Antibacterial peptide P3 with extended-spectrum splitting activity and application thereof

Abstract

The invention discloses an antibacterial peptide P3 with extended-spectrum splitting activity and application of the antibacterial peptide P3. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO. 1. The antibacterial peptide P3 disclosed by the invention has strong bactericidal activity on more than thirty pathogenic bacteria, including staphylococcus aureus and acinetobacter baumannii, which are harmful to human health, animal breeding and agricultural production in vitro. Meanwhile, the antibacterial peptide has no cytotoxicity, shows good safety and effectiveness in in-vivo animal experiments, and has wide application prospects in research and development of antibacterial and anti-infection biological agents.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, in particular to an antimicrobial peptide P3 with ultra-broad-spectrum lytic activity and applications thereof. Background Art

[0002] With the widespread use of antibiotics, antibiotic resistance has become a major challenge facing global public health. In particular, the emergence of drug-resistant strains not only poses a serious threat to human health, but also puts tremendous pressure on agriculture and animal husbandry. These pathogens show significant resistance to multiple antibiotics, which brings great difficulties to clinical treatment. Therefore, there is an urgent need to develop new antibacterial strategies, especially alternative antibacterial molecules that can effectively combat multidrug-resistant bacteria (MDR) and have a lower risk of resistance. In recent years, antimicrobial peptides (AMPs) have become a hot topic in anti-infection treatment research due to their unique mechanism of action and broad antibacterial spectrum. Antimicrobial peptides usually exert their bactericidal effects by destroying the bacterial cell membrane and have a low incidence of drug resistance.

[0003] However, the current sources of antimicrobial peptides are relatively limited to animals and plants. For example, the antimicrobial peptide disclosed in CN116535481A is derived from leeches, the antimicrobial peptide disclosed in CN117430672A is derived from zebrafish, and the antimicrobial peptide disclosed in CN117866047A is derived from the scutellaria raptor. According to APD3 (https: / / aps.unmc.edu / facts), an authoritative database on antimicrobial peptides, over 89% of antimicrobial peptides have been found to be derived from animals and plants. Therefore, the development of antimicrobial peptides from other sources is urgently needed to address the crisis of drug-resistant bacteria in various fields. Summary of the Invention

[0004] Based on the exploration of antimicrobial peptides in phage lytic enzymes, the present invention obtained an antimicrobial peptide P3 with ultra-broad-spectrum lytic activity, which exhibits a wide range of antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria that are harmful to human health, animal husbandry and agricultural production.

[0005] The present invention adopts a variety of bioinformatics methods to identify an antimicrobial peptide P3 with ultra-broad-spectrum lytic activity from phage lytic enzymes.

[0006] The amino acid sequence of the antimicrobial peptide P3 has at least 70% similarity to SEQ ID NO. 1. SEQ ID NO. 1: FGYRRRFGRMHYGIDLSVNR

[0007] Further preferably, the amino acid sequence of the antimicrobial peptide P3 is shown as SEQ ID NO.1.

[0008] The present invention also provides the use of the antimicrobial peptide P3 with ultra-broad-spectrum bactericidal activity in the preparation of antimicrobial agents, preservatives, antiseptics, pharmaceuticals, pesticides, and feed additives for lysing Gram-positive and Gram-negative bacteria. Furthermore, the pharmaceuticals may be antimicrobial and anti-infective biological preparations.

[0009] Preferably, the Gram-positive bacteria is any one of Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria grayi, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus nepalensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus suis.

[0010] Preferably, the Gram-negative bacteria are Acinetobacter baumannii, Citrobacter amalonaticus, Dickea dadantii, Dickea fangzhongdai, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pectobacterium urioidearum, Pectobacterium polaris, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas koreanus, Ralstonia solanacearum, Salmonella enterica, Salmonella paratyphi. paratyphi, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Vibrio parahaemolyticus, Vibrio vulnificus.

[0011] Beneficial effects of the present invention:

[0012] Compared with the existing technology, the present invention obtains an antimicrobial peptide P3 with ultra-broad-spectrum lytic activity by mining and screening phage-encoded phage lytic enzymes. The antimicrobial peptide P3 exhibits broad and potent antimicrobial activity against more than 30 Gram-positive and Gram-negative bacteria that are harmful to human health, animal husbandry and agricultural production, and is non-cytotoxic. It has shown good safety and efficacy in in vivo animal experiments and has broad application prospects in the research and development of antimicrobial agents, preservatives, preservatives, drugs, pesticides, and feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Attachment Figure 1 This is the charge distribution diagram of the antimicrobial peptide P3 in Example 1 of the present invention.

[0014] Attachment Figure 2 This is a graph showing the antibacterial activity of the antimicrobial peptide P3 against Staphylococcus aureus and Acinetobacter baumannii in Example 2 of the present invention.

[0015] Attachment Figure 3 This is a graph showing the bactericidal kinetics of the antimicrobial peptide P3 against Staphylococcus aureus and Acinetobacter baumannii in Example 3 of the present invention.

[0016] Attachment Figure 4 This is a graph showing the activity of the antimicrobial peptide P3 in Example 4 of the present invention in killing different Gram-positive and Gram-negative bacteria.

[0017] Attachment Figure 5 This is a graph showing the cytotoxicity of the antimicrobial peptide P3 to different cells in Example 5 of the present invention.

[0018] Attachment Figure 6 This is the effect of the antimicrobial peptide P3 in Example 6 of the present invention in an in vivo experiment. DETAILED DESCRIPTION

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0020] Unless otherwise specified, the reagents, strains and equipment used in the present invention are commercially available or can be obtained by existing methods.

[0021] Bacillus cereus (WHG21001) is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0022] Enterococcus faecalis, WHS30002, WHS30006, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0023] Enterococcus faecium, WHS30003, WHS30005, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0024] Listeria grayi, CGMCC 1.2989, was commercially available from the China General Microbiological Culture Collection Center.

[0025] Listeria monocytogenes (WHG50001) is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0026] Staphylococcus aureus, N315, T23, WHS11097, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0027] Staphylococcus epidermidis, WHS10011, is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0028] Staphylococcus nepalensis, WHS11098, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0029] Streptococcus agalactiae, S12, is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0030] Streptococcus pneumoniae, WHS20011, WHS20013, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0031] Streptococcus pyogenes, ATCC 49399, commercially available.

[0032] Streptococcus suis, WHS21001, is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0033] Acinetobacter baumannii, WHG40035, WHG40064, and WHG40137, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0034] Citrobacter amalonaticus, WHG10063, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0035] Dickeya dadantii, WHG10105, is a strain preserved in the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0036] Dickea fangzhongdai, WHG10102 and WHG10103, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0037] Enterobacter cloacae, WHG10064, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0038] Escherichia coli, O157, O97, WHG10002, WHG10003, are strains preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0039] Klebsiella pneumoniae, WHG11004 and WHG11009, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0040] Pectobacterium aroidearum, WHG10100, WHG10101, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0041] Pectobacterium polaris, WHG10104, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0042] Proteus mirabilis (WHG10061) is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0043] Pseudomonas aeruginosa, WHG50014, WHG50021, WHG50033, WHG50035, are strains preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0044] Pseudomonas koreensis, WHG10106, is a strain preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0045] Ralstonia solanacearum, GIM1.74, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0046] Salmonella enterica, ATCC 13076, CICC (B) 21497, commercially available.

[0047] Salmonella paratyphi, CICC(B)50094, commercially available.

[0048] Salmonella typhimurium, CICC(B)50115, commercially available.

[0049] Shigella flexneri, M90T, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0050] Shigella sonnei, WHG10065, is a strain preserved at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0051] Vibrio parahaemolyticus, ATCC 17802, commercially available.

[0052] Vibrio vulnificus, ATCC 27562, commercially available.

[0053] Example 1

[0054] Charge distribution of antimicrobial peptide P3

[0055] After extensive experimental analysis, the inventors identified the antimicrobial peptide P3 from a phage-encoded lytic enzyme. The antimicrobial peptide P3 is derived from a truncated fragment of the Porphyromonas gingivalis phage lytic enzyme (NCBI sequence number ERJ66656.1).

[0056] As shown in Table 1, the antimicrobial peptide P3 has an amino acid similarity of less than 45% with the experimentally characterized antimicrobial peptides in the public database APD3. Figure 1 The results showed that the antimicrobial peptide P3 has basic amino acids on one side and hydrophobic amino acids on the other side, which is consistent with the characteristics of α-helical antimicrobial peptides. The antimicrobial peptide P3 was synthesized by solid-phase synthesis and purified to a purity of >95%.

[0057] Table 1

[0058]

[0059] Table 1 shows the amino acid similarity between the antimicrobial peptide P3 in Example 1 of the present invention and experimentally characterized antimicrobial peptides in the public database APD3.

[0060] Example 2

[0061] Antimicrobial activity of antimicrobial peptide P3 against Staphylococcus aureus and Acinetobacter baumannii

[0062] The corresponding strains of Staphylococcus aureus and Acinetobacter baumannii were cultured to the logarithmic phase (OD600nm = 0.4-0.6), the precipitates were collected by low-temperature centrifugation, washed twice with HEPES buffer, and then diluted to about 10 in HEPES buffer. 6 CFU / mL, and the corresponding bacterial solution was obtained. Antimicrobial peptide P3 was mixed with the above bacterial solution to make the final concentration of antimicrobial peptide 2.5μg / mL, 5μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL. At the same time, a mixture of equal amount of buffer and the above bacterial solution was used as a negative control. After incubation at 37℃ for 1 hour, the plate was counted and the results were shown in the table. Figure 2 .

[0063] from Figure 2 The results show that the antimicrobial peptide P3 has strong killing activity against Staphylococcus aureus and Acinetobacter baumannii, indicating that the antimicrobial peptide P3 has ultra-broad spectrum against Gram-positive and Gram-negative bacteria.

[0064] Example 3

[0065] Bactericidal kinetics of antimicrobial peptide P3 against Staphylococcus aureus and Acinetobacter baumannii

[0066] Staphylococcus aureus and Acinetobacter baumannii were cultured to the logarithmic phase (OD600nm = 0.4-0.6), and the precipitates were collected by low-temperature centrifugation and washed twice with HEPES buffer, and then diluted to about 10 in HEPES buffer. 6 CFU / mL, and the corresponding bacterial solution was obtained. Antimicrobial peptide P3 was mixed with the above bacterial solution to make the final concentration of antimicrobial peptide 10μg / mL. At the same time, a mixture of equal amount of buffer and the above bacterial solution was used as a negative control. After incubation at 37℃ for 0, 1, 5, 10, 15, 30, 45, and 60 minutes, the plate was counted. The results are shown in Figure 3 .

[0067] from Figure 3 The results show that the antimicrobial peptide P3 can kill Staphylococcus aureus and Acinetobacter baumannii in a short time and has rapid bactericidal kinetics.

[0068] Example 4

[0069] Activity of antimicrobial peptide P3 against different Gram-positive and Gram-negative bacteria

[0070] Various strains of Gram-positive and Gram-negative bacteria that are harmful to human health, animal husbandry, and agricultural production were cultured to the logarithmic phase (OD600nm = 0.4-0.6), the precipitates were collected by low-temperature centrifugation, washed twice with HEPES buffer, and then diluted to about 10 in HEPES buffer. 6CFU / mL, and the corresponding bacterial solution was obtained. The antimicrobial peptide P3 was mixed with the above bacterial solution to make the final concentration of the antimicrobial peptide 10μg / mL. At the same time, a mixture of equal amounts of buffer and the above bacterial solution was used as a negative control. After incubation at 37℃ for 1 hour, the plate was counted and the results were shown in Figure 4 .

[0071] from Figure 4The results showed that the antimicrobial peptide P3 had an active effect on Acinetobacter baumannii, Bacillus cereus, Citrobacter amalonaticus, Dickea dadantii, Dickea fangzhongdai, Enterobactercloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Listeria grayi, Listeria monocytogenes, Pectobacterium aroidearum, Pectobacterium polaris, Proteus mirabilis, and Pectobacterium thuringiensis. mirabilis, Pseudomonas aeruginosa, Pseudomonas koreensis, Ralstonia solanacearum, Salmonella enterica, Salmonella paratyphi, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus nepalensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes pyogenes, Streptococcus suis, Vibrio parahaemolyticus, and Vibrio vulnificus all had potent bactericidal activity, indicating that the antimicrobial peptide P3 had potent bactericidal activity and ultra-broad spectrum against Gram-positive and Gram-negative bacteria.

[0072] Example 5

[0073] Cytotoxicity of antimicrobial peptide P3 on different cells

[0074] Cultured RAW 264.7 cells and A549 cells were seeded into 96-well plates and cultured for 24 hours to allow them to adhere to the surface, after which the culture medium was removed. Antimicrobial peptide P3 was taken, sterilized by filtration, and added to the above-mentioned well plate to co-culture with the cells. The final concentrations of antimicrobial peptide P3 were 0, 25, 50, 100, 200, 300, 400, 500, 800, and 1000 μg / mL. Normal culture medium without antimicrobial peptide P3 was used as a positive control (PC), and culture medium without antimicrobial peptide P3 and normal cells were used as negative controls (NC). Relative cell viability was calculated using the following formula: % relative viability = (ODsample-ODNC) / (ODPC-ODNC)] × 100%; the results are shown in Figure 2. Figure 5 .

[0075] from Figure 5 The results showed that the antimicrobial peptide P3 had no cytotoxicity to RAW 264.7 cells and A549 cells.

[0076] Example 6

[0077] Effects of antimicrobial peptide P3 in vivo

[0078] Female BALB / c mice aged 6-8 weeks were divided into two groups: Acinetobacter baumannii group and Staphylococcus aureus group. Mice in the Acinetobacter baumannii group were injected intraperitoneally with 10 7 The mice in the Staphylococcus aureus group were injected intraperitoneally with 10 CFU of Acinetobacter baumannii. 8 CFU doses of Staphylococcus aureus were administered. 50 μL of different samples were administered 0.5, 2.5, and 4.5 hours after infection. The antimicrobial peptide P3 (0.9 mg) group received intraperitoneal injections of 0.5 mg, 0.2 mg, and 0.2 mg of antimicrobial peptide P3 at 0.5, 2.5, and 4.5 hours, respectively. The antimicrobial peptide P3 (0.4 mg) group received intraperitoneal injections of 0.2 mg, 0.1 mg, and 0.1 mg of antimicrobial peptide P3 at 0.5, 2.5, and 4.5 hours, respectively. The control group received injections of HEPES buffer containing antimicrobial peptide P3 at the same time points. Mice were sacrificed by cervical dislocation 12 hours after infection. Heart, liver, spleen, lung, kidney, and blood were removed, weighed, and homogenized using a tissue cell disruptor. Samples from the Acinetobacter baumannii group were diluted and plated on LB plates containing 4 μg / mL gentamicin and 2 μg / mL meropenem, while samples from the Staphylococcus aureus group were diluted and plated on LB plates containing 8 μg / mL penicillin. LB plates were incubated at 37 degrees overnight and counted. Figure 6 .

[0079] from Figure 6 The results show that the antimicrobial peptide P3 has a high killing activity against both Acinetobacter baumannii and Staphylococcus aureus in experiments on mice, and can significantly reduce the bacterial load in various organs of mice.

[0080] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. An antimicrobial peptide P3 with ultra-broad-spectrum lytic activity, characterized by: The amino acid sequence of the antimicrobial peptide P3 has at least 70% similarity to SEQ ID NO.1; SEQ ID NO.1: FGYRRRFGRMHYGIDLSVNR.

2. The antimicrobial peptide P3 with ultra-broad-spectrum lytic activity according to claim 1, characterized in that: The amino acid sequence of the antimicrobial peptide P3 is shown in SEQ ID NO.

1.

3. Use of the antimicrobial peptide P3 with ultra-broad-spectrum lytic activity as claimed in claim 1 in the preparation of antimicrobial agents, preservatives, antiseptics, medicines and pesticides for lysing Gram-positive and Gram-negative bacteria.

4. The use according to claim 3, characterized in that: The medicine is an antibacterial and anti-infective biological preparation.

5. The use according to claim 3, characterized in that: The Gram-positive bacteria is any one of Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria grayi, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus nepalensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus suis.

6. The use according to claim 3, characterized in that: The Gram-negative bacteria are Acinetobacter baumannii, Citrobacter amalonaticus, Dickea dadantii, Dickea fangzhongdai, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pectobacterium aroidearum, Pectobacterium polaris, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas koreensis, Ralstonia solanacearum, Salmonella enterica, Salmonella paratyphi, and the like. paratyphi, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Vibrio parahaemolyticus, Vibrio vulnificus.

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