CN118359684A - Broad-spectrum antibacterial peptides bsa and bsa086 and application thereof - Google Patents

Abstract

The invention discloses broad-spectrum antibacterial peptides bsa049 and bsa086 and application thereof, and relates to the field of molecular biology. In the invention, the antibacterial peptide has broad-spectrum antibacterial activity, can inhibit gram-positive bacteria such as staphylococcus aureus and gram-negative bacteria such as escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and pseudomonas aeruginosa, and has higher antibacterial activity. Has stronger inhibiting effect on clinically separated drug-resistant strains with multiple drug resistance. In addition, the antibacterial peptide has low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight and easy chemical synthesis. The internal safety is ensured while the sterilization is efficient.

Description

Broad-spectrum antimicrobial peptides bsa049 and bsa086 and their applications

Technical Field

The present invention relates to the field of molecular biology, and in particular to broad-spectrum antimicrobial peptides BSA049 and BSA086 and applications thereof.

Background technique

Antimicrobial peptides are polypeptides with antimicrobial activity, usually with strong alkalinity, and play an important role in innate immunity. Antimicrobial peptides exist in almost all species of life and exert biological activities such as antibacterial, antiviral, antifungal and antiparasitic. Compared with antibiotics, antimicrobial peptides are green and environmentally friendly, and do not cause drug resistance in target bacteria, so they have broad application prospects.

The antimicrobial strength is generally expressed by the minimum inhibitory concentration (MIC). If the MIC value of the antimicrobial peptide is too high, a large dose is required to achieve effectiveness, and such a product is worthless. In addition, increasing the drug concentration will also cause problems such as cytotoxicity and cell hemolysis. As a polypeptide substance, antimicrobial peptides are not particularly resistant to high temperatures and acid and alkali. Antimicrobial peptides are biologically active polypeptides. During the biological fermentation process, the host bacteria will produce a lot of proteases. These proteases have the activity of degrading polypeptides, causing the antimicrobial peptides to be degraded and lost during the fermentation process, losing their production value. At the same time, in the application of antimicrobial peptides, there is also the phenomenon of multidrug resistance. Therefore, there is an urgent need for antimicrobial peptides with low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight, and easy chemical synthesis.

Summary of the invention

Therefore, in order to solve the above-mentioned deficiencies, the present invention provides broad-spectrum antimicrobial peptides bsa049 and bsa086 and their applications.

In order to achieve the above object, the present invention adopts the following technical scheme: broad-spectrum antimicrobial peptides bsa049 and bsa086, wherein the antimicrobial peptides are provided in two groups, wherein one group of antimicrobial peptides is the antimicrobial peptide bsa049 having the amino acid sequence shown in SEQ ID: RLWRIVVIRVWR;

Another group of antimicrobial peptides is the antimicrobial peptide bsa086 having an amino acid sequence shown in SEQ ID: KKLAGLAKKWAGLAKKLAGLA.

Application of broad-spectrum antimicrobial peptides BSA049 and BSA086, wherein the antimicrobial peptides are used to prepare inhibitors against bacterial infections, and the antimicrobial peptides are used to prepare inhibitors against multiple bacterial infections.

As a further embodiment of the present invention, the bacteria are Staphylococcus aureus (ATCC 25923) streaked and inoculated in TSB medium, Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 43816), Acinetobacter baumannii (ATCC19606) and Pseudomonas aeruginosa (ATCC 27853).

As a further embodiment of the present invention, the minimum inhibitory concentration of the antimicrobial peptide bsa049 against Staphylococcus aureus is 7.81 μg/mL, the minimum inhibitory concentration against Escherichia coli is 7.81 μg/mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 125 μg/mL, the minimum inhibitory concentration against Acinetobacter baumannii is 3.91 μg/mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 31.25 μg/mL.

As a further embodiment of the present invention, the minimum inhibitory concentration of the antimicrobial peptide bsa086 against Escherichia coli is 7.81 μg/mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 125 μg/mL, the minimum inhibitory concentration against Acinetobacter baumannii is 1.95 μg/mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 31.25 μg/mL.

As a further embodiment of the present invention, the multi-antibacterial agent is Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa.

As a further scheme of the present invention, the minimum inhibitory concentration of the antimicrobial peptide bsa049 against multidrug-resistant strains of Escherichia coli reached 15.6 μg/mL, the minimum inhibitory concentration against three multidrug-resistant strains of Acinetobacter baumannii was 15.6 μg/mL; the minimum inhibitory concentration against three multidrug-resistant strains of Klebsiella pneumoniae was 15.6 μg/mL.

As a further solution of the present invention, the minimum inhibitory concentration of the antimicrobial peptide bsa086 against multidrug-resistant strains of Escherichia coli reached 7.8 μg/mL, the minimum inhibitory concentration against multidrug-resistant strains of Acinetobacter baumannii reached 3.9 μg/mL, and the minimum inhibitory concentration against multidrug-resistant strains of Klebsiella pneumoniae was 31.3 μg/mL.

Compared with the prior art, the present invention provides broad-spectrum antimicrobial peptides bsa049 and bsa086 and their applications, which have the following beneficial effects:

In the present invention, the antimicrobial peptide has a broad spectrum of antimicrobial activity, can inhibit Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, and all have high antimicrobial activity. It has a strong inhibitory effect on multi-drug resistant strains isolated from clinical sites. In addition, the antimicrobial peptide has low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight, and is easy to chemically synthesize. It ensures in vivo safety while efficiently killing bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the drug sensitivity test results of 116 clinical drug-resistant bacteria of the present invention;

FIG2 is a schematic diagram of the cytotoxicity of the antimicrobial peptide bsa049 of the present invention;

FIG3 is a schematic diagram of the cytotoxicity of the antimicrobial peptide bsa086 of the present invention;

FIG4 is a schematic diagram of the hemolytic activity of the antimicrobial peptide bsa049 of the present invention;

FIG5 is a schematic diagram of the hemolytic activity of the antimicrobial peptide bsa086 of the present invention.

Detailed ways

In order to further explain the technical solution of the present invention, it is described in detail below through specific embodiments.

Example 1

1. Synthesis of antimicrobial peptides

The antimicrobial peptide is prepared by a chemical organic solid phase synthesis method, the sequence is condensed from the C-terminus to the N-terminus in sequence to complete the peptide chain, the polypeptide is cut off from the resin with a cutting liquid, and the side chain protecting group is cut off at the same time, the crude peptide is precipitated with ice ether, and the crude polypeptide is purified by a preparative high performance liquid chromatography to a purity of more than 95% and freeze-dried to prepare the antimicrobial peptide. The antimicrobial peptide is an antimicrobial peptide bsa049 having an amino acid sequence shown in SEQ ID: RLWRIVVIRVWR, and another antimicrobial peptide is an antimicrobial peptide bsa086 having an amino acid sequence shown in SEQ ID: KKLAGLAKKWAGLAKKLAGLA.

2. Determination of Antimicrobial Peptide Activity

Determination of minimum inhibitory concentration of standard strains

Bacteria: Staphylococcus aureus (ATCC 25923) was streaked into TSB medium, Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC 43816), Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa (ATCC27853) were streaked into LB medium, and placed in a 37°C CO ₂ incubator for inversion culture for 24 hours. After a single colony is formed on the plate, pick a single colony and transfer it to the corresponding liquid culture medium, Staphylococcus aureus to TSB liquid culture medium, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa to LB liquid culture medium. Place in a 37°C incubator and shake culture for 4 hours. When OD ₆₀₀ = 0.6, dilute the bacterial solution into CAMHB liquid culture medium, adjust the bacterial volume to 10 ⁵ CFU/mL, and keep it for later use;

Dissolve 1 mg of antimicrobial peptide in 1 mL of saline to a concentration of 1 mg/mL. Add 100 μL of the two-fold gradient dilution of antimicrobial peptide to a 96-well plate, and then add 100 μL of bacterial solution with a concentration of 10 ⁵ CFU/mL. At this time, the concentrations of antimicrobial peptides are 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. Record the OD600 at this time. A mixture of 100 μL of bacterial solution and 100 μL of saline is used as a negative control. Place the 96-well plate in a CO ₂ incubator at 37°C and culture for 20 hours, and record OD ₆₀₀ again. The minimum inhibitory concentration (MIC) is the lowest concentration at which the growth of the strain is completely inhibited. The formula for calculating the antimicrobial peptide inhibition rate is as follows:

The results in Table 1 show that the antimicrobial peptide bsa049 of the present invention has a significant inhibitory effect on Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. The minimum inhibitory concentration for Staphylococcus aureus is 7.81 μg/mL, the minimum inhibitory concentration for Escherichia coli is 7.81 μg/mL, the minimum inhibitory concentration for Klebsiella pneumoniae is 125 μg/mL, the minimum inhibitory concentration for Acinetobacter baumannii is 3.91 μg/mL, and the minimum inhibitory concentration for Pseudomonas aeruginosa is 31.25 μg/mL. It can be seen that the antibacterial effect of the antimicrobial peptide of the present invention on Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii is particularly significant;

In addition, the results in Table 1 also show that the antimicrobial peptide bsa086 of the present invention has a significant inhibitory effect on Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. The minimum inhibitory concentration for Escherichia coli is 7.81 μg/mL, the minimum inhibitory concentration for Klebsiella pneumoniae is 125 μg/mL, the minimum inhibitory concentration for Acinetobacter baumannii is 1.95 μg/mL, and the minimum inhibitory concentration for Pseudomonas aeruginosa is 31.25 μg/mL. It can be seen that the antibacterial effect of the antimicrobial peptide of the present invention on Escherichia coli and Acinetobacter baumannii is particularly significant;

Table 1 Minimum inhibitory concentration of antimicrobial peptides against standard strains

Determination of Minimum Inhibitory Concentration of Multidrug-resistant Strains

In order to verify the antibacterial effect against multi-drug resistant bacteria, 4 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa were isolated from the clinic, totaling 16 multi-drug resistant bacteria. Their resistance is shown in Figure 1:

The minimum inhibitory concentration determination method was used to determine the minimum inhibitory concentration of the antimicrobial peptide bsa049 against the above 16 clinically isolated multidrug-resistant bacteria. The results are shown in the following table:

Table 2 Minimum inhibitory concentration for multidrug-resistant bacteria

Combined with the above results, it can be seen that the antimicrobial peptide bsa049 of the present invention has excellent antibacterial effects on different clinical multidrug-resistant bacteria: the minimum inhibitory concentration for multidrug-resistant Escherichia coli strains reached 15.6 μg/mL, the minimum inhibitory concentration for three multidrug-resistant strains of Acinetobacter baumannii was 15.6 μg/mL; the minimum inhibitory concentration for three multidrug-resistant strains of Klebsiella pneumoniae was 15.6 μg/mL;

The minimum inhibitory concentration determination method was used to determine the minimum inhibitory concentration of the antimicrobial peptide bsa086 against the above 16 clinically isolated multidrug-resistant bacteria. The results are shown in the following table:

Table 3 Minimum inhibitory concentration for multidrug-resistant bacteria

Combined with the above results, it can be seen that the antimicrobial peptide bsa086 of the present invention has excellent antibacterial effects on different clinical multidrug-resistant bacteria: the minimum inhibitory concentration for multidrug-resistant Escherichia coli strains reached 7.8 μg/mL, the minimum inhibitory concentration for multidrug-resistant Acinetobacter baumannii strains reached 3.9 μg/mL, and the minimum inhibitory concentration for multidrug-resistant Klebsiella pneumoniae strains was 31.3 μg/mL.

Example 2

Cytotoxicity assay of antimicrobial peptides

Prepare 100 μL of cell suspension in a 96-well plate (10 ⁴ cells per well). Pre-culture the culture plate in an incubator for 24 hours (at 37°C, 5% CO ₂ ). Add 100 μL of the peptide to be tested with a concentration range of 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL to the culture plate. Incubate in an incubator for 24 hours. Add 10 μL of CCK-8 solution to each well. Incubate the culture plate in an incubator for 4 hours. Measure the absorbance at 450 nm with an enzyme-labeled instrument. The formula for cell activity detection is as follows;

A (drug added): absorbance of wells with cells, CCK-8 solution, and drug solution;

A (blank): absorbance of wells with culture medium and CCK-8 solution but no cells;

A(0 drug addition): absorbance of the wells with cells and CCK-8 solution but no drug solution;

Cell viability: cell proliferation activity or cytotoxic activity;

Cytotoxicity test is an experimental method widely used in the field of biological research to evaluate the degree of toxicity of compounds to cells in organisms. This test aims to reveal whether the antimicrobial peptides of the present invention will affect the structure and function of cells, so as to evaluate the potential risks of antimicrobial peptides to the health of organisms, thereby providing important data for drug development and safety assessment.

As shown in Figures 2 and 3, the IC50 (half-inhibitory concentration of the measured antagonist) value of the antimicrobial peptide bsa049 of the present invention is 641.3 μg/mL, that is, the cell proliferation activity at this concentration is 50%, which is significantly different from its MIC value and has a wider clinical application range. The IC50 (half-inhibitory concentration of the measured antagonist) value of the antimicrobial peptide bsa086 of the present invention is 1195 μg/mL, that is, the cell proliferation activity at this concentration is 50%, which is significantly different from its MIC value and has a wider clinical application range.

Example 3

Hemolytic activity assay of antimicrobial peptides

Take a 96-well plate and add 100 μL of the peptide to be tested in the concentration range of 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL. Pipette 100 μL of 2% red blood cells into the 96-well plate and add to the solution and mix well. Incubate at 37℃ for 1 hour, centrifuge at 5000 rpm/min for 5 minutes; carefully pipette 100 μL of supernatant into the enzyme-linked well, set up four parallel control wells, and measure OD 570nm with an enzyme-linked well reader. The negative control is saline, and the positive control is 10% Triton-saline. Physiological saline was selected as the negative control and TritionX-100 as the positive control. The results met the experimental requirements: TritionX-100 (positive control) has the effect of increasing the permeability of the cell membrane, making the system non-isotonic, causing the red blood cells to rupture, hemolysis to occur, and a clear red solution to appear; while the physiological saline solution control tube (negative control tube) had no hemolysis or red blood cell agglutination. After comparing each experimental well with the negative control tube and the positive control tube, the red blood cells in all experimental wells sank, the supernatant was colorless and clear, no hemolysis occurred, and the precipitated red blood cells dispersed after shaking, and no red blood cell agglutination occurred. The absorbance of the supernatant was detected by an enzyme marker at OD570nm, and the hemolysis rate of the polypeptide was calculated. The formula is as follows:

Hemolysis refers to reactions such as extravascular or intravascular hemolysis and erythrocyte aggregation caused by drug preparations. This experiment aims to investigate whether the antimicrobial peptides of the present invention can cause hemolysis and erythrocyte aggregation in vitro. The active ingredients of the drug and its metabolites, excipients, related substances and physicochemical properties (such as pH value, osmotic pressure, etc.) may cause hemolysis, and severe toxicity will affect the safety and effectiveness of the drug. Therefore, before the clinical application of the drug, the local and/or systemic toxicity caused by the preparation after use at the administration site should be studied to indicate the possible toxic reactions, toxic target organs, safety range, clinical research monitoring indicators during clinical application, and provide reference for clinical detoxification or rescue measures to ensure the safety and effectiveness of clinical drug use.

As shown in Figures 4 and 5, the HC50 (half hemolytic value) of the antimicrobial peptide bsa049 of the present invention is 317360 μg/mL, that is, the hemolytic rate is 50% at this concentration, which is significantly different from its MIC value, and has a wider clinical application range. The HC50 (half hemolytic value) of the antimicrobial peptide bsa086 of the present invention is 4529 μg/mL, that is, the hemolytic rate is 50% at this concentration, which is significantly different from its MIC value, and has a wider clinical application range.

In summary, antimicrobial peptides bsa049 and bsa086 have broad-spectrum antibacterial activity, can inhibit Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, and both have high antibacterial activity. They have a strong inhibitory effect on multidrug-resistant strains isolated from clinical sites. In addition, antimicrobial peptides have low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight, and are easy to chemically synthesize. They ensure in vivo safety while being highly effective in sterilization.

The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (9)

1. Broad-spectrum antimicrobial peptides bsa049 and bsa086, characterized in that: the antimicrobial peptides are provided in two groups, one of which is the antimicrobial peptide bsa049 having the amino acid sequence shown in SEQ ID: RLWRIVVIRVWR; Another group of antimicrobial peptides is the antimicrobial peptide bsa086 having an amino acid sequence shown in SEQ ID: KKLAGLAKKWAGLAKKLAGLA. 2. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 as claimed in claim 1, characterized in that the antimicrobial peptides are used to prepare inhibitors against bacterial infections. 3. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 as claimed in claim 1, characterized in that the antimicrobial peptides are used to prepare inhibitors against multiple bacterial infections. 4. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 according to claim 2, characterized in that the bacteria are Staphylococcus aureus (ATCC 25923) streaked in TSB medium, Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 43816), Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa (ATCC 27853). 5. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 according to claim 2, characterized in that the minimum inhibitory concentration of the antimicrobial peptide bsa049 against Staphylococcus aureus is 7.81 μg/mL, the minimum inhibitory concentration against Escherichia coli is 7.81 μg/mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 125 μg/mL, the minimum inhibitory concentration against Acinetobacter baumannii is 3.91 μg/mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 31.25 μg/mL. 6. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 according to claim 2, characterized in that the minimum inhibitory concentration of the antimicrobial peptide bsa086 against Escherichia coli is 7.81 μg/mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 125 μg/mL, the minimum inhibitory concentration against Acinetobacter baumannii is 1.95 μg/mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 31.25 μg/mL. 7. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 according to claim 3, characterized in that the multiple antibacterials are Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. 8. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 according to claim 3, characterized in that the minimum inhibitory concentration of the antimicrobial peptide bsa049 against multidrug-resistant strains of Escherichia coli reaches 15.6 μg/mL, the minimum inhibitory concentration against three multidrug-resistant strains of Acinetobacter baumannii is 15.6 μg/mL; the minimum inhibitory concentration against three multidrug-resistant strains of Klebsiella pneumoniae is 15.6 μg/mL. 9. The use of the broad-spectrum antimicrobial peptides bsa049 and bsa086 according to claim 3, characterized in that the minimum inhibitory concentration of the antimicrobial peptide bsa086 against multidrug-resistant strains of Escherichia coli reaches 7.8 μg/mL, the minimum inhibitory concentration against multidrug-resistant strains of Acinetobacter baumannii reaches 3.9 μg/mL, and the minimum inhibitory concentration against multidrug-resistant strains of Klebsiella pneumoniae is 31.3 μg/mL.