Broad-spectrum antibacterial peptide bsa138 and application thereof

Abstract

The invention relates to a broad-spectrum antibacterial peptide bsa138 and application thereof, and relates to the technical field of biology. The antibacterial peptide has broad-spectrum antibacterial activity, can inhibit gram-negative bacteria such as escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and pseudomonas aeruginosa and fungi such as candida auriculata and candida albicans, and has high antibacterial activity. The compound has a relatively strong inhibition effect on clinically separated drug-resistant strains with multiple drug resistance. In addition, the antibacterial peptide is low in hemolytic activity, small in toxicity, short in synthetic sequence, small in molecular weight and easy to chemically synthesize. And the in-vivo safety is ensured while efficient sterilization is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a broad-spectrum antimicrobial peptide, bsa138, and its applications. Background Technology

[0002] Antibiotic resistance is one of the serious challenges facing modern medicine. In recent decades, the number of pathogenic microorganisms that develop antibiotic resistance has continued to increase, and multidrug-resistant bacteria and super-drug-resistant bacteria have emerged one after another. Every year, the number of deaths caused by infections caused by drug-resistant bacteria reaches 700,000 worldwide.

[0003] As the fight against antibiotics continues, antimicrobial peptides have emerged as a powerful weapon against drug-resistant bacterial infections due to their mechanism of action. Antimicrobial peptides are small peptides with antimicrobial activity widely found in nature. They possess advantages such as low resistance to drug development and rapid action, while also being easily degraded and causing no persistent environmental pollution. Antimicrobial peptides can inhibit pathogenic microorganisms through various mechanisms of action. Common mechanisms include binding to the cell membrane of pathogens, disrupting cell membrane structure, or directly forming micropores in the cell membrane, allowing cell contents to leak out and ultimately killing the pathogens. In recent years, antimicrobial peptides that can resist multidrug-resistant bacteria without easily inducing resistance have been considered next-generation antimicrobial agents to replace traditional antibiotics.

[0004] Currently reported antimicrobial peptides suffer from drawbacks such as low bioactivity, high toxicity, and poor stability. Antimicrobial potency is generally expressed using the minimum inhibitory concentration (MIC). If the MIC value of an antimicrobial peptide is too high, large doses are required to achieve effectiveness, rendering the product worthless. Furthermore, increasing drug concentration can lead to cytotoxicity and hemolysis. As polypeptides, antimicrobial peptides are not particularly resistant to high temperatures or acids and alkalis. Antimicrobial peptides are bioactive polypeptides, and during bio-fermentation, host bacteria often produce many proteases. These proteases have degrading activity, causing the antimicrobial peptides to be degraded and lost during fermentation, rendering them worthless. Naturally occurring or genetically engineered antimicrobial peptides constitute a very small proportion of the separation and purification matrix, containing a large amount of impurities such as proteins and other substances, resulting in high separation and purification costs. Therefore, developing antimicrobial peptides with simple structures and high bioactivity, synthesized using solid-phase chemical methods through artificial intelligence design, is currently an urgent priority in combating the emergence of drug-resistant bacteria. Summary of the Invention

[0005] (1) Technical problems solved

[0006] To address the aforementioned technical problems, this invention provides a broad-spectrum antimicrobial peptide, bsa138, and its applications.

[0007] (II) Technical Solution

[0008] Based on this, the present invention provides the following technical solution: a broad-spectrum antimicrobial peptide bsa138, wherein the antimicrobial peptide is an antimicrobial peptide bsa138 having the amino acid sequence shown in SEQ ID: TRSRWRIRITQRSS;

[0009] The antimicrobial peptide has a sequence length of 14, a charge number of 4.996, a hydrophilicity / hydrophobicity of 0.14, and an isoelectric point of 12.00.

[0010] Preferably, the design steps are as follows:

[0011] Step S1: Synthesis of antimicrobial peptides. Using a chemical organic solid-phase synthesis method, the sequence is condensed sequentially from the C-terminus to the N-terminus to complete the peptide chain. The peptide is removed from the resin using a cleavage fluid, and the side chain protecting groups are removed at the same time. The crude peptide is precipitated with ice-cold ether. The crude peptide is purified to a purity of over 95% using a preparative high-performance liquid chromatography system and then freeze-dried.

[0012] Application of a broad-spectrum antimicrobial peptide, bsa138, wherein the antimicrobial peptide is used to prepare an inhibitor against bacterial infection, wherein the bacteria are Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, and the minimum inhibitory concentration (MIC) of the antimicrobial peptide bsa138 against Escherichia coli is 15.63 μg / mL, against Klebsiella pneumoniae is 15.63 μg / mL, against Acinetobacter baumannii is 15.63 μg / mL, and against Pseudomonas aeruginosa is 7.81 μg / mL.

[0013] Preferably, the antimicrobial peptide is used to prepare an inhibitor of antifungal infection, wherein the fungus is Candida auris or Candida albicans, and the minimum inhibitory concentration of the antimicrobial peptide bsa138 against Candida auris is 7.81 μg / mL and the minimum inhibitory concentration against Candida albicans is 7.81 μg / mL.

[0014] (3) Beneficial effects

[0015] Compared with the prior art, the present invention provides a broad-spectrum antimicrobial peptide bsa138 and its applications, which have the following beneficial effects:

[0016] This invention relates to a broad-spectrum antimicrobial peptide, bsa138, and its applications. The antimicrobial peptide bsa138 has the amino acid sequence shown in SEQ ID: TRSRWRIRITQRSS. This antimicrobial peptide exhibits broad-spectrum antimicrobial activity, inhibiting Gram-negative bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, and *Pseudomonas aeruginosa*, as well as fungi such as *Candida auris* and *Candida albicans*, with high antimicrobial activity against clinically isolated multidrug-resistant strains. Furthermore, the antimicrobial peptide exhibits low hemolytic activity, low toxicity, a short synthetic sequence, and a small molecular weight, making it easy to synthesize chemically. It ensures in vivo safety while achieving highly effective bactericidal action. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the drug susceptibility test results of the clinically resistant strain of the present invention;

[0018] Figure 2 This is a schematic diagram illustrating the cytotoxicity of the antimicrobial peptides of the present invention.

[0019] Figure 3 This is a schematic diagram illustrating the hemolytic properties of the antimicrobial peptide of the present invention. Detailed Implementation

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Example 1

[0022] A broad-spectrum antimicrobial peptide bsa138, wherein the antimicrobial peptide is an antimicrobial peptide bsa138 having the amino acid sequence shown in SEQ ID: TRSRWRIRITQRSS; the antimicrobial peptide has a sequence length of 14, a charge number of 4.996, a hydrophilicity / hydrophobicity of 0.14, and an isoelectric point of 12.00.

[0023] Example 2

[0024] A broad-spectrum antimicrobial peptide, bsa138, is synthesized using the following steps:

[0025] The antimicrobial peptide sequence in this invention is: TRSRWRIRITQRSS;

[0026] The antimicrobial peptides were synthesized using a chemical organic solid-phase synthesis method. The sequence was sequentially condensed from the C-terminus to the N-terminus to complete the peptide chain. The peptide was then removed from the resin using a cleavage fluid, along with the side chain protecting groups. The crude peptide was precipitated with ice-cold ether and purified to a purity of over 95% using a preparative high-performance liquid chromatography (HPLC) system before being lyophilized.

[0027] Example 3

[0028] Assay for the activity of antimicrobial peptides

[0029] 1. Determination of minimum inhibitory concentration of standard strains.

[0030] Bacteria: *Escherichia coli* (ATCC 25922), *Klebsiella pneumoniae* (ATCC 43816), *Acinetobacter baumannii* (ATCC 19606), and *Pseudomonas aeruginosa* (ATCC 27853) were streaked onto LB agar and incubated upside down in a 37°C CO2 incubator for 24 hours. After single colonies formed on the plates, single colonies were picked and transferred to LB liquid medium. The medium was then incubated at 37°C with shaking for 4 hours until OD (dose retardation) was reached. 600 When the concentration is 0.6, dilute the bacterial culture into CAMHB liquid medium and adjust the bacterial count to 10. 5 CFU / mL, keep for later use;

[0031] Fungi: *Candida auris* (ATCC 12766) and *Candida albicans* (ATCC 10231). The strains were inoculated onto Sabouraud dextrose agar (SDA) plates and incubated at 25°C for 24 hours, activating the culture at least twice. Single colonies were then picked and cultured in Sabouraud dextrose liquid medium (SDB) overnight at 37°C with shaking at 200 rpm. The overnight culture was then diluted 10-fold twice with Sabouraud dextrose liquid medium (SDB). A hemocytometer was moistened with 75% ethanol, dried with lens paper, and covered with a coverslip. 10 μL of homogenized bacterial suspension was drawn from the diluted culture and injected through the lower edge of the coverslip, ensuring the suspension filled the counting area while avoiding air bubbles. After standing for 1 minute, under a 40x objective microscope, the bacterial cells were found to have largely stopped drifting in the liquid. Using a manual counter, the number of spores (N) was counted in the four central squares (top left, bottom left, top right, bottom right, and center) and the five central squares along the diagonal. (For budding spores, a bud reaching half the size of the parent spore is counted as two spores.) Bacterial concentration = N / 5 × 25 × 10⁻⁶ 4 CFU / mL. The bacterial concentration was calculated, and the bacterial suspension was serially diluted up to tenfold using RPMI-1640 medium to adjust the concentration to 2 × 10⁻⁶. 3 CFU / ml. Since the bacterial suspension doubles in volume when added to the susceptibility testing plate, it is equivalent to being diluted by half. Therefore, the final concentration of the bacterial suspension in the plate is 1×10⁻⁶. 3 CFU / m;

[0032] Dissolve 1 mg of antimicrobial peptide in 1 mL of physiological saline to a concentration of 1 mg / mL. Add 100 μL of the serially diluted antimicrobial peptide (2-fold) to a 96-well plate, then add 100 μL of a 10% concentration... 5 The bacterial culture was prepared at CFU / mL. The antimicrobial peptide concentrations at these concentrations were 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, 7.81 μg / mL, 3.91 μg / mL, and 1.95 μg / mL, respectively. The OD values ​​at these concentrations were recorded. 600A mixture of 100 μL bacterial culture and 100 μL physiological saline was used as a negative control. The 96-well plate was placed in a CO2 incubator at 37°C and incubated for 20 h. OD was recorded again. 600 The minimum inhibitory concentration (MIC) is the lowest concentration at which the growth of a bacterial strain is completely inhibited. The formula for calculating the inhibition rate of antimicrobial peptides is as follows;

[0033]

[0034] Table 1 shows that the antimicrobial peptides of this invention have significant inhibitory effects on bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) and fungi (Candida auris and Candida albicans). The minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida auris, and Candida albicans are all 15.63 μg / mL. Therefore, the antimicrobial peptides of this invention exhibit particularly significant inhibitory effects against Pseudomonas aeruginosa, Candida auris, and Candida albicans.

[0035] Table 1. Minimum inhibitory concentrations of antimicrobial peptides against standard strains.

[0036]

[0037] (2) Determination of minimum inhibitory concentration for multidrug-resistant strains

[0038] To verify the antibacterial efficacy against multidrug-resistant bacteria, four strains each of *Escherichia coli*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, and *Pseudomonas aeruginosa* were isolated from clinical samples, totaling 16 multidrug-resistant bacteria. Their drug resistance characteristics are as follows: Figure 1 As shown;

[0039] The minimum inhibitory concentration (MIC) assay was used to determine the MIC of the antimicrobial peptides against the 16 clinically isolated multidrug-resistant bacteria. The results are shown in the table below:

[0040] Table 2 Minimum inhibitory concentrations against multidrug-resistant bacteria

[0041]

[0042] Based on the above results, it is evident that the antimicrobial peptides of the present invention exhibit excellent antibacterial effects against various clinically multidrug-resistant bacteria: the minimum inhibitory concentration (MIC) against multidrug-resistant Escherichia coli reaches 15.6 μg / mL, the MIC against multidrug-resistant Pseudomonas aeruginosa is 15.6 μg / mL, and the MIC against multidrug-resistant Klebsiella pneumoniae is 7.8 μg / mL.

[0043] Example 4

[0044] Cytotoxicity assay of antimicrobial peptides

[0045] Prepare 100 μL of cell suspension in each well of a 96-well plate (10 cells per well). 4 Pre-culture the plate in an incubator for 24 hours (at 37°C and 5% CO2). Add 100 μL of the test peptide (100 μg / mL) to the plate. Incubate for 24 hours. Add 10 μL of CCK-8 solution to each well. Incubate the plate for 4 hours. Measure the absorbance at 450 nm using a microplate reader. The cell viability assay formula is as follows;

[0046]

[0047] A (Drug Addition): Absorbance of the pores containing cells, CCK-8 solution, and drug solution;

[0048] A (blank): Absorbance of pores containing culture medium and CCK-8 solution but without cells;

[0049] A(0 drug added): Absorbance of the pore containing cells and CCK-8 solution but no drug solution;

[0050] Cell viability: cell proliferation activity or cytotoxic activity;

[0051] Cytotoxicity assays are widely used experimental methods in biological research to assess the toxicity of compounds to cells in vivo. This assay aims to reveal whether the antimicrobial peptides of this invention affect cell structure and function, thereby assessing the potential risks of the antimicrobial peptides to the health of organisms and providing important data for drug development and safety assessment.

[0052] Specifically, such as Figure 2 As shown, the antimicrobial peptide of the present invention exhibits 100% cell proliferation activity at a concentration of 100 μg / mL, consistent with the results of cell culture in DMEM medium (the negative control cell culture medium), indicating that the antimicrobial peptide has no toxic effect on cells at this concentration.

[0053] hemolytic assay of antimicrobial peptides

[0054] Take a 96-well plate and add 100 μL of the test peptide at a concentration of 100 μg / mL. Add 100 μL of 2% red blood cells to the 96-well plate and mix well. Incubate at 37℃ for 1 h, centrifuge at 5000 rpm / min for 5 min; carefully aspirate 100 μL of the supernatant into the enzyme-linked immunosorbent assay (ELISA) wells, setting up four parallel control wells. Measure the OD at 570 nm using an ELISA reader. The negative control is physiological saline, and the positive control is 10% Triton-physiological saline. Using physiological saline as the negative control and Triton X-100 as the positive control yielded the following results: Triton X-100 (positive control) increases cell membrane permeability, causing the system to become isotonic, leading to red blood cell rupture and hemolysis, resulting in a clear red solution; while the physiological saline control tubes (negative control tubes) showed no hemolysis or red blood cell agglutination. After comparison with the negative and positive control tubes, all red blood cells in the experimental wells precipitated, and the supernatant was colorless and clear, indicating no hemolysis. Furthermore, the precipitated red blood cells dispersed upon shaking, and no red blood cell aggregation was observed. The hemolysis rate of the peptide was calculated by measuring the absorbance of the supernatant at OD570 nm using a microplate reader, using the following formula:

[0055]

[0056] Hemolysis refers to reactions such as extravascular or intravascular hemolysis and erythrocyte aggregation caused by drug formulations. This experiment aims to investigate whether the antimicrobial peptide of this invention can induce reactions such as hemolysis and erythrocyte aggregation in vitro. The active ingredient and its metabolites, excipients, related substances, and physicochemical properties (such as pH value, osmotic pressure, etc.) of a drug can all potentially cause hemolysis. Severe toxicity can affect the safety and efficacy of medication. Therefore, before clinical application, the local and / or systemic toxicity caused by the formulation after administration should be studied to indicate potential toxic reactions, target organs of toxicity, safety margins, clinical research monitoring indicators, and to provide a reference for clinical detoxification or rescue measures, ensuring the safety and efficacy of clinical drug use.

[0057] like Figure 3 As shown, the antimicrobial peptide of the present invention has a hemolysis rate of 0% at a concentration of 100 μg / mL, which is consistent with the results of culturing red blood cells in physiological saline as a negative control, that is, the antimicrobial peptide does not cause hemolysis at this concentration.

[0058] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A broad-spectrum antimicrobial peptide, bsa138, characterized in that: The antimicrobial peptide is bsa138, which has the amino acid sequence shown in SEQ ID: TRSRWRIRITQRSS. The antimicrobial peptide has a sequence length of 14, a charge number of 4.996, a hydrophilicity / hydrophobicity of 0.14, and an isoelectric point of 12.

00.

2. An application of the broad-spectrum antimicrobial peptide bsa138 as described in claim 1, characterized in that: The antimicrobial peptide is used to prepare inhibitors against bacterial infections.

3. An application of the broad-spectrum antimicrobial peptide bsa138 as described in claim 1, characterized in that: The antimicrobial peptide is used to prepare inhibitors against fungal infections.

4. The application of the broad-spectrum antimicrobial peptide bsa138 according to claim 2, characterized in that: The bacteria mentioned are Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

5. The application of the broad-spectrum antimicrobial peptide bsa138 according to claim 3, characterized in that: The fungi mentioned are Candida auris and Candida albicans.

6. The application of the broad-spectrum antimicrobial peptide bsa138 according to claim 2, characterized in that: The minimum inhibitory concentration (MIC) of the antimicrobial peptide BSA138 against Escherichia coli is 15.63 μg / mL, against Klebsiella pneumoniae is 15.63 μg / mL, against Acinetobacter baumannii is 15.63 μg / mL, and against Pseudomonas aeruginosa is 7.81 μg / mL.

7. The application of the broad-spectrum antimicrobial peptide BSA138 according to claim 3, characterized in that: The minimum inhibitory concentration (MIC) of the antimicrobial peptide bsa138 against Candida auris is 7.81 μg / mL, and the minimum inhibitory concentration (MIC) against Candida albicans is 7.81 μg / mL.

8. The broad-spectrum antimicrobial peptide bsa138 according to claim 1, characterized in that: Step S1: Synthesis of antimicrobial peptides. Using a chemical organic solid-phase synthesis method, the sequence is condensed sequentially from the C-terminus to the N-terminus to complete the peptide chain. The peptide is removed from the resin using a cleavage fluid, and the side chain protecting groups are removed at the same time. The crude peptide is precipitated with ice-cold ether. The crude peptide is purified to a purity of over 95% using a preparative high-performance liquid chromatography system and then freeze-dried.

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