CN115925872B - Antibacterial peptide SKL17-2 targeting pseudomonas deformans and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide SKL17-2 targeting pseudomonas deformans and application thereof. The full sequence of the antibacterial peptide SKL17-2 is Ser-Ala-Leu-Lys-Gly-Leu-Arg-Lys-Lys-Met-Lys-Arg-Leu-Lys-Gln-Arg-Leu, which comprises 17 amino acid residues, has a molecular weight of 2054.6 Da and an isoelectric point of 12.4, and belongs to alkaline polypeptides. The antibacterial peptide SKL17-2 has narrow-spectrum bactericidal activity, can specifically kill pseudomonas deformans at low concentration, and has weaker antibacterial activity on other gram-positive bacteria and gram-negative bacteria. Meanwhile, SKL17-2 has weak hemolytic activity and small cytotoxicity, can replace antibiotics, and is expected to be applied to prevention and treatment of fish visceral ichthyophthiriasis caused by pseudomonas deformans.

Description

An antimicrobial peptide SKL17-2 targeting Pseudomonas aeruginosa and its application

Technical Field

The present invention relates to the field of biotechnology, and in particular to an antimicrobial peptide SKL17-2 targeting Pseudomonas aeruginosa and an application thereof.

Background Art

Antimicrobial peptides are small molecule polypeptides with antimicrobial activity, which participate in the body's resistance to microbial infection. Antimicrobial peptides mainly exert their bactericidal effects through membrane targeting and non-membrane targeting. During the membrane targeting process, cationic antimicrobial peptides interact with the negative charges on the bacterial outer membrane, and then the hydrophobic region of the antimicrobial peptides is inserted into the cell membrane, causing changes in the cell membrane conformation and forming holes, leading to the excretion of intracellular ions or nutrients in bacteria, and ultimately leading to bacterial lysis and death. In the non-membrane targeting process, after the interaction between antimicrobial peptides and microbial cell membranes, antimicrobial peptides can penetrate into the cells, but this process does not change the permeability of the cell membrane. When antimicrobial peptides enter the cells, they can bind to intracellular molecules, resulting in the accumulation of a large number of antimicrobial peptides in the cytoplasm, thereby inhibiting the synthesis of bacterial DNA, RNA, protein and cell wall, causing bacterial death.

Antimicrobial peptides have high efficiency and broad-spectrum bactericidal effects and have broad application prospects. However, naturally occurring antimicrobial peptides have a series of problems such as weak biological activity, high toxicity, and poor stability, which seriously limit and hinder their application. Therefore, based on in-depth research on the structural-functional relationship of antimicrobial peptides, the development of new artificial synthetic peptides through artificial design is the best way to solve the bottleneck of antimicrobial peptide application. Since antimicrobial peptides can exert bactericidal activity by destroying bacterial cell membranes, antimicrobial peptides can not only kill pathogens but also kill probiotics in the body during use. In order to reduce the killing activity of antimicrobial peptides on probiotics in the body during use, artificially designed antimicrobial peptides should have narrow-spectrum bactericidal activity, that is, antimicrobial peptides can specifically kill target pathogenic microorganisms and have no or very low killing activity on other microorganisms.

Pseudomonas plecoglossicida is a Gram-negative bacterium with a rod-shaped morphology and polar flagella that can infect fish such as large yellow croaker, grouper and rainbow trout. When Pseudomonas plecoglossicida infects fish, it can cause white nodules in the spleen, kidney and liver of the fish, so the disease is called visceral white spot disease. Visceral white spot disease has caused huge economic losses to the aquaculture industry and seriously restricted the development of aquaculture. The development of antimicrobial peptides that can specifically kill Pseudomonas plecoglossicida can provide potential therapeutic drugs for the prevention and treatment of visceral white spot disease in aquaculture animals.

Summary of the invention

In order to obtain an antimicrobial peptide that can specifically kill Pseudomonas aeruginosa and has good biosafety, the present invention designs an antimicrobial peptide SKL17-2, which has a targeted selection ability for target bacteria.

The purpose of the present invention is achieved through the following technologies:

The present invention first provides an antimicrobial peptide SKL17-2, the amino acid sequence of the antimicrobial peptide SKL17-2 being: Ser-Ala-Leu-Lys-Gly-Leu-Arg-Lys-Lys-Met-Lys-Arg-Leu-Lys-Gln-Arg-Leu.

The present invention further provides a method for preparing the antimicrobial peptide SKL17-2, which is as follows:

(1) Using the large yellow croaker IFN-γrel protein sequence as a template, a 17-amino acid linear peptide was extracted to obtain the peptide SKL17. The amino acid sequence of the peptide SKL17 is: Ser-Ala-Leu-Lys-Gly-Leu-Glu-Lys-Lys-Met-Lys-Glu-Leu-Lys-Gln-Arg-Leu;

(2) The negatively charged glutamic acid in the polypeptide SKL17 is replaced with the positively charged arginine, so that the amino acid sequence of the antimicrobial peptide SKL17-2 is Ser-Ala-Leu-Lys-Gly-Leu-Arg-Lys-Lys-Met-Lys-Arg-Leu-Lys-Gln-Arg-Leu;

(3) The antimicrobial peptide SKL17-2 was synthesized by solid phase chemical synthesis.

The present invention also provides the use of the antimicrobial peptide SKL17-2 in the preparation of a drug targeting Pseudomonas aeruginosa.

The present invention also provides the use of the antimicrobial peptide SKL17-2 in the preparation of feed additives.

The antimicrobial peptide SKL17-2 of the present invention has the following advantages and beneficial effects: the antimicrobial peptide SKL17-2 prepared by the present invention has a narrow-spectrum bactericidal activity, can specifically kill Pseudomonas aeruginosa at low concentrations, and has weak antibacterial activity against other Gram-positive bacteria and Gram-negative bacteria. At the same time, the antimicrobial peptide SKL17-2 also has the characteristics of low hemolytic activity and low cytotoxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the mass spectrum of the antimicrobial peptide SKL17-2.

FIG. 2 is a graph showing the bactericidal activity of the antimicrobial peptide SKL17-2 against Pseudomonas aeruginosa.

FIG3 is a graph showing the growth curves of Gram-positive and Gram-negative bacteria after treatment with the antimicrobial peptide SKL17-2.

FIG. 4 is a graph showing the hemolytic activity of the antimicrobial peptide SKL17-2 on red blood cells.

FIG. 5 is a graph showing the cytotoxicity of the antimicrobial peptide SKL17-2 to LYC-FM cells.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments:

Example 1: Design and synthesis of antimicrobial peptide SKL17-2, the specific steps are:

(1) Using the large yellow croaker IFN-γrel protein sequence as a template, a 17-amino acid linear peptide was extracted to obtain the peptide SKL17. The antimicrobial peptide SKL17-2 was obtained by replacing the 7th and 12th Glu of the peptide SKL17 with Arg;

The amino acid sequence of the linear peptide SKL17 is:

Ser-Ala-Leu-Lys-Gly-Leu-Glu-Lys-Lys-Met-Lys-Glu-Leu-Lys-Gln-Arg-Leu.

The amino acid sequence of the antimicrobial peptide SKL17-2 is:

Ser-Ala-Leu-Lys-Gly-Leu-Arg-Lys-Lys-Met-Lys-Arg-Leu-Lys-Gln-Arg-Leu.

Compared with SKL17, the theoretical molecular weight of SKL17-2 changed from 2000.46 to 2054.6, the net charge increased from +4 to +8, and the isoelectric point changed from 10.8 to 12.4.

The mass spectrum of the antimicrobial peptide SKL17-2 is shown in Figure 1 .

(2) SKL17-2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the solid-phase chemical synthesis method.

Example 2: Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide SKL17-2, the specific steps are:

(1) Pick the test strain into liquid culture medium, shake and culture until the logarithmic growth phase, and adjust the bacterial liquid concentration to 2×10 ⁵ CFU/mL with culture medium;

(2) Take 20 µL of the antimicrobial peptide SKL17-2 with a starting concentration of 1280 µM and mix it with 80 µL of 1×PBS (pH=7.4), then take 50 µL of the diluted antimicrobial peptide SKL17-2 and dilute it serially to the final concentrations of 1, 2, 4, 8, 16, 32, and 64 µM;

(3) 50 µL of the diluted antimicrobial peptide SKL17-2 solution (1–64 µM) was added to columns 1-7 of the 96-well plate, and then 50 µL of bacterial solution was added to each well. 50 µL of bacterial solution and 50 µL of 1× PBS (pH=7.4) were added to the positive control in column 8, and 50 µL of culture medium and 50 µL of 1× PBS (pH=7.4) were added to the negative control in column 9. Three replicates were set up for each well. The above process was completed within 15 min.

(4) Place the 96-well plate in an incubator for 12-18 h. After taking out the plate, the minimum antimicrobial peptide SKL17-2 concentration at which no turbidity is visible at the bottom of the well is the minimum inhibitory concentration.

(5) Place the 96-well plate in a spectrophotometer and read the absorbance at 600 nm. Use the concentration of the antimicrobial peptide SKL17-2 as the horizontal axis and the absorbance value as the vertical axis to draw the growth curves of different test bacteria.

As shown in Figure 2, the antimicrobial peptide SKL17-2 has strong antibacterial activity against Pseudomonas aeruginosa, with the minimum inhibitory concentration and the minimum bactericidal concentration being 2 μM and 4 μM, respectively. As shown in Figure 3, the antimicrobial peptide SKL17-2 has no antibacterial activity against other tested Gram-positive and Gram-negative bacteria, or shows very weak antibacterial activity. This indicates that the antimicrobial peptide SKL17-2 of the present invention can target Pseudomonas aeruginosa to exert antibacterial activity.

Example 3: Determination of antibacterial activity of antimicrobial peptide SKL17-2, the specific steps are:

(1) Add Pseudomonas aeruginosa in the logarithmic growth phase to a solid culture medium at a temperature of about 50°C. The final concentration of the culture solution is 1×10 ⁵ CFU/mL. Each mixed culture plate contains about 12 mL of culture medium. Place the mixed culture plate horizontally to cool and solidify.

(2) After the mixed bacterial plate solidifies, use a 1 mL pipette tip to make a hole;

(3) The antimicrobial peptide SKL17-2 was diluted to 520 µM with sterile water, and 60 µL was added to each well. Sterile water was used as a negative control.

(4) Place the mixed bacterial plate in a constant temperature incubator for 12 to 24 hours.

As shown in Figure 2, a transparent inhibition zone was shown around the wells to which the antimicrobial peptide SKL17-2 was added, indicating that the antimicrobial peptide SKL17-2 had good antibacterial activity against Pseudomonas aeruginosa.

Example 4: Determination of hemolytic activity of antimicrobial peptide SKL17-2, the specific steps are:

(1) Anticoagulant was added to the blood of large yellow croaker and centrifuged at 500 g for 10 min;

(2) Discard the supernatant, resuspend in 1× PBS (pH=7.4), and centrifuge at 500 g for 10 min. Repeat three times.

(3) Resuspend the cells in 1× PBS (pH=7.4) and adjust the cell concentration to 1.5×10 ⁸ cells/mL;

(4) Add 120 μL of cell suspension to a 96-well plate, add 80 μL of different concentrations of antimicrobial peptide SKL17-2 to a final concentration of 1, 2, 4, 8, 16, 32, 64, and 128 μM, and add 80 μL of 5% Triton-X 100 and 80 μL of 1× PBS (pH = 7.4) to the control wells as controls for 100% and 0% hemolysis, respectively. Set up 3 replicates for each group;

(5) Incubate the cells at 28°C for 1 h, centrifuge and transfer 100 µL of the supernatant to a 96-well plate, and read the absorbance at 405 nm.

(6) Calculation of hemolytic activity:

Hemolytic activity = [(Apeptide - A0% lysis)/(A100% lysis - A0% lysis)] × 100%, A is the absorbance at 405 nm.

The test results are shown in FIG4 . The hemolytic rates of the antimicrobial peptide SKL17-2 at each test concentration on the red blood cells of large yellow croaker are all lower than 0.5%, indicating that the antimicrobial peptide SKL17-2 of the present invention has little hemolytic effect on red blood cells.

Example 5: Cytotoxicity assay of antimicrobial peptide SKL17-2, the specific steps are as follows:

(1) Adjust the concentration of large yellow croaker macrophage LYC-FM cells to 2×10 ⁵ cells/mL, add 100 µL of cell suspension to each well of a 96-well plate, and culture at 28°C overnight;

(2) Remove the culture medium and add 100 µL of fresh culture medium containing the antimicrobial peptide SKL17-2 to each well. The final concentration of the antimicrobial peptide SKL17-2 is 1, 2, 4, 8, 16, 32, 64, and 128 µM. Add 60 µL of blank culture medium mixed with 40 µL of 5% Triton X-100 and 60 µL of blank culture medium mixed with 40 µL of 1× PBS (pH=7.4) to the control wells as controls for 100% and 0% cytotoxicity. Set up 3 replicates for each group.

(3) After 24 h of static incubation at 28°C, add 10 µL of CCK-8 solution to each well, mix well, continue incubation at 28°C for 4 h, and read the absorbance at 450 nm.

(4) Calculate the live cell ratio:

Live cell ratio = [(Apeptide – A100% lysis)/(A0% lysis - A100% lysis)] × 100%, where A is the absorbance at 450 nm.

The test results are shown in Figure 5. The cell survival rate of the antimicrobial peptide SKL17-2 at each test concentration on large yellow croaker macrophage LYC-FM was higher than 70%, indicating that the antimicrobial peptide SKL17-2 of the present invention has little cytotoxicity.

In summary, the antimicrobial peptide SKL17-2 of the present invention has a narrow spectrum bactericidal activity, can specifically kill Pseudomonas aeruginosa at low concentrations, and has weak antibacterial activity against other Gram-positive bacteria and Gram-negative bacteria. At the same time, the antimicrobial peptide SKL17-2 also has the characteristics of low hemolytic activity and low cytotoxicity. Therefore, the antimicrobial peptide SKL17-2 of the present invention is expected to be preferably used in the preparation of anti-Pseudomonas aeruginosa drugs and feed additives.

The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims (3)

1. An antimicrobial peptide SKL17-2, characterized in that its amino acid sequence is: Ser-Ala-Leu-Lys-Gly-Leu-Arg-Lys-Lys-Met-Lys-Arg-Leu-Lys-Gln-Arg-Leu. 2. Use of the antimicrobial peptide SKL17-2 according to claim 1 in the preparation of a drug for killing Pseudomonas aeruginosa. 3. Use of the antimicrobial peptide SKL17-2 according to claim 1 in the preparation of feed additives.