Xenopus laevis engineered antimicrobial peptide and application thereof

Abstract

The application discloses an X. laevis modified antibacterial peptide and application thereof, and belongs to the technical field of biological medicine. The X. laevis modified antibacterial peptide is pFG-11K or pFG-11K-8K; the amino acid sequence of pFG-11K is shown as SEQ ID NO. 2; and the amino acid sequence of pFG-11K-8K is shown as SEQ ID NO. 3. The modified antibacterial peptide obtained by modifying the antibacterial peptide pFG of X. laevis has broad-spectrum antibacterial property (especially pFG-11K-8K), has strong bactericidal and bacteriostatic effects on methicillin-resistant Staphylococcus aureus in particular, has almost no hemolytic toxicity on defibrillated horse red blood cells, and can be used for preparing antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antimicrobial peptide derived from an African Xenopus laevis and its applications. Background Technology

[0002] With the widespread use of broad-spectrum antibiotics and antimicrobial drugs, a large number of drug-resistant bacteria have emerged. The World Health Organization has published a list of multidrug-resistant bacteria, collectively known as ESKAPE bacteria. This necessitates the urgent advancement of antimicrobial drug development. Carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa are the two most drug-resistant groups among the listed Gram-negative bacteria. Carbapenem antibiotics are used as a "last resort" when all other options fail, but they are highly likely to lead to the emergence and spread of antimicrobial resistance (AMR). Among the three listed Gram-positive bacteria, methicillin-resistant and vancomycin-resistant Staphylococcus aureus have the most serious drug resistance situation globally.

[0003] Antimicrobial peptides (AMPs), as a type of novel antimicrobial drug, possess advantages over traditional antimicrobial drugs, including high safety, broad-spectrum antimicrobial activity, strong stability, and low likelihood of inducing bacterial resistance. They show great potential in combating bacterial infections and are expected to become a promising antimicrobial treatment strategy. Furthermore, antimicrobial peptides exhibit multiple antimicrobial mechanisms, such as disrupting bacterial cell membranes and even inducing reactive oxygen species (ROS) generation. This mechanism makes it difficult for bacteria to develop resistance through a single mutation. Therefore, the development of antimicrobial peptides from plants or animals is of great significance for addressing the problem of bacterial resistance. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an African Xenopus mutant antimicrobial peptide and its application. The mutant antimicrobial peptides pFG-11K and pFG-11K-8K obtained by modifying the antimicrobial peptide pFG derived from the African Xenopus have broad-spectrum antimicrobial activity (especially pFG-11K-8K), and have particularly strong bactericidal and bacteriostatic effects against methicillin-resistant Staphylococcus aureus. They also have almost no hemolytic toxicity to defibrinated horse erythrocytes and can be used to prepare antimicrobial drugs.

[0005] This invention is achieved through the following technical solution: In a first aspect, the present invention provides an antimicrobial peptide derived from Xenopus laevis, wherein the antimicrobial peptide derived from Xenopus laevis is pFG-11K or pFG-11K-8K. The amino acid sequence of pFG-11K is shown in SEQ ID NO.2; The amino acid sequence of pFG-11K-8K is shown in SEQ ID NO.3.

[0006] In a second aspect, the present invention provides the application of the aforementioned Xenopus laevis-modified antimicrobial peptide in the preparation of antimicrobial drugs.

[0007] Furthermore, the antibacterial drug is used to inhibit Gram-positive and Gram-negative bacteria.

[0008] Furthermore, the Gram-positive bacteria are one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Enterococcus faecalis; the Gram-negative bacteria are one or more of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0009] Furthermore, the aforementioned antibacterial drug is used to inhibit methicillin-resistant Staphylococcus aureus.

[0010] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The modified Xenopus laevis antimicrobial peptides pFG-11K and pFG-11K-8K (especially pFG-11K-8K) of this invention exhibit good broad-spectrum antimicrobial activity, showing certain antimicrobial activity against both Gram-negative and Gram-positive bacteria, particularly against methicillin-resistant Staphylococcus aureus. Furthermore, the modified antimicrobial peptide pFG-11K-8K exhibits low hemolytic activity against sterile defibrinated horse erythrocytes and low hepatotoxicity to mice. The modified antimicrobial peptides pFG-11K and pFG-11K-8K of this invention can serve as potential broad-spectrum antimicrobial drugs. Attached Figure Description

[0011] Figure 1 The sterilization curves of pFG, pFG-11K, and pFG-11K-8K are shown. Figure 2 Figure 1 shows the results of alanine aminotransferase (ALT) tests in the blood of mice in different treatment groups. Figure 3 Figure 1 shows the results of creatinine (CREA) testing in the blood of mice in different treatment groups. Figure 4 Figure 1 shows the results of total bilirubin (TBIL) tests in the blood of mice in different treatment groups. Figure 5 Figure 1 shows the results of blood urea nitrogen (BUN) tests in mice from different treatment groups. Figure 6 Figure 1 shows the results of blood aspartate aminotransferase (AST) tests in mice from different treatment groups. Figure 7 Figure 1 shows the results of alkaline phosphatase (ALP) tests in the blood of mice in different treatment groups. Detailed Implementation

[0012] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0014] Example 1 Based on the antimicrobial peptide pGQL (Accession: NP_001091262) derived from Xenopus laevis, the complete sequence of the original protein was obtained by searching the NCBI proteome database using the online tool BLAST. An antimicrobial peptide was then obtained from the original protein sequence and named pFG. The amino acid sequence of pFG (SEQ ID NO.1) is: FLSFLLGPLIDLISKG; To improve the antibacterial activity of the antimicrobial peptide pFG while reducing its hemolytic toxicity, pFG was modified by replacing the aspartic acid at position 11 with lysine, resulting in pFG-11K, with the amino acid sequence (SEQ ID NO.2) being: FLSFLLGKLIKLISKG. Based on the antibacterial activity and hemolytic toxicity results, further modification was performed by replacing the proline at position 8 with lysine in pFG-11K. This improved the antibacterial activity while reducing the hemolytic toxicity to a safe level, resulting in pFG-11K-8K, with the amino acid sequence (SEQ ID NO.3) being: FLSFLLGKLIKLISKG.

[0015] Example 2 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial peptide pFG, the modified antimicrobial peptide pFG-11K, and pFG-11K-8K in Example 1 were determined. The bacteria used in this example were Gram-negative and Gram-positive bacteria, respectively. The Gram-negative bacteria were: *Escherichia coli* NCTC 13846 (… E. coli 13846), NCTC 10418 ( E. coli 10418), ATCC 25922 ( E. coli 25922), ATCC 2340 ( E. coli 2340), Pseudomonas aeruginosa ATCC 27853 ( Pa27853), Klebsiella pneumoniae (ATCC 43816) K. pneumoniae 43816); Gram-positive bacteria include: Staphylococcus aureus (ATCC 6538) S.auraus 6538), NCTC 10788 ( S.auraus 10788), Methicillin-resistant Staphylococcus aureus (NCTC 12493) MRSA 12493 ), and Enterococcus faecalis ATCC 29212 ( E.faecalis 29212), NCTC 12697 ( E.faecalis 12697). All the above standard strains were purchased from the American Type Culture Collection (ATCC) or the National Culture Collection (NCTC) of the United Kingdom. All the above standard strains were inoculated into nutrient broth (NB) medium and cultured.

[0016] (1) Preparation of bacterial culture: The above-mentioned Gram-negative and Gram-positive bacteria were cultured in a dual-function air bath constant temperature shaker (ZD-85, Changzhou Jintan Liangyou Instrument Co., Ltd.) at 37℃ and 180 rpm for 18 h. The absorbance was measured using a UV-Vis spectrophotometer, and the concentration was adjusted to achieve the desired OD value for the Gram-positive bacteria. 550 The OD value for Gram-negative bacteria was 0.23. 550 The value is 0.4, at which point the bacterial concentration is 1×10⁻⁴. 8 CFU / mL, then the bacterial solution was diluted 200 times with culture medium; (2) Using DMSO as the solvent, the storage concentrations of pFG, pFG-11K, and pFG-11K-8K were 12800 µM. They were diluted according to a concentration gradient to 6400 µM, 3200 µM, 1600 µM, 800 µM, 400 µM, 200 µM, and 100 µM. 1 µL of the corresponding peptide was spotted in each well of a 96-well plate. The antimicrobial peptide concentration was then diluted 100-fold by adding bacterial culture medium (i.e., the concentrations were 128 µM, 64 µM, 32 µM, 16 µM, 8 µM, 4 µM, 2 µM, and 1 µM). This was repeated in three wells. A bacterial culture without the drug was used as a positive control, and a blank culture medium was used as a negative control. Vancomycin, ciprofloxacin, and ceftazidime pentahydrate were also used. Polymyxin E sulfate (dimePentahydrate) and Colistin Sulfate were used as positive controls. The samples were incubated at 37°C for 18 hours. OD values ​​were measured using a multi-functional microplate reader. 550 The minimum inhibitory concentration (MIC) was determined by the readings, and the results are shown in Table 1 below. For concentrations of MIC and above, 10 µL of each well from three replicates of each concentration was taken and spotted onto the corresponding agar plate. The plates were then incubated at 37°C for 18 h. The minimum bactericidal concentration at which no bacterial growth was observed by visual inspection was defined as MBC, and the results are shown in Table 2 below.

[0017] Table 1. Test values ​​of pFG, pFG-11K, and pFG-11K-8K for different strains. Note: NA indicates no significant activity at 128 μM. Table 2. Test results of MBC (μM) values ​​of pFG, pFG-11K, and pFG-11K-8K for different strains. Note: NA indicates no significant activity at 128 μM. As shown in Tables 1 and 2 above, pFG has no antibacterial activity against Gram-positive and Gram-negative bacteria. After modification, pFG-11K exhibits enhanced antibacterial activity against both Gram-negative and Gram-positive bacteria. pFG-11K-8K shows the strongest antibacterial effect, possessing broad-spectrum antibacterial activity, particularly against methicillin-resistant Staphylococcus aureus, with both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIC) at 4 μM.

[0018] (3) Based on the MIC values ​​of pFG-11K and pFG-11K-8K against MRSA 12493, 2 μL of antimicrobial peptides of 400, 800, and 1600 μM were added to 96-well plates, respectively, and 198 μL of diluted bacterial solution (MRSA 12493) were added and mixed. The concentrations at this time were 1×MIC, 2×MIC, and 4×MIC. A blank control group without drug was set up, i.e., 200 μL of diluted bacterial solution. A solvent control group was set up, i.e., 2 μL of DMSO was mixed with 198 μL of diluted bacterial solution. 90 μL of PBS was added to each well of a 96-well plate beforehand. At 0 min, 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, and 180 min, 10 μL of the 200 μL system was taken from each well and added to 90 μL of PBS, then mixed thoroughly by pipetting. The mixture was then serially diluted 10-fold, 100-fold, 1000-fold, and 10000-fold. 10 μL of the 1000-fold and 10000-fold diluted bacterial solutions were spotted onto the corresponding agar plates, repeated three times. After incubation overnight at 37°C, viable cell counts were performed. A bactericidal curve was plotted with drug action time on the x-axis and colony count on the y-axis. The results are shown below. Figure 1 As shown. By Figure 1 It can be seen that the bactericidal curves corresponding to concentrations of 1×MIC, 2×MIC, and 4×MIC overlap, and pFG-11K and pFG-11K-8K completely killed MRSA 12493 within 5 min.

[0019] Example 3 Hemolytic activity assays of pFG, pFG-11K, and pFG-11K-8K against sterile defibrinated horse blood cells: (1) Add PBS to sterile defibrinated horse blood cells (Beijing Solarbio Science & Technology Co., Ltd.), shake gently, centrifuge at 1000 rpm for 10 min, discard dead blood cells, and obtain the supernatant; repeat the above steps until the supernatant is colorless or pale yellow, and the precipitate retained at this time is sterile defibrinated horse blood live cells; add PBS to the precipitate to prepare a 4% sterile defibrinated horse blood live cell suspension.

[0020] (2) The concentration of antimicrobial peptides (pFG, pFG-11K, and pFG-11K-8K) was prepared to 1024 µM, and then serially diluted with PBS to 512, 256, 128, 64, 32, 16, 8, 4, and 2 µM. After mixing, three replicates were prepared for each concentration, with 100 μL in each tube. 100 µL of 4% sterile defibrinated horse blood cell suspension was added to each of the above tubes and mixed. At this time, the final concentration of the peptides was 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 µM. Sterile defibrinated horse blood cell suspension treated with 1% Triton-100 was used as a positive control, and sterile defibrinated horse blood cell suspension treated with PBS was used as a negative control. Three replicates were prepared for each control. The cells were incubated in a 37°C incubator for 2 h. After incubation, the cells were centrifuged at 1000 rpm for 10 min and 100 μL of the solution was collected. µL of supernatant was added to a 96-well plate according to the above concentration gradient, and the absorbance was measured at 570 nm. The hemolytic rate of the peptide on erythrocytes was calculated using the following formula: Among them, A s A0 is the average absorbance value of the antimicrobial peptide supernatant, and A0 is the average absorbance value of the blank control. p The average absorbance value for the positive control is given by three independent assays.

[0021] Calculate HC 50 The concentration of the drug required to cause 50% of red blood cells to rupture (hemolysis) and the therapeutic index (TI). The TI value is calculated according to HC. 50 / GM calculation. When HC 50 When the value exceeds 128 μM, 256 μM is used to calculate the TI value. Similarly, when no antibacterial activity is detected at a concentration of 128 μM, a concentration of 256 μM is used to calculate the TI value. The geometric mean (GM) is a statistical indicator used to measure the central tendency of a set of positive numbers. For a set of n positive numbers x1, x2, ..., x... n The formula for calculating its geometric mean GM is: Gram + and Gram - These are two main types of bacterial classification based on Gram staining. + Represents Gram-positive bacteria. - Represents Gram-negative bacteria; The test results are shown in Table 3 below: Table 3. Results of hemolytic activity tests for pFG, pFG-11K, and pFG-11K-8K TI>10, high selectivity and good safety; 5<TI ≤ 10, above-average selectivity; 1<TI ≤ 5, moderate selectivity and average safety; TI ≤ 1, hemolytic toxicity ≥ antimicrobial activity; TI<0.1, strong hemolytic toxicity. The results of hemolytic activity show that pFG-11K-8K has a high therapeutic index for blood cells, good safety, low toxicity, and is suitable for in vivo experiments.

[0022] Example 4 Toxicity of pFG-11K-8K to liver and kidney of mice ICR mice (6 mice in each group) were divided into three administration dose groups of 0 mg / kg, 5 mg / kg and 10 mg / kg, and intraperitoneal injection was performed on the mice according to body weight. After a single injection, the animals' behavior and body weight changes were monitored for four consecutive days. Mice in all three groups remained active and exhibited normal behavior. Body weight remained stable or increased slightly over time. After four consecutive days of monitoring, whole blood was collected from the mice via orbital blood collection, serum was separated, and the levels of creatinine (CREA), blood urea nitrogen (BUN), total bilirubin (TBIL), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in serum were detected, so as to evaluate the effect of the test substance pFG-11K-8K on liver and kidney functions of mice and its in vivo toxicity.

[0023] The detection results of the contents of CREA, BUN, TBIL, ALT, ALP and AST in the serum of mice in different treatment groups are respectively shown in Figures 2-7 , and all three dose groups are within the safe range (i.e., the reference interval, which is usually developed based on the distribution of 95% healthy population and is between the two dashed lines in the figure); the above results show that pFG-11K-8K has no toxicity to the liver and kidney of mice.

Claims

1. An antimicrobial peptide derived from an African Xenopus laevis, characterized in that, The modified Xenopus laevis antimicrobial peptide is pFG-11K or pFG-11K-8K; The amino acid sequence of pFG-11K is shown in SEQ ID NO.2; The amino acid sequence of pFG-11K-8K is shown in SEQ ID NO.

3.

2. The use of the African Xenopus modified antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs.

3. The application according to claim 2, characterized in that, The aforementioned antibacterial drug is used to inhibit Gram-positive and Gram-negative bacteria.

4. The application according to claim 3, characterized in that, The Gram-positive bacteria are one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Enterococcus faecalis; the Gram-negative bacteria are one or more of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

5. The application according to claim 4, characterized in that, The aforementioned antibacterial drug is used to inhibit methicillin-resistant Staphylococcus aureus.

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