Antibacterial peptide MAP34B-1 and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide MAP34B-1 and application thereof. The amino acid sequence of the antibacterial peptide MAP34B-1 provided by the invention is as shown in SEQ ID NO: 1, and the C terminal of the antibacterial peptide MAP34B-1 can be subjected to amidation modification. The antibacterial peptide MAP34B-1 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-1 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-1 and its applications. Background Technology

[0002] Escherichia coli ( Escherichia coli , E. coli Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium with flagella, capable of motility, and is a facultative anaerobe. It is also an opportunistic pathogen, primarily transmitted through direct human-to-human and animal-to-human contact; fecal-oral transmission; and contaminated water and food, causing peritonitis, appendicitis, and pneumonia. Acinetobacter baumannii , A.baumannii It is a non-fermenting Gram-negative bacterium; it has flagella; it is motile; it is a strict aerobic bacterium; it is an important pathogen of hospital-acquired infections; it has strong adhesiveness and easily adheres to object surfaces; it has strong resistance; and it can cause bacteremia, urinary tract infections, secondary meningitis, and surgical site infections.

[0003] Current technologies typically employ antibiotics to inhibit bacterial cell wall formation, suppress protein synthesis (by acting on ribosomes), inhibit DNA synthesis, damage DNA, inhibit folic acid synthesis, and inhibit RNA synthesis. However, bacteria can produce enzymes that hydrolyze β-lactam antibiotics, and alterations in outer membrane proteins and reduced outer membrane permeability lead to mutations in the antibiotic targets of bacteria, resulting in the emergence of drug-resistant bacteria.

[0004] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of structurally diverse short peptides, typically composed of fewer than 100 amino acids. Due to their high efficiency, broad spectrum, thermostability, and low tendency to induce microbial resistance, they have become a hot topic in anti-infective drug research. However, not all small molecule peptides possess antimicrobial activity. While some natural antimicrobial peptides exhibit bacterial killing effects, their hemolytic activity and cytotoxicity prevent their application in clinical treatment, severely limiting the development of antimicrobial peptides. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide MAP34B-1 and its application, which has good bactericidal and bacteriostatic effects on Gram-negative bacteria, including Escherichia coli and Acinetobacter baumannii, especially in that it can destroy type IV fimbriae, thereby damaging bacterial growth and cell membrane synthesis.

[0006] This invention provides an antimicrobial peptide MAP34B-1, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Preferably, the C-terminus of the antimicrobial peptide MAP34B-1 is modified by amidation.

[0008] The application provides application of the antibacterial peptide MAP34B-1 in the preparation of a bactericide and / or a bacteriostatic agent.

[0009] Preferably, the bactericide and / or the bacteriostatic agent is directed against gram-negative bacteria.

[0010] Preferably, the gram-negative bacteria include Escherichia coli (E.coli) Escherichia coli ) and / or Acinetobacter baumannii (A.baumannii) Acinetobacter baumnnii ).

[0011] The application provides application of the antibacterial peptide MAP34B-1 in the preparation of a medicine for preventing and / or treating a bacterial infectious disease.

[0012] Preferably, the bacteria include gram-negative bacteria.

[0013] Preferably, the gram-negative bacteria include Escherichia coli and / or Acinetobacter baumannii.

[0014] The application provides a bactericide and / or a bacteriostatic product, and an active ingredient includes the antibacterial peptide MAP34B-1.

[0015] Preferably, the active ingredient further includes other bacteriostatic and bactericidal components; the other bacteriostatic and bactericidal components include antibiotics.

[0016] Beneficial effects: The antibacterial peptide MAP34B-1 provided by the application has a small 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 bacteriostatic and bactericidal role. Moreover, the antibacterial peptide MAP34B-1 provided by the application has low hemolytic activity and no cytotoxicity, can be applied to the preparation of a product with a bacteriostatic and bactericidal role, developed as an antibacterial peptide bacteriostatic and bactericidal agent, and prepared into a medicine for preventing and / or treating a bacterial infectious disease. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0018] Figure 1 A bactericidal kinetic curve of the antibacterial peptide MAP34B-1 on Escherichia coli; Figure 2 A bactericidal kinetic curve of the antibacterial peptide MAP34B-1 on Acinetobacter baumannii; Figure 3 A cytotoxicity detection result of the antibacterial peptide MAP34B-1 on human immortalized keratinocytes Hacat; Figure 4The cytotoxicity of the antibacterial peptide MAP34B-1 on mouse fibroblast L929 cells; Figure 5 The hemolytic activity of the antibacterial peptide MAP34B-1 on human red blood cells; Figure 6 The influence of the antibacterial peptide MAP34B-1 on the outer membrane permeability of E. coli cells; Figure 7 The influence of the antibacterial peptide MAP34B-1 on the outer membrane permeability of A. baumannii cells; Figure 8 The influence of the antibacterial peptide MAP34B-1 on the inner membrane permeability of E. coli cells; Figure 9 The influence of the antibacterial peptide MAP34B-1 on the inner membrane permeability of A. baumannii cells; Figure 10 The influence of the antibacterial peptide MAP34B-1 on the twitching movement of E. coli cells; Figure 11 The influence of the antibacterial peptide MAP34B-1 on the twitching movement of A. baumannii cells; wherein, represents P <0.05; represents P <0.01; represents P <0.001; represents P＜ 0.0001. DETAILED DESCRIPTION

[0019] The present application provides an antibacterial peptide MAP34B-1, and the amino acid sequence is shown as SEQ ID NO: 1.

[0020] As an embodiment, the C-terminal of the antibacterial peptide MAP34B-1 is modified by amidation. Compared with the unmodified antibacterial peptide, the antibacterial peptide MAP34B-1 modified by amidation has the advantage of improving the stability of the antibacterial peptide.

[0021] The present application provides the application of the antibacterial peptide MAP34B-1 in the preparation of bactericides and / or bacteriostatic agents.

[0022] As an embodiment, the bactericides and / or bacteriostatic agents of the present application are directed against gram-negative bacteria. As an embodiment, the gram-negative bacteria of the present application include E. coli and / or A. baumannii.

[0023] The present application provides the application of the antibacterial peptide MAP34B-1 in the preparation of drugs for preventing and / or treating bacterial infectious diseases.

[0024] 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.

[0025] This invention provides a bactericidal and / or bacteriostatic product, the active ingredient of which includes the antimicrobial peptide MAP34B-1 described in the above-mentioned scheme.

[0026] 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.

[0027] 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.

[0028] To further illustrate the present invention, the following detailed description of an antimicrobial peptide MAP34B-1 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.

[0029] Example 1 The polypeptide sequence described in SEQ ID NO:1 (KVKRMGWRIKWIFR) was synthesized artificially and its carboxyl terminus was amidated, designated as MAP34B-1. The molecular weight was 1904.35 Da, the isoelectric point (pI) was 12.31, and the secondary structure was predicted to be an α-helix.

[0030] 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.

[0031] 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.

[0032] 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-1 obtained in Example 1, and the test sample in rows D, E, and F is ampicillin).

[0033] 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.

[0034] 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.

[0035] 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-1 against Escherichia coli ATCC 25922 was 18.75 μg / mL, and the MIC against Acinetobacter baumannii ATCC 19606 was also 18.75 μg / mL.

[0036] Table 1. MIC (μg / mL) of the drug against the bacterial strain

[0037] Example 3 1. Sterilization kinetics detection Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 were used as test strains. MAP34B-1 obtained in Example 1 was used as the test polypeptide. The positive drug was ampicillin at the same concentration, and the negative control was physiological saline.

[0038] 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 the peptide / 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. 3Take 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.

[0039] The results are as follows Figure 1 and Figure 2 As shown, compared to the positive control drug ampicillin, which cannot completely kill bacteria within 2 hours, the bactericidal kinetic curve of MAP34B-1 shows that it can efficiently and thoroughly kill bacteria within two hours.

[0040] 2. 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 the culture wells (1 / 2MIC, MIC, 2×MIC, and 4×MIC concentration wells) in Example 2.

[0041] (2) Spread the above culture medium onto fresh LB plates without drugs and incubate the spread plates at 37°C for 24 h.

[0042] (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-1 against Escherichia coli ATCC 25922 is 1×MIC, and the minimum bactericidal concentration against Acinetobacter baumannii ATCC 19606 is 4×MIC.

[0043] Table 2. MBC of the drug against the strain

[0044] Example 4 Cytotoxicity assay The toxicity of the peptide MAP34B-1 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. 4 Cells / mL. MAP34B-1 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 MAP34B-1 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 another 4 h. The absorbance at OD450 nm was measured using a microplate reader.

[0045] The results are as follows Figure 3 and Figure 4 As shown, at 8×MIC, the survival rate of both cell types was above 60%, indicating that the peptide MAP34B-1 has a certain degree of safety.

[0046] Example 5 hemolysis experiment Human red blood cells were used as a blood sample. 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 then 500 μL of different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC) of the Example 1 polypeptide MAP34B-1 solution 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 in a constant temperature shaker. 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%.

[0047] The results are as follows Figure 5 As shown, at 16×MIC, the hemolytic activity was consistently less than 15%, indicating that the peptide MAP34B-1 has a certain degree of safety.

[0048] 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-1 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 100Calculate the penetration rate by multiplying (-FI0) by 100%.

[0049] The results are as follows Figure 6 and Figure 7 As shown, the permeability of peptide MAP34B-1 gradually increased over time and with increasing concentration, approaching the results of the positive control. This indicates that peptide MAP34B-1 uses the disruption of bacterial outer membrane permeability as one of its bactericidal mechanisms, and this effect is reflected in the concentration.

[0050] 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, 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 100 μL of antibacterial solutions (MAP34B-1 peptide and ampicillin from Example 1) at different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC) to 96-well plates. 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 (%) was calculated using the formula: Remaining biofilm percentage (%) = (OD measured value - OD blank group) / (OD positive group - OD blank group) × 100%.

[0051] The results are as follows Figure 8 and Figure 9 As shown, the inhibitory effect of peptide MAP34B-1 on biofilm gradually increases with increasing concentration, indicating that MAP34B-1 uses the inhibition of bacterial biofilm formation as one of its bactericidal mechanisms, and this effect is concentration-dependent.

[0052] Example 8 Bacterial Motility Experiment Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 were cultured overnight at 37℃ 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 1×10⁻⁶. 9cfu / mL. MAP34B-1 (Example 1) and ampicillin were added to the bacterial suspension to achieve final concentrations of 1×MIC and 2×MIC, respectively. 2 μL of the bacterial motility diameter was measured after incubating the bacterial suspension at 37°C for 20 h on the bottom of a twitching agar plate (0.5% TSA).

[0053] The results are as follows Figure 10 and 11 As shown, the peptide MAP34B-1 significantly inhibits bacterial motility. Escherichia coli and Acinetobacter baumannii possess type IV pili, and twitching is their specific type of motility. Therefore, it is reasonable to infer that the peptide MAP34B-1 inhibits twitching while destroying type IV pili.

[0054] 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-1, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

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

3. The use of the antimicrobial peptide MAP34B-1 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-1 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-1 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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