Aggregatilineales antimicrobial peptide and its application

Abstract

The present invention relates to the technical field of antimicrobial peptides, and discloses an Aggregatilineales antimicrobial peptide and its application, wherein the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1. The antimicrobial peptide obtained by the present invention has good antibacterial effects on Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, in particular to an Aggregatilineales antimicrobial peptide and applications thereof. Background Art

[0002] Pathogenic microorganisms are pathogenic microorganisms that can cause illness in humans, animals, and plants. Microorganisms are groups of organisms with an individual diameter generally less than 1 mm. They have a simple structure, most are single-celled, and some do not even have a cell structure. Common pathogenic microorganisms in the prior art include Staphylococcus aureus ( Staphylococcus aureus, S. aureus ), Klebsiella pneumoniae, and Acinetobacter baumannii. Among them, Staphylococcus aureus often parasitizes the skin, nasal cavity, throat, stomach, carbuncle, and purulent sores of humans and animals, and is also ubiquitous in the air, sewage, and other environments. Under appropriate conditions, Staphylococcus aureus can produce enterotoxins, causing food poisoning and compromising consumer safety. Klebsiella pneumoniae can cause pneumonia, and Acinetobacter baumannii commonly causes bacteremia, pneumonia, meningitis, peritonitis, endocarditis, as well as urinary tract and skin infections.

[0003] Because food is rich in nutrients, improper handling during food production, storage, or transportation can lead to the rapid proliferation of pathogenic bacteria, which, when consumed by consumers, can cause food poisoning, gastroenteritis, and even serious systemic infections. Antimicrobial peptides (AMPs) are important components of prokaryotes and eukaryotes. These small peptides, typically composed of 12-50 amino acids, possess broad-spectrum antibacterial, antifungal, and antiviral properties and are highly valuable in pharmacological treatment and prevention applications. Therefore, there is an urgent need to develop an antimicrobial peptide that can be applied during food production, storage, or transportation to reduce the harm of pathogens to consumers. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an Aggregatilineales antimicrobial peptide and its application. The antimicrobial peptide obtained by the present invention has good antibacterial effect on Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0005] The present invention provides an Aggregatilineales antimicrobial peptide, the amino acid sequence of the antimicrobial peptide is shown as SEQ ID NO: 1.

[0006] The present invention also provides an antimicrobial composition comprising an antimicrobial peptide having an amino acid sequence such as SEQ ID NO: 1.

[0007] The present invention also provides a gene encoding the antimicrobial peptide.

[0008] The present invention also provides a recombinant vector, an expression cassette, a transgenic cell line, a transgenic plant or a recombinant microorganism containing the encoding gene.

[0009] The present invention also provides the use of the antimicrobial peptide in preparing medicines for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0010] The present invention also provides the use of the antibacterial composition in preparing medicines for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0011] The present invention also provides the use of the antimicrobial peptide in preparing an additive, wherein the additive is any one of a cosmetic, feed or food additive.

[0012] The present invention also provides the use of the antimicrobial peptide in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii for non-disease treatment and / or non-disease diagnosis.

[0013] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide for inhibiting Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration for inhibiting Klebsiella pneumoniae is 200 μM, and the minimum inhibitory concentration for inhibiting Acinetobacter baumannii is 100 μM.

[0014] The embodiments of the present invention have the following technical effects:

[0015] The present invention obtains a new antimicrobial peptide, which has good antibacterial effect on Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is the genomic spectrum of the antimicrobial peptide provided in the embodiment of the present invention.

[0018] Figure 2 It is a three-dimensional structure diagram of the antimicrobial peptide provided in an embodiment of the present invention.

[0019] Figure 3 is a diagram showing the antibacterial effect of the antimicrobial peptide provided in an embodiment of the present invention, wherein Figure 3 (a) is the inhibition zone of the antimicrobial peptide of the present invention against Staphylococcus aureus, Figure 3(b) is the inhibition zone of the antimicrobial peptide of the present invention against Klebsiella pneumoniae, Figure 3 (c) is the inhibition zone of the antimicrobial peptide of the present invention against Acinetobacter baumannii.

[0020] Figure 4 This is a graph showing the minimum inhibitory concentration of antimicrobial peptides provided in an embodiment of the present invention.

[0021] Figure 5 The antimicrobial peptide provided in the embodiment of the present invention is applied to chicken.

[0022] Figure 6 The antimicrobial peptide provided by the embodiment of the present invention is applied to milk. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some 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.

[0024] In a first aspect, some embodiments of the present invention provide an Aggregatilineales antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0025] In a second aspect, some embodiments of the present invention further provide an antimicrobial composition comprising an antimicrobial peptide having an amino acid sequence such as SEQ ID NO: 1.

[0026] In a third aspect, some embodiments of the present invention further provide a gene encoding the antimicrobial peptide.

[0027] In a fourth aspect, some embodiments of the present invention further provide a recombinant vector, an expression cassette, a transgenic cell line, a transgenic plant or a recombinant microorganism containing the encoding gene.

[0028] In a fifth aspect, some embodiments of the present invention further provide use of the antimicrobial peptide in the preparation of a drug for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0029] In a sixth aspect, some embodiments of the present invention further provide use of the antibacterial composition in the preparation of a medicament for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0030] In a seventh aspect, some embodiments of the present invention further provide the use of the antimicrobial peptide in the preparation of an additive, wherein the additive is any one of a cosmetic, feed or food additive.

[0031] In an eighth aspect, some embodiments of the present invention further provide use of the antimicrobial peptide in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii for non-disease treatment and / or non-disease diagnosis.

[0032] In some embodiments, the antimicrobial peptide has a minimum inhibitory concentration of 12.5 μM for inhibiting Staphylococcus aureus, a minimum inhibitory concentration of 200 μM for inhibiting Klebsiella pneumoniae, and a minimum inhibitory concentration of 100 μM for inhibiting Acinetobacter baumannii.

[0033] The following is elaborated in conjunction with the embodiments and comparative examples:

[0034] Example 1: Obtaining microbial genomes based on metagenomic component-related technologies:

[0035] CRR807596, derived from a hot spring environment, was downloaded from the National Biotechnology Information Center (NGDC) public database. Quality control was performed using Trim Galore (v. 0.5.0) to remove adapter sequences and low-quality reads (quality score < 20) to obtain clean reads. Clean reads were assembled using MEGAHIT. Seqtk was used to filter the assembled contigs based on sequence length (retaining a length of 1500 bp). Clean reads were mapped to the corresponding contigs using Bowtie2. Mapping results were converted to BAM format using Samtools. The BAM files were sorted and indexed using SAMtools, and then the genome was generated using CONCOCT. The resulting genome was 133.CRR807596_concoct_sub. The completeness and contamination levels of the genome were analyzed using CheckM2, which were 59.99% and 3.63%, respectively, meeting MIMAG genome requirements. Species annotation was performed using GTDB-Tk, and the results showed that the genome was CP2-2F sp938005675 of the order Aggregatilineales of bacteria (e.g. Figure 1 Detailed annotation information is as follows:

[0036] d__Bacteria;p__Chloroflexota;c__Anaerolineae;o__Aggregatilineales;f__UBA2796;g__CP2-2F;s__CP2-2F sp938005675.

[0037] The amino acid sequence of the antimicrobial peptide (antimicrobial peptide 744) formed by the genome is shown in SEQ ID NO: 1. SEQ ID NO: 1 is: NSKLAAKIAKLIGVKLKKLKVG.

[0038] According to the amino acid sequence of the obtained antimicrobial peptide, the antimicrobial peptide is synthesized:

[0039] (1) According to the first amino acid at the C-terminus of the peptide sequence, 0.5 mmol of the corresponding Fmoc-protected amino acid - WangResin was selected and added to the solid phase reactor. After adding DCM to swell the resin for 30 minutes, it was drained and washed three times with DMF. A 20% by volume solution of hexahydropyridine in DMF was added and the reaction was continued for 5 minutes. A 20% by volume solution of hexahydropyridine in DMF was added again and the reaction was continued for 10 minutes. The resin was washed once with DMF in the middle. After the reaction was completed, the resin was drained and washed three times with DMF.

[0040] (2) According to the sequence of the peptide from C-terminus to N-terminus, the condensation reaction and Fmoc removal reaction were carried out alternately according to the reaction amount of 1.5 mmol of amino acids. According to the amino acid sequence of the antimicrobial peptide, all amino acids were condensed on the resin. After the condensation of the last amino acid, a 20% by volume solution of hexahydropyridine in DMF was added and the reaction was continued for 5 minutes. A 20% by volume solution of hexahydropyridine in DMF was added again and the reaction was continued for 10 minutes. The resin was washed once with DMF in the middle. After the reaction was completed, the resin was drained and washed 3 times with DMF.

[0041] (3) Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 minutes. Use ninhydrin to detect whether the reaction is complete. After the reaction is complete, wash it three times with DMF and DMC alternately, and then wash and shrink it with methanol to obtain a dry polypeptide-resin.

[0042] (4) Place the polypeptide-resin in a round-bottom flask, slowly add the prepared lysis solution at 0°C (the volume ratio of the lysis solution is TFA: thioanisole: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2) and stir slowly. React at low temperature for 0.5 hours and at room temperature for 2 hours. Filter to obtain the lysis solution, slowly add the lysis solution into anhydrous ice ether and stir, filter and separate the crude polypeptide, wash with ice ether 3 times to obtain the crude peptide.

[0043] (5) Use mass spectrometry to check whether the molecular weight of the crude product is correct. If correct, purify and separate it using high performance liquid chromatography, freeze-dry it, and then obtain the pure polypeptide.

[0044] The antimicrobial peptide obtained in Example 1 was tested:

[0045] (1) Use AlphaFold2 to predict its three-dimensional structure. The results are as follows Figure 2 shown.

[0046] (2) Determination of the antibacterial spectrum of antimicrobial peptides: The pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella enteritidis, Salmonella typhimurium, Listeria monocytogenes) stored previously were taken out of the -80℃ refrigerator, and a small amount of bacterial liquid was dipped with an inoculation loop. Three zones were drawn on LB solid culture medium (commercially available), and cultured in a 37℃ constant temperature box for 16-24 hours.

[0047] After a single colony grows on the plate, use a sterilized inoculation loop to pick a single colony and inoculate it into 5 mL of LB liquid culture medium and place it in a 37°C constant temperature shaker for activation for 16-24 hours.

[0048] 1 mL of the activated pathogenic bacteria culture solution was taken and added to 100 mL of LB liquid medium (commercially available), cultured at 37°C and 200 rpm for 16-24 h, and the biomass was measured using a microplate reader to obtain the final biomass OD 600 Reach between 1.0-1.2.

[0049] Take 40 μL of the cultured bacterial liquid and spread it evenly on the LB solid culture medium with a spreading rod. Place 32 sterilized filter paper pieces (the diameter of the filter paper pieces is 6 mm) at the same intervals on the plate covered with pathogenic bacteria. Take 20 μL of the antimicrobial peptide solution with a concentration of 400 μM (the antimicrobial peptide is dispersed in sterilized water) and spot it on the filter paper pieces. After spotting, culture the LB solid culture medium at 37°C for 16-24 hours.

[0050] The LB solid medium after culture was taken and the inhibition zone formed was observed and measured. The test was repeated three times for each strain to be tested, with three replicates each time. Each inhibition zone was measured three times and the average value was taken. Finally, the average value of all the inhibition zone diameters measured represented the final inhibition zone diameter of the antimicrobial peptide for the strain. The results showed that the antimicrobial peptide of the present invention formed inhibition zones against Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii. Figure 3 shown.

[0051] (3) Determination of minimum inhibitory concentration (MIC) of antimicrobial peptides:

[0052] An LB culture medium solution containing antimicrobial peptide was prepared, and the concentration of the antimicrobial peptide was 400 μM.

[0053] 500 μL of the above LB culture medium solution containing antimicrobial peptides was added to 500 μL of LB culture medium to prepare a culture medium solution containing antimicrobial peptides at a concentration of 200 μM.

[0054] Similarly, the culture medium solution containing the antimicrobial peptide was diluted to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM in sequence, and 6 mL of the culture medium solution containing the antimicrobial peptide at each concentration was prepared.

[0055] Similarly, a LB medium solution containing the natural antimicrobial peptide nisin was prepared at a concentration of 400 μM. Following the above steps, the natural antimicrobial peptide nisin was diluted sequentially to 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM solutions.

[0056] The pathogenic bacteria Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii stored previously were taken out from the -80°C refrigerator and activated and cultured on LB solid medium at 37°C for 16-24 hours.

[0057] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid medium. Place it in a 37°C constant temperature shaker for activation for 16-24 hours, and use a microplate reader to measure the biomass. The final biomass OD 600 When the concentration reaches between 1.0-1.2, dilute the activated bacterial solution 100 times and place it in a 2 mL sterile centrifuge tube for later use.

[0058] 200 μL of culture medium solutions containing different concentrations of antimicrobial peptides were respectively placed in a sterile 96-well culture plate, and three parallel experiments were performed for each concentration. Then, 10 μL of diluted Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii cultures were added to the culture medium solutions containing different concentrations of antimicrobial peptides, and the culture medium solution containing antimicrobial peptides without the addition of pathogenic bacteria was used as a blank control group.

[0059] Following the above method, Nisin control was performed in the same manner.

[0060] The 96-well plate was placed in a shaker at 37°C and 200 rpm for 16-24 h, and the biomass was measured using a microplate reader to determine the minimum inhibitory concentration (MIC).

[0061] (4) Application of antimicrobial peptides in chicken preservation:

[0062] Cut raw chicken into 1cm pieces 3 The square pieces (about 0.27g) were first sterilized by soaking in 0.5% sodium hypochlorite for 1 minute, then washed three times with PBS (phosphate buffered saline), and then sterilized by soaking in 75% ethanol for 1 minute, washed three times with PBS, and evenly irradiated with ultraviolet light for 1.2 hours to make the raw chicken sterile.

[0063] Taking Staphylococcus aureus as an example, the previously stored Staphylococcus aureus was taken out from the -80°C refrigerator and activated and cultured on LB solid culture medium at 37°C for 16-24 hours.

[0064] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid medium. Place it in a 37°C constant temperature shaker for activation for 16-24 hours, and use a microplate reader to measure the biomass. The final biomass OD 600 Reach between 1.0-1.2.

[0065] The activated bacterial solution was diluted to a concentration of 5×10 3 CFU / mL, the diluted bacterial solution was evenly spread on the surface of the chicken cubes with a sterile cotton swab and placed in a 25℃ constant temperature box for 2 hours to infect the chicken.

[0066] The antimicrobial peptide with a concentration of 12.5 μM was evenly spread on the surface of the chicken cubes with a sterile cotton swab and cultured in a 25°C incubator for 32 hours. PBS buffer solution was used as a PBS control.

[0067] The chicken pieces were homogenized and centrifuged, and the supernatant was diluted and spread on the corresponding selective medium to enumerate the remaining viable bacteria.

[0068] Formula for PBS buffer solution (pH 7.2): Take 100 mL of 0.2 mol / L potassium dihydrogen phosphate solution and 70 mL of 0.2 mol / L sodium hydroxide solution, dilute to 400 mL with freshly boiled cold water, and adjust the pH to between 7.2 and 7.4.

[0069] (5) Application of antimicrobial peptides in milk preservation:

[0070] Aliquot 30 mL of commercially available fresh milk into heat-resistant 50 mL Erlenmeyer flasks and seal the flasks with sterilized film. Sterilize the fresh milk in an autoclave at 115°C for 30 minutes. After sterilization, slowly cool the milk and immediately refrigerate at 4°C.

[0071] Taking Staphylococcus aureus as an example, the previously stored Staphylococcus aureus was taken out from the -80°C refrigerator and activated and cultured on LB solid culture medium at 37°C for 16-24 hours.

[0072] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid medium. Place it in a 37°C constant temperature shaker for activation for 16-24 hours, and use a microplate reader to measure the biomass. The final biomass OD 600 Reach between 1.0-1.2.

[0073] The activated bacterial solution was diluted to a concentration of 5×10 3 CFU / mL, add the diluted bacterial solution into the above-mentioned conical flask containing sterilized milk, place it in a 25℃ constant temperature box and culture it for 2 hours to infect the milk.

[0074] The antimicrobial peptide with a concentration of 12.5 μM was added to the conical flask containing the bacterial solution and milk, and the mixture was placed in a 25° C. incubator for 32 h. Sterile water was used as a blank control.

[0075] Each of the above milks was diluted and spread on the corresponding selective culture medium to count the remaining viable bacteria.

[0076] Results and Analysis:

[0077] exist Figure 2 The antimicrobial peptide of the present invention was found to possess a typical α-helical structure. Antimicrobial peptide 744 (NSKLAAKIAKLIGVKLKKLKVG) is a peptide with potential antimicrobial activity. Results showed that the peptide possessed a high structural confidence (pLDDT = 90.5) and a good match with the experimental structure (pTM = 0.204). Its electrostatic charge of 7 indicates that the peptide possesses a positive charge under physiological conditions, favoring interaction with negatively charged bacterial membranes. Its normalized hydrophobic moment of 0.87 and amphipathic index of 1.17 indicate that the peptide possesses moderate hydrophobicity and amphipathicity, enabling interaction with the lipid bilayer of bacterial membranes. Its isoelectric point penetration depth of 15 further demonstrates its strong penetrating ability near the isoelectric point. The peptide possesses a typical α-helical structure with a helix number of 5 and a triple helix propensity of 0.88. These structural features facilitate its binding and insertion into bacterial membranes, thereby exerting its antimicrobial activity.

[0078] Figure 3 It can be found that the antimicrobial peptides obtained by the present invention can inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii. Figure 3 (a) It can be found that the diameter of the inhibition zone of Staphylococcus aureus is 7 mm, the diameter of the inhibition zone of Klebsiella pneumoniae is 7 mm, and the diameter of the inhibition zone of Acinetobacter baumannii is 12 mm.

[0079] Figure 4 In the results, it can be found that the antimicrobial peptide obtained by the present invention has a MIC of 12.5μM against Staphylococcus aureus, and its inhibitory effect on Staphylococcus aureus is significantly better than Nisin in the prior art. In addition, the antimicrobial peptide of the present invention has a MIC of 200μM against Klebsiella pneumoniae and a MIC of 100μM against Acinetobacter baumannii, while Nisin in the prior art has no inhibitory effect on Klebsiella pneumoniae and Acinetobacter baumannii. It can be seen that the antimicrobial peptide of the present invention has significant advantages in inhibiting Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

[0080] Figure 5 It can be found that the antimicrobial peptide obtained by the present invention can be used to inhibit Staphylococcus aureus in chicken, and its inhibitory effect on Staphylococcus aureus within 32 hours is significant.

[0081] Figure 6 It can be found that the antimicrobial peptide obtained by the present invention can be used to inhibit Staphylococcus aureus in milk, and its inhibitory effect on Staphylococcus aureus within 32 hours is significant.

[0082] In summary, the antimicrobial peptides obtained in the present invention can inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii, and the antibacterial effect is significantly better than that of the existing technology.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. An Aggregatilineales antimicrobial peptide, characterized in that The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. An antibacterial composition, characterized in that The antimicrobial composition comprises an antimicrobial peptide with an amino acid sequence such as SEQ ID NO:

1.

3. A coding gene, characterized in that The coding gene is used to encode the antimicrobial peptide according to claim 1.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant microorganism containing the encoding gene according to claim 3.

5. Use of the antimicrobial peptide according to claim 1 in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii for non-disease treatment and / or non-disease diagnosis.

6. Use of the antimicrobial peptide according to claim 1 in the preparation of a medicament for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

7. Use of the antibacterial composition according to claim 2 in the preparation of a medicament for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

8. Use of the antimicrobial peptide according to claim 1 in the preparation of an additive, characterized in that: The additive is any one of a cosmetic additive and a feed additive.

9. The use according to claim 5, characterized in that The minimum inhibitory concentration of the antimicrobial peptide for inhibiting Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration for inhibiting Klebsiella pneumoniae is 200 μM, and the minimum inhibitory concentration for inhibiting Acinetobacter baumannii is 100 μM.

Images