Broad-spectrum antibacterial peptide SS-7 and application thereof

Abstract

The invention discloses a broad-spectrum antibacterial peptide SS-7 and application thereof, and belongs to the field of biological medicine. The amino acid sequence of the broad-spectrum antibacterial peptide SS-7 disclosed by the invention is VRRVRFVVLRRIKKWRRI, and the broad-spectrum antibacterial peptide SS-7 has a remarkable inhibition effect on various gram-positive bacteria and gram-negative bacteria such as drug-resistant strains such as methicillin-resistant staphylococcus aureus (ATCC43300) and shows low toxicity and no hemolytic toxicity on eukaryotic cells and red blood cells of mammals; lPS and lipoteichoic acid on the surfaces of bacteria can be combined to achieve the effect of neutralizing the bacteria, and a very good treatment and protection effect is achieved on animals infected by the bacteria. The antibacterial peptide SS-7 can be used for preparing an antibacterial agent and preparing a medicine for preventing or treating bacterial infection, has the advantages of being wide in antibacterial spectrum, low in drug-resistant mutation frequency, low in toxicity and low in production cost, can effectively solve the current bacterial drug resistance problem, and has wide application prospects and important clinical value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a broad-spectrum antimicrobial peptide SS-7 and application thereof. Background Art

[0002] Since the advent of antibiotics, they have been widely used in many fields such as medicine, food and agriculture. The problem of bacterial resistance has become increasingly serious due to the irrational use of antibiotics, which seriously threatens human and animal health. With the advent of the post-antibiotic era, there is an embarrassing situation that there is no effective drug treatment for multidrug-resistant pathogens. In the past two decades, the research and development of antibiotics has been slow, and the speed of bacterial resistance has far exceeded the speed of new drug development. Therefore, there is an urgent need to develop new drugs or antibiotic substitutes.

[0003] Antimicrobial peptides play an important role in the host immune defense process. They are widely present in prokaryotes and eukaryotes and can be extracted from nature or synthesized artificially by chemical means. Antimicrobial peptides do not target the common targets of classical antibiotics, but mainly target bacterial membranes, destroying the integrity of bacterial membranes through electrostatic interactions, making it difficult for bacteria to develop resistance to traditional antibiotics as they do to traditional antibiotics. However, the cytotoxicity and immunogenicity of natural antimicrobial peptides may have adverse effects on host cells, especially when used in high concentrations, which increases potential toxic side effects. In contrast, amino acid substitution-modified peptides have multiple advantages. By modifying natural antimicrobial peptides with amino acids, the stability of the peptides can be enhanced, making them more resistant to degradation in the body, thereby prolonging their duration of action. At the same time, amino acid substitution modification can reduce the toxicity and immunogenicity of the peptides and improve their selectivity for host cells. In addition, this modification can also optimize the antibacterial spectrum and activity of the peptides, making them more effective against multiple drug-resistant strains. Through these modifications, the prospects for drug development of antimicrobial peptides have been further expanded, making them effective alternatives to broad-spectrum antibiotics for anti-infective treatment. Summary of the invention

[0004] The object of the present invention is to provide a novel antimicrobial peptide SS-7 having broad-spectrum antimicrobial activity, low toxicity and no drug-resistant mutation, and its application in the antimicrobial field.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The novel antimicrobial peptide SS-7 provided by the present invention has broad-spectrum antimicrobial activity, low toxicity and no drug resistance mutation. It is based on suis streptolysin as a template (GenBank accession number JF813299), and a sequence RFVVLRREKK (SEQ ID NO.1) with antimicrobial peptide characteristics is selected and recorded as SS-1, and then a short sequence with a high degree of α helix is ​​added to the C segment and N segment of the sequence, and glutamic acid in the sequence is replaced by isoleucine, which is recorded as SS-7, and its amino acid sequence is VRRVRFVVLRRIKKWRRI (SEQ ID NO.7). The helical wheel projection of the antimicrobial peptide SS-7 is as follows: Figure 1 As shown. In vitro antibacterial experiments, cytotoxicity experiments, hemolytic activity experiments, drug-resistant mutation frequency experiments and bacterial membrane component LPS and lipoteichoic acid neutralization experiments showed that the antimicrobial peptide SS-7 has a broad-spectrum antibacterial activity and can effectively inhibit the growth of Gram-positive and Gram-negative bacteria. In addition, the antimicrobial peptide SS-7 exhibits low toxicity and no hemolytic toxicity to mammalian eukaryotic cells and erythrocytes, and the antimicrobial peptide SS-7 achieves a neutralization effect by binding to the negatively charged LPS and lipoteichoic acid on the bacterial surface. Animal experiments have shown that the antimicrobial peptide SS-7 has a good therapeutic and protective effect on animals infected with multidrug-resistant Streptococcus suis SC19, and is superior to the commonly used clinical drug ampicillin.

[0007] The present invention finds that the antimicrobial peptide SS-7 has a broad-spectrum antibacterial effect, low toxicity and no hemolytic toxicity, and has a therapeutic and protective effect on mice infected with Streptococcus suis SC19. Based on this, the present invention provides the following applications of the antimicrobial peptide SS-7:

[0008] Application of antimicrobial peptide SS-7 in antibacterial activities.

[0009] Application of antimicrobial peptide SS-7 in the preparation of broad-spectrum antimicrobial agents.

[0010] The invention discloses an application of antimicrobial peptide SS-7 in the preparation of a broad-spectrum drug for preventing or treating bacterial infection. The antimicrobial peptide SS-7 realizes the application by antibacterial effect and inhibiting the production of inflammatory factors caused by bacterial infection.

[0011] A broad-spectrum antibacterial agent comprises the antibacterial peptide SS-7 and may further comprise pharmaceutically acceptable excipients.

[0012] A broad-spectrum drug for preventing or treating bacterial infection, comprising the antimicrobial peptide SS-7, and further comprising pharmaceutically acceptable excipients.

[0013] The bacteria or germs include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria include Staphylococcus aureus, Listeria, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, etc., and the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, and Salmonella, etc.

[0014] The present invention has the following advantages and beneficial effects:

[0015] (1) The antimicrobial peptide SS-7 of the present invention exhibits significant broad-spectrum antibacterial activity against a variety of pathogens, including Gram-positive bacteria and Gram-negative bacteria.

[0016] (2) The antimicrobial peptide SS-7 of the present invention exhibits low host cell toxicity while maintaining high antimicrobial activity, thereby reducing potential adverse effects on host cells and improving the safety of clinical application.

[0017] (3) The antimicrobial peptide SS-7 treated mice infected with Streptococcus suis, reduced the inflammatory response and tissue bacterial load, and significantly improved the survival rate of mice.

[0018] (4) The antimicrobial peptide SS-7 is suitable for large-scale synthetic production through optimized design. Compared with other antimicrobial peptides, its preparation cost is lower and it has good production cost-effectiveness, which is conducive to wide clinical promotion and application.

[0019] (5) The present invention provides a new strategy for the research of antibiotic alternatives, especially in combating multidrug-resistant bacteria, and has important clinical value. It is expected to become an important supplement or alternative in the field of antibacterial therapy in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the helical wheel projection diagram of the antimicrobial peptide SS-7, showing that it has good amphiphilicity.

[0021] Figure 2 The cytotoxicity of the antimicrobial peptide SS-7 in Example 2 to two mammalian eukaryotic cells: A: mouse macrophage RAW264.7; B: green monkey kidney cell Vero (ATCC CCL-81).

[0022] Figure 3 The hemolytic toxicity of the antimicrobial peptide SS-7 in Example 3 to defibrinated sheep erythrocytes.

[0023] Figure 4 This is a graph showing the results of the drug resistance analysis of Streptococcus pneumoniae and Staphylococcus aureus to the antimicrobial peptide SS-7 in Example 4.

[0024] Figure 5 It is a kinetic reaction curve diagram of the antimicrobial peptide SS-7 in Example 5 and the bacterial membrane components lipoteichoic acid LTA (A) and LPS (B).

[0025] Figure 6 This is a survival curve of mice infected with Streptococcus suis in Example 6.

[0026] Figure 7 These are graphs showing the results of detecting the bacterial load in the liver and spleen of mice infected with Streptococcus suis (A, B) and TNF-α and IL-6 in the serum (C, D) in Example 7. DETAILED DESCRIPTION

[0027] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0028] The antimicrobial peptide SS-7 used in the following examples was synthesized by commissioning GenScript Corporation using the Fmoc solid phase synthesis scheme; the statistical analysis of the results of the examples was performed using a two-tailed unpaired t-test, *: P<0.05, **: P<0.01, ***: P<0.001.

[0029] Example 1

[0030] By using the antimicrobial peptide database CAMPR3 to predict the antimicrobial peptides of SLY protein sequences, it was found that the sequence RFVVLRREKK (SS-1) has the potential to develop into an antimicrobial peptide. This sequence consists of amino acid residues 69-78 of the SLY protein sequence. After different modifications, a total of 7 peptides were obtained. According to the CLSI antimicrobial drug sensitivity test operation standards, the minimum inhibitory concentrations of the 7 antimicrobial peptides against Staphylococcus aureus and Escherichia coli were determined by the classic micro-continuous two-fold dilution method. The experiment was repeated 3 times in parallel. The results are shown in Table 1. The minimum inhibitory concentration of SS-7 is much lower than that of other antimicrobial peptides.

[0031] Table 1 Antibacterial activity of different peptides

[0032]

[0033]

[0034] Example 2

[0035] According to the CLSI antimicrobial susceptibility test operating standards, the classic micro-serial two-fold dilution method was used to determine the minimum inhibitory concentration of the antimicrobial peptide SS-7 against Gram-positive bacteria (Staphylococcus aureus, Listeria, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Salmonella). The experiment was repeated three times in parallel. The results are shown in Table 2.

[0036] Table 2 Minimum inhibitory concentration of antimicrobial peptide SS-7 against common strains

[0037]

[0038] Example 3

[0039] The cytotoxicity experiment of antimicrobial peptide SS-7 on mammalian cells was conducted on RAW264.7 and Vero cells using CCK8 assay. The cells were cultured and activated in DMEM medium containing 10% fetal bovine serum, transferred to a 96-well culture plate, 0.2 mL per well, and cultured for 24 hours at 5% carbon dioxide and 37°C for later use; when the cells filled the 96-well plate, the cells were washed with PBS, and fresh culture medium containing 0, 2, 4, 8, 16, 32, 64, and 128 μg / mL antimicrobial peptide was added to continue culturing for 24 hours. At the same time, culture medium containing 2.5% Triton X-100 or culture medium was used as positive and negative controls for 24 hours, and culture medium without cells was used as a blank control. The cell culture medium was replaced with a culture medium containing 10% CCK8, and the OD was read using a spectrophotometer after continuing to culture for 2 hours. 450 The absorbance at 100 nm was measured, and three biological replicates were set. The calculation formula is: Cell survival rate = [(absorbance of experimental well - absorbance of blank well) / (absorbance of negative control well - absorbance of blank well)] × 100%.

[0040] Results Figure 2 The antimicrobial peptide SS-7 had no significant effect on the viability of both cells within the concentration range of 2-128 μg / mL. This shows that the antimicrobial peptide SS-7 has no obvious toxicity to mammalian cells.

[0041] Example 4

[0042] The following experiment uses fresh defibrinated sheep red blood cells for hemolysis experiment to detect the hemolytic toxicity of antimicrobial peptide SS-7. Fresh sheep red blood cells are aspirated, washed with PBS, and centrifuged at 1000r / min and 4°C for 10 minutes. Different concentrations of antimicrobial peptides are added to the suspension containing 2% fresh defibrinated sheep red blood cells, and 2.5% Triton X-100 is added as a positive hemolysis control, and red blood cells containing only PBS are used as a negative control. Incubate at 37°C for 6 hours, then centrifuge at 1000r / min for 10 minutes, aspirate the supernatant and measure the absorbance at 543nm, and set up three biological replicates. The hemolysis rate is calculated according to the following formula: Hemolysis rate (%) = [(OD 543 Antimicrobial peptide-OD 543 Negative control) / (OD 543 2.5% Triton X-100-OD 543 negative control)] × 100%.

[0043] Results Figure 3Compared with the blank control without drug, the antimicrobial peptides did not cause obvious hemolysis of erythrocytes within the concentration range of 2-128 μg / mL, and the hemolytic toxicity produced was negligible.

[0044] Example 5

[0045] The frequency of drug-resistant mutations was evaluated by continuously monitoring the MIC values ​​of the antimicrobial peptide SS-7 against Streptococcus pneumoniae ATCC 49619 and Staphylococcus aureus ATCC25923. Streptococcus pneumoniae ATCC 49619 and Staphylococcus aureus ATCC25923 were activated overnight and transferred at a ratio of 1:100. The bacterial volume was adjusted to 5×10 5 CFU / mL, sub-MIC concentrations of ampicillin and antimicrobial peptide SS-7 were co-incubated with bacteria, and the surviving bacteria were tested for MIC after 24 h of culture. Then, a portion of the bacteria were co-incubated with the newly measured sub-MIC concentrations of ampicillin or SS-7. The MIC was tested in this way for 30 consecutive days, and the resistance curve was drawn based on the change in the MIC value.

[0046] Results Figure 4 After 30 generations, Streptococcus pneumoniae and Staphylococcus aureus did not acquire resistance to SS-7. In contrast, after 30 consecutive passages, the MIC values ​​of ampicillin against Streptococcus and Staphylococcus aureus were 32 times and 64 times higher than the original values, respectively. The results showed that SS-7 showed a lower resistance development trend compared with traditional antibiotics.

[0047] Example 6

[0048] The equilibrium dissociation constants of antimicrobial peptide SS-7 and two bacterial membrane components, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), were determined by isothermal titration calorimetry (ITC) technology to prove the interaction between antimicrobial peptide SS-7 and two bacterial membrane components. First, the antimicrobial peptide SS-7 and the two bacterial membrane components were degassed, and the nano ITC software was opened for preheating. The instrument was cleaned with 300 mL of Dikang detergent, ultrapure water and PBS buffer respectively. The instrument setting parameters were as follows: temperature 25℃, number of sample injections 20, 2.5 μL per injection. 300 μL of degassed 50 μmol / L LPS or LTA was drawn with an injection needle and slowly added to the reaction cell to avoid the generation of bubbles. At the same time, 300 μL of ultrapure water was added to the reference cell as a control. 50 μL of 500 μmol / L antimicrobial peptide SS-7 was drawn with a titration needle, and titration was performed after the instrument was balanced. The titration curve was analyzed by Nano Analyzer, and the binding capacity and binding curve were analyzed by Independent model.

[0049] Results Figure 5 The equilibrium binding constant (KD) of the antimicrobial peptide SS-7 and LPS was 2.25 μM, and the stoichiometric ratio was 1.206:1, while the equilibrium binding constant (KD) of the antimicrobial peptide SS-7 and LTA was 12.13 μM, and the stoichiometric ratio was 1.863:1, which indicated that the antimicrobial peptide SS-7 had a strong binding affinity with LPS and LTA.

[0050] Example 7

[0051] All animal experiments were conducted under the guidance of the Animal Experiment Protection and Supervision Committee of Huazhong Agricultural University (HZAUMO-2021-0008). Six-week-old 16±2g BALB / c female mice (purchased from Huazhong Agricultural University) were randomly divided into four groups, with 10 mice in each group. 200 μL of 1×10 9 CFU / mL of Streptococcus suis SC19 were used for infection. One hour after infection, PBS, antimicrobial peptide SS-7 (10 mg / kg) or ampicillin (10 mg / kg) were intraperitoneally administered for treatment, and the survival rate of mice was recorded within 7 days.

[0052] Results Figure 6 All untreated mice died three days after infection, while the survival rate of infected mice treated with the antimicrobial peptide SS-7 reached 70%, while the survival rate of the group treated with the antibiotic ampicillin was only 50%.

[0053] Example 8

[0054] Six-week-old BALB / c female mice (5 mice per group) with a weight of 16 ± 2 g were intraperitoneally injected with 200 μL of 2 × 10 8 CFU / mL of SC19, the treatment procedure was the same as in Example 7. 12 hours after injection, the heart blood was collected from the anesthetized mice to detect the levels of IL-6 and TNF-α in the serum; the liver and spleen were ground, diluted in multiples, and then applied to TSA plates containing 5% fetal bovine serum. The samples were cultured overnight at 37°C, and then the bacteria were counted.

[0055] Results Figure 7 , the antimicrobial peptide SS-7 effectively reduced the bacterial load in the spleen and liver of infected mice, and compared with the untreated group, the bacterial load in the liver and spleen of the SS-7 treated group decreased by 1.7 and 2.1 log values, respectively. At the same time, compared with the untreated group and the ampicillin treated group, the antimicrobial peptide SS-7 can significantly inhibit the increase of pro-inflammatory factors in infected mice, indicating that the antimicrobial peptide SS-7 can not only exert antibacterial effects in vivo, but also effectively inhibit the production of inflammatory factors caused by bacteria.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An antimicrobial peptide SS-7, characterized in that: The amino acid sequence is VRRVRFVVLRRIKKWRRI.

2. The use of the antimicrobial peptide SS-7 according to claim 1 in antibacterial treatment, characterized in that: The application is for non-disease treatment purposes.

3. Use of the antimicrobial peptide SS-7 according to claim 1 in the preparation of an antimicrobial agent.

4. Use of the antimicrobial peptide SS-7 according to claim 1 in the preparation of a drug for preventing or treating bacterial infection.

5. The use according to any one of claims 2 to 4, characterized in that: The bacteria include Gram-positive bacteria and Gram-negative bacteria.

6. The use according to claim 5, characterized in that: The Gram-positive bacteria include Staphylococcus aureus, Listeria, Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus suis; the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, and Salmonella.

7. An antibacterial agent, characterized in that: The invention comprises the antimicrobial peptide SS-7 according to claim 1.

8. A drug for preventing or treating bacterial infection, characterized in that: The invention comprises the antimicrobial peptide SS-7 according to claim 1.

9. The antibacterial agent according to claim 7 or the medicine according to claim 8, characterized in that: Also contains pharmaceutically acceptable excipients.

10. The antibacterial agent according to claim 7 or the medicine according to claim 8, characterized in that: The bacteria include Gram-positive bacteria and Gram-negative bacteria.

Images