Broad-spectrum antibacterial peptide SS-7 and application thereof

Abstract

The application discloses a broad-spectrum antibacterial peptide SS-7 and application thereof, and belongs to the field of biological medicines. The amino acid sequence of the broad-spectrum antibacterial peptide SS-7 is VRRVRFVVLRRIKKWRRI, the antibacterial peptide SS-7 has a significant inhibiting effect on drug-resistant strains such as methicillin-resistant Staphylococcus aureus (ATCC43300) and various gram-positive bacteria and gram-negative bacteria, shows low toxicity and no hemolytic toxicity to eukaryotic cells and red blood cells of mammals, can combine LPS and lipophosphomannan on the surface of bacteria to achieve the effect of neutralizing bacteria, and has a good therapeutic protection effect on animals infected with bacteria. The antibacterial peptide SS-7 can be used for preparing antibacterial agents and drugs for preventing or treating bacterial infections, has the advantages of a wide antibacterial spectrum, a low drug resistance mutation frequency, low toxicity and a low production cost, can effectively cope with the current bacterial drug resistance problem, and has a wide application prospect and important clinical value.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to a broad-spectrum antibacterial peptide SS-7 and application thereof. BACKGROUND

[0002] Since the advent of antibiotics, antibiotics have been widely used in medicine, food, agriculture and other fields. The problem of bacterial drug resistance is becoming more and more serious due to the large-scale and unreasonable use of antibiotics, which seriously threatens human and animal health. The advent of the post-antibiotic era has made it difficult to find effective drugs to treat multi-drug resistant pathogens. In the past two decades, the development of antibiotics has been slow, and the speed of bacterial drug resistance is much faster than the speed of new drug development. Therefore, it is urgent to develop new drugs or antibiotic substitutes.

[0003] Antibacterial peptides play an important role in host immune defense and are widely present in prokaryotes and eukaryotes. They can be extracted from nature or artificially synthesized by chemical means. Antibacterial peptides do not target the common targets of classic antibiotics, but mainly target bacterial membranes, destroy the integrity of bacterial membranes through electrostatic interactions, and thus make it difficult for bacteria to develop resistance to traditional antibiotics. However, the cytotoxicity and immunogenicity of natural antibacterial peptides may have adverse effects on host cells, especially when used at high concentrations, which increases the potential toxic side effects. In contrast, amino acid substitution modified peptides have many advantages. By modifying natural antibacterial peptides through amino acid substitution, the stability of the peptides can be enhanced, making them more resistant to degradation in the body and thus prolonging their action time. 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 optimize the antibacterial spectrum and activity of the peptides, making them more effective against a variety of drug-resistant strains. Through these modifications, the drug development prospects of antibacterial peptides are further expanded, becoming effective broad-spectrum anti-infection treatment antibiotic substitutes. SUMMARY

[0004] The purpose of the present application is to provide a novel antibacterial peptide SS-7 with broad-spectrum antibacterial activity, low toxicity and no drug resistance mutation, and application thereof in the field of antibacterial.

[0005] The purpose of the present application is achieved by 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-resistant mutations. It is based on Streptococcus suis hemolysin as a template (GenBank accession number JF813299). A sequence RFVVLRREKK (SEQ ID NO. 1) with antimicrobial peptide properties is selected and designated as SS-1. Short sequences with high α-helical activity are added to the C and N segments of the sequence, and glutamic acid in the sequence is replaced by isoleucine, designated as SS-7. The amino acid sequence of the antimicrobial peptide SS-7 is VRRVRFVVLRRIKKWRRI (SEQ ID NO. 7). The helical wheel projection of the antimicrobial peptide SS-7 is shown in FIG. Figure 1 As shown. In vitro antibacterial experiments, cytotoxicity experiments, hemolytic activity experiments, drug-resistant mutation frequency experiments, and neutralization experiments with bacterial membrane components LPS and lipoteichoic acid showed that the antimicrobial peptide SS-7 has broad-spectrum antimicrobial 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 discovered that the antimicrobial peptide SS-7 has broad-spectrum antimicrobial activity, low toxicity, and no hemolytic toxicity, and has a therapeutic and protective effect against 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 relates to an application of the antimicrobial peptide SS-7 in the preparation of a broad-spectrum drug for preventing or treating bacterial infection. The antimicrobial peptide SS-7 achieves this application by having antimicrobial effects 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 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.

[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 antimicrobial activity against a variety of pathogens, including Gram-positive 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 widespread clinical promotion and application.

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

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

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

[0022] Figure 3 This is 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 This is a graph showing the kinetic reaction between the antimicrobial peptide SS-7 and the bacterial membrane components lipoteichoic acid LTA (A) and LPS (B) in Example 5.

[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 bacterial load in the liver and spleen of mice infected with Streptococcus suis (A, B) and the levels of 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 GenScript using an Fmoc solid-phase synthesis protocol. The results of the examples were statistically analyzed using a two-tailed unpaired t-test, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0029] Example 1

[0030] Using the antimicrobial peptide database CAMPR3 to predict antimicrobial peptides from SLY protein sequences, the sequence RFVVLRREKK (SS-1) was identified as having the potential to develop into an antimicrobial peptide. This sequence, consisting of amino acid residues 69-78 of the SLY protein sequence, was modified in various ways to generate seven peptides. The minimum inhibitory concentrations (MICs) of these seven peptides against Staphylococcus aureus and Escherichia coli were determined using the classic serial two-fold microdilution method according to the CLSI antimicrobial susceptibility testing protocol. The experiments were repeated three times in parallel. The results are shown in Table 1. The MIC of SS-7 was significantly lower than that of the 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 microserial 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 monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, and 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 96-well culture plates, 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 and cultured 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 culture medium containing 10% CCK8, and after culturing for 2 hours, the OD was read using a spectrophotometer. 450 The absorbance at 100 nm was measured, and three biological replicates were set. The calculation formula was: Cell viability = [(absorbance of experimental well - absorbance of blank well) / (absorbance of negative control well - absorbance of blank well)] × 100%.

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

[0041] Example 4

[0042] The following experiment uses fresh defibrinated sheep erythrocytes for hemolysis testing of the hemolytic toxicity of the antimicrobial peptide SS-7. Fresh sheep erythrocytes are aspirated, washed with PBS, and centrifuged at 1000 r / min at 4°C for 10 minutes. Different concentrations of antimicrobial peptides are then added to a suspension containing 2% fresh defibrinated sheep erythrocytes. 2.5% Triton X-100 is added as a positive hemolysis control, and erythrocytes containing only PBS are used as a negative control. The suspension is incubated at 37°C for 6 hours, then centrifuged at 1000 r / min for 10 minutes. The supernatant is aspirated and the absorbance at 543 nm is measured. Three biological replicates are set. The hemolysis rate is calculated using 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] See the results Figure 3Compared with the blank control without drug, the antibacterial peptide did not cause obvious hemolysis of red blood cells in the concentration range of 2-128 μg / mL, and the hemolytic toxicity was negligible.

[0044] Example 5

[0045] The resistance mutation frequency of the antibacterial peptide SS-7 was evaluated by continuously monitoring the MIC values of S. pneumoniae ATCC 49619 and S. aureus ATCC 25923. S. pneumoniae ATCC 49619 and S. aureus ATCC 25923 were activated overnight, and then subcultured at a ratio of 1:100. The bacterial amount was adjusted to 5x10 5 CFU / mL. Ampicillin and the antibacterial peptide SS-7 at sub-MIC concentrations were incubated with the bacteria, respectively. After 24 h of culture, the surviving bacteria were subjected to MIC determination. Then, a portion of the bacteria was incubated with the newly determined sub-MIC concentration of ampicillin or SS-7. In this way, the MIC determination was continuously performed for 30 days. The change multiples of the obtained MIC values were plotted to generate the resistance curve.

[0046] The results are shown in Figure 4 After 30 generations, S. pneumoniae and S. aureus did not acquire resistance to SS-7. In contrast, after 30 continuous generations of bacteria, the MIC values of ampicillin for S. pneumoniae and S. aureus were 32 times and 64 times the original value, respectively. The results showed that SS-7 exhibited a lower resistance development trend compared to traditional antibiotics.

[0047] Example 6

[0048] The equilibrium dissociation constant of the antibacterial peptide SS-7 and the two bacterial membrane components, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), was determined by isothermal titration calorimetry (ITC) technology to prove the interaction of the antibacterial peptide SS-7 with the two bacterial membrane components. First, the antibacterial 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 Dikang lotion, ultrapure water, and PBS buffer, respectively. The instrument setting parameters were as follows: temperature 25℃, number of sample needles 20, and volume per needle 2.5 μL. 300 μL of degassed 50 μmol / L LPS or LTA was slowly added to the reaction pool with a sample needle to avoid the generation of bubbles, and 300 μL of ultrapure water was added to the reference pool as a control. 50 μL of 500 μmol / L antibacterial peptide SS-7 was taken with a titration needle, and the titration was performed after the instrument was balanced. The titration curve was analyzed by Nano Analyzer, and the Independent model was used for binding force and binding curve analysis.

[0049] The results are shown in Figure 5 The equilibrium binding constant (KD) between antimicrobial peptide SS-7 and LPS was 2.25 μM, and the stoichiometric ratio was 1.206:1, while the equilibrium binding constant (KD) between antimicrobial peptide SS-7 and LTA was 12.13 μM, and the stoichiometric ratio was 1.863:1, indicating that the antimicrobial peptide SS-7 has strong binding ability 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 of 10 mice each. 200 μL of 1×10 9 The mice were infected with Streptococcus suis SC19 at a concentration of 100 CFU / mL. 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 the mice was recorded over 7 days.

[0052] See the 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 16±2g BALB / c female mice (5 per group) were intraperitoneally injected with 200 μL of 2×10 8 CFU / mL of SC19 was used. The treatment procedure was the same as in Example 7. Twelve hours after injection, cardiac blood was collected from anesthetized mice, and serum IL-6 and TNF-α levels were measured. The liver and spleen were ground, diluted in series, and then plated on TSA plates containing 5% fetal bovine serum. The samples were incubated overnight at 37°C, and then the bacteria were counted.

[0055] See the results Figure 7 The antimicrobial peptide SS-7 effectively reduced bacterial loads in the spleen and liver of infected mice. Compared to untreated mice, the bacterial loads in the liver and spleen of SS-7-treated mice decreased by 1.7 and 2.1 log values, respectively. Furthermore, compared to untreated and ampicillin-treated mice, the antimicrobial peptide SS-7 significantly suppressed the increase in pro-inflammatory cytokines in infected mice, indicating that while the antimicrobial peptide SS-7 exerts its antibacterial effects in vivo, it can also effectively inhibit the production of bacterial-induced inflammatory factors.

[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 considered as equivalent replacement methods and are included in the scope of protection 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; the bacteria are Staphylococcus aureus, Listeria, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, Escherichia coli, Klebsiella pneumoniae, and Salmonella.

3. Use of the antimicrobial peptide SS-7 according to claim 1 in the preparation of an antimicrobial agent, characterized in that: The bacteria are Staphylococcus aureus, Listeria, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, Escherichia coli, Klebsiella pneumoniae and Salmonella.

4. Use of the antimicrobial peptide SS-7 according to claim 1 in the preparation of a medicament for preventing or treating bacterial infection, characterized in that: The bacteria are Staphylococcus aureus, Listeria, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, Escherichia coli, Klebsiella pneumoniae and Salmonella.

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

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

7. The antibacterial agent according to claim 5 or the medicine according to claim 6, characterized in that: Also contains pharmaceutically acceptable excipients.

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