drug against the bacterial strain

[0036] Example 3 Minimum bactericidal concentration (MBC) determination 1. Using Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 as the test strains, 50 µL of culture medium was taken from each of the culture wells (1 / 2MIC, MIC, 2×MIC, and 4×MIC concentration wells) in Example 2.

[0037] 2. Spread the above culture medium onto fresh LB agar plates that do not contain the drug, and incubate the spread plates at 37°C for 24 h.

[0038] 3. Observe the number of colonies on the plate. The minimum drug concentration that kills 99.9% of bacteria is the MBC. The results are shown in Table 2. The minimum bactericidal concentration of polypeptide MAP34B-2 against Escherichia coli ATCC 25922 is 2×MIC, and the minimum bactericidal concentration against Acinetobacter baumannii ATCC 19606 is >4×MIC.

[0039] Table 2. MBC of the drug against the strain

[0040] Example 4 Cytotoxicity assay The toxicity of the peptide MAP34B-2 obtained in Example 1 to human immortalized keratinocytes (HaCat) and mouse fibroblasts (L929) was determined using the CCK-8 colorimetric method. The specific steps are 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. 4Cells / mL. MAP34B-2 was then diluted to different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC for E. coli ATCC 25922), with 5 replicates per group. After cell adhesion, different concentrations of MAP34B-2 dilutions were added. After cell interaction, 110 µL of 10% CCK8 solution (i.e., a 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 4 hours. The absorbance at OD 450 nm was measured using a microplate reader.

[0041] The results are as follows Figure 1 and Figure 2 As shown, at 2×MIC, the survival rate of both cell types was above 50%, indicating that the antimicrobial peptide MAP34B-2 has a certain degree of safety.

[0042] Example 5 hemolysis experiment 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 MAP34B-2 solution (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, and 8×MIC for *E. coli* ATCC 25922) of different concentrations were added. PBS was used as the negative control, and 1% (v / v) Triton-100 was used as the positive control. The mixture was thoroughly mixed and 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%.

[0043] The results are as follows Figure 3 As shown, at 16×MIC, the hemolytic activity was consistently less than 5%, indicating that the antimicrobial peptide MAP34B-2 has a certain degree of safety.

[0044] Example 6 Bacterial outer membrane permeability test Colonies of *Escherichia coli* ATCC 25922 and *Acinetobacter baumannii* ATCC 19606 were transferred to LB broth and cultured at 37°C with shaking until the logarithmic growth phase. The cultures were 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, the peptide MAP34B-2 from Example 1 was added to the bacterial suspension to final concentrations of 1×MIC, 2×MIC, 4×MIC, and 8×MIC (FI). peptide ), with PBS as the negative control (FI0) and 0.2 mg / mL polymyxin B as the positive control (FI). 100 ), and recorded the changes in bacterial outer membrane permeability over time by fluorescence intensity, according to the formula: Permeability (%) = (FI) peptide -FI0) / (FI 100 Calculate the penetration rate by multiplying (-FI0) by 100%.

[0045] The results are as follows Figure 4 and Figure 5 As shown, as the concentration gradually increases, the permeability of the antimicrobial peptide MAP34B-2 also gradually increases, approaching the result of the positive control. This indicates that MAP34B-2 uses the disruption of bacterial outer membrane permeability as one of its bactericidal mechanisms, and this effect is reflected in the concentration.

[0046] Example 7 Biomembrane inhibition function Escherichia coli ATCC 25922 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, the culture was diluted with fresh medium to a concentration of 2 × 10⁻⁶. 7CFU / mL. 100 μL of bacterial culture was added to 100 μL of antimicrobial solutions (Example 1 peptide MAP34B-2 and ampicillin) at different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC) in a 96-well plate. The positive control (PC) was bacterial culture with sterile water, and the negative control (NC) was 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 crystal violet staining solution was added 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 using the formula: Remaining biofilm percentage (%) = (OD measured value - OD blank group) / (OD positive group - OD blank group) × 100%.

[0047] The results are as follows Figure 6 As shown, the inhibitory effect of the antimicrobial peptide MAP34B-2 on biofilm gradually increases with increasing concentration, indicating that MAP34B-2 uses the inhibition of bacterial biofilm formation as one of its bactericidal mechanisms, and this effect is concentration-dependent.

[0048] 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 MAP34B-2, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The antimicrobial peptide MAP34B-2 according to claim 1, characterized in that, The C-terminus of the antimicrobial peptide MAP34B-2 is modified by amidation.

3. The use of the antimicrobial peptide MAP34B-2 according to claim 1 or 2 in the preparation of bactericides and / or bacteriostatic agents.

4. The application according to claim 3, characterized in that, The bactericides and / or bacteriostatic agents are targeted at Gram-negative bacteria.

5. The application according to claim 4, characterized in that, The Gram-negative bacteria include Escherichia coli (Escherichia coli) Escherichia coli ) and / or Acinetobacter baumannii ( Acinetobacter baumnnii ).

6. The use of the antimicrobial peptide MAP34B-2 according to claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of bacterial infectious diseases.

7. The application according to claim 6, characterized in that, The bacteria include Gram-negative bacteria.

8. The application according to claim 7, characterized in that, The Gram-negative bacteria include Escherichia coli and / or Acinetobacter baumannii.

9. A bactericidal and / or bacteriostatic product, characterized in that, The active ingredient includes the antimicrobial peptide MAP34B-2 as described in claim 1 or 2.

10. The bactericidal and / or bacteriostatic product according to claim 9, characterized in that, The active ingredients also include other antibacterial and bactericidal components; the other antibacterial and bactericidal components include antibiotics.

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