A novel antimicrobial peptide of Calorimonas adulescens and its application

Abstract

This invention relates to the field of antimicrobial peptide technology, and discloses a novel antimicrobial peptide, Calorimonas adulescens, and its applications. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1. The antimicrobial peptide obtained by this invention exhibits good antibacterial effects against Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and in particular to a novel antimicrobial peptide, Calorimonas adulescens, and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus, S. aureus Staphylococcus aureus, also known as "Staphylococcus aureus," belongs to the genus Staphylococcus and is a representative Gram-positive bacterium, a common foodborne pathogen. Its optimal growth temperature is 37°C, and its optimal pH is 7.4. It is tolerant of high salt concentrations, able to grow in environments with salt concentrations approaching 10%. Staphylococcus aureus commonly resides on the skin, nasal cavity, throat, gastrointestinal tract, boils, and abscesses of humans and animals, and is also ubiquitous in the air and sewage. Under suitable conditions, Staphylococcus aureus can produce enterotoxins, causing food poisoning and affecting consumer safety.

[0003] Klebsiella pneumoniae is the most important bacterium in the genus Klebsiella of the family Enterobacteriaceae (commonly known as pneumonia bacillus), and it accounts for more than 95% of Klebsiella infections. It exists in the human upper respiratory tract and intestines. When the body's resistance is reduced, it enters the lungs through the respiratory tract and causes confluent consolidation of lobules or lobes, which is more common in the upper lobe.

[0004] Acinetobacter baumannii (scientific name: Acinetobacter baumannii Acinetobacter baumannii (commonly known as AB bacteria), also called Acinetobacter baumannii, is a Gram-negative bacterium. It is a strictly aerobic, non-lactose-fermenting, conditionally pathogenic bacterium, lacking flagella and exhibiting low mobility, but possessing extremely strong vitality and widespread presence in nature. This bacterium is a common type of Acinetobacter in hospital-acquired infections and is also a pathogen in aquaculture animals. It typically causes bacteremia, pneumonia, meningitis, peritonitis, endocarditis, as well as urinary tract and skin infections.

[0005] Because food is rich in nutrients, improper handling during food production, storage, or transportation can lead to the rapid proliferation of pathogenic bacteria, causing food poisoning, gastroenteritis, or even serious systemic infections in consumers. 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 capabilities, and are highly valuable in pharmacological therapeutic and preventative applications. Therefore, there is an urgent need to obtain an antimicrobial peptide that can be applied to the production, storage, or transportation of food to reduce the harm of pathogenic bacteria to consumers. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a novel antimicrobial peptide, Calorimonas adulescens, and its application. The antimicrobial peptide obtained by this invention exhibits good antibacterial effects against Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

[0007] This invention provides a novel antimicrobial peptide of Calorimonas adulescens, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] The present invention also provides an antimicrobial composition comprising an antimicrobial peptide having an amino acid sequence such as SEQ ID NO:1.

[0009] The present invention also provides a gene encoding the antimicrobial peptide.

[0010] The present invention also provides recombinant vectors, expression cassettes, transgenic cell lines, transgenic plants or recombinant microorganisms containing the said coding gene.

[0011] The present invention also provides the use of the antimicrobial peptide in the preparation of drugs that inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0012] The present invention also provides the use of the antibacterial composition in the preparation of medicaments that inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0013] The present invention also provides the application of the antimicrobial peptide in the preparation of additives, wherein the additives are any one of cosmetic, feed or food additives.

[0014] The present invention also provides the use of the antimicrobial peptide in the inhibition of Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii in non-disease treatment and / or non-disease diagnosis.

[0015] Furthermore, the minimum inhibitory concentration (MIC) of the antimicrobial peptide against Staphylococcus aureus is 3.125 μM, against Klebsiella pneumoniae is 50 μM, and against Acinetobacter baumannii is 6.25 μM.

[0016] The embodiments of the present invention have the following technical effects:

[0017] This invention yields a novel antimicrobial peptide that exhibits good antibacterial effects against Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is the genomic spectrum of the antimicrobial peptide provided in the embodiments of the present invention.

[0020] Figure 2 This is a three-dimensional structural diagram of the antimicrobial peptide provided in an embodiment of the present invention.

[0021] Figure 3 This is a diagram illustrating the antibacterial effect of the antimicrobial peptide provided in an embodiment of the present invention. Figure 3 (a) shows the inhibition zone of the antimicrobial peptide of the present invention against Staphylococcus aureus. Figure 3 (b) shows the inhibition zone of the antimicrobial peptide of the present invention against Klebsiella pneumoniae. Figure 3 (c) shows the inhibition zone of the antimicrobial peptide of the present invention against Acinetobacter baumannii.

[0022] Figure 4 This is a graph showing the minimum inhibitory concentration (MIC) data of the antimicrobial peptides provided in the embodiments of the present invention.

[0023] Figure 5 The antimicrobial peptide provided in this embodiment of the invention is applied to chicken.

[0024] Figure 6 The antimicrobial peptides provided in this embodiment of the invention are applied to milk. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0026] In a first aspect, some embodiments of the present invention provide a novel antimicrobial peptide, Calorimonas adulescens, the amino acid sequence of which is shown in SEQ ID NO:1.

[0027] Secondly, some embodiments of the present invention also provide antimicrobial compositions comprising an antimicrobial peptide with an amino acid sequence such as SEQ ID NO:1.

[0028] Thirdly, some embodiments of the present invention also provide a gene encoding the antimicrobial peptide.

[0029] Fourthly, some embodiments of the present invention also provide recombinant vectors, expression cassettes, transgenic cell lines, transgenic plants, or recombinant microorganisms containing the coding gene.

[0030] Fifthly, some embodiments of the present invention also provide the use of the antimicrobial peptide in the preparation of drugs that inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0031] Sixthly, some embodiments of the present invention also provide the use of the antibacterial composition in the preparation of medicaments that inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0032] In a seventh aspect, some embodiments of the present invention also provide the application of the antimicrobial peptide in the preparation of additives, wherein the additives are any one of cosmetic, feed, or food additives.

[0033] Eighthly, some embodiments of the present invention also provide the use of the antimicrobial peptide in the inhibition of Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii in non-disease treatment and / or non-disease diagnosis.

[0034] In some embodiments, the minimum inhibitory concentration (MIC) of the antimicrobial peptide against Staphylococcus aureus is 3.125 μM, against Klebsiella pneumoniae is 50 μM, and against Acinetobacter baumannii is 6.25 μM.

[0035] The following description, in conjunction with examples and comparative models, illustrates the points:

[0036] Example 1: Obtaining microbial genomes based on metagenomic correlation technology:

[0037] SRR23823812, originating from the subsurface environment, was downloaded from the NCBI public database. Trim Galore (v.0.5.0) was used for quality control, removing adapter sequences and low-quality reads (quality score <20) to obtain clean reads. MEGAHIT was used to assemble the clean reads. seqtk was used to filter the assembled contigs based on sequence length (retaining a length of 1500bp). Bowtie2 was used to map the clean reads to their corresponding contigs. Samtools was used to convert the mapping results to BAM format. SAMtools was used to sort and index the BAM file, and then CONCOCT was used to generate the genome. The resulting genome was 5.SRR23823812_concoct. CheckM2 analysis showed that the genome integrity and contamination were 91.19% and 1.9%, respectively, meeting the MIMAG requirements. GTDB-Tk was used for species annotation, showing that the genome is... Calorimonas adulescens (like Figure 1 (As shown), detailed annotation information is as follows:

[0038] d__Bacteria;p__Bacillota_A;c__Thermoanaerobacteria;o__Thermoanaerobacterales;f__UBA4877;g__Calorimonas;s__Calorimonas adulescens.

[0039] The amino acid sequence of the antimicrobial peptide (antimicrobial peptide 746) formed by this genome is shown in SEQ ID NO:1, which is: KVYVIKAPRFIKKLLSVFIKK.

[0040] Based on the obtained amino acid sequence of the antimicrobial peptide, the antimicrobial peptide was synthesized:

[0041] (1) Based on the first amino acid at the C-terminus of the polypeptide sequence, select 0.5 mmol of the corresponding Fmoc protected amino acid - WangResin, add it to the solid-phase reactor, add DCM swelling resin for 30 min, dry it, wash it three times with DMF, add a 20% hexahydropyridine DMF solution, react for 5 min, add another 20% hexahydropyridine DMF solution and react for 10 min, wash with DMF once in the middle, dry it after the reaction is finished, and wash it with DMF 3 times.

[0042] (2) Following the sequence of the peptide from C-terminus to N-terminus, condensation reaction and Fmoc removal reaction were carried out alternately with an amino acid reaction amount of 1.5 mmol. According to the amino acid sequence of the antimicrobial peptide, all amino acids were condensed onto the resin. After the last amino acid was condensed, a 20% hexahydropyridine DMF solution was added and reacted for 5 min. Then, another 20% hexahydropyridine DMF solution was added and reacted for 10 min. DMF was washed once in the middle. After the reaction was completed, the solution was dried and washed with DMF 3 times.

[0043] (3) Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 minutes. Use ninhydrin to check whether the reaction is complete. After the reaction is complete, wash three times with DMF and DMC alternately, and then wash with methanol to shrink and obtain dried polypeptide-resin.

[0044] (4) Place the polypeptide-resin into a round-bottom flask and slowly add the prepared lysis buffer at 0°C (the volume ratio of the reagent formula used in the lysis buffer is TFA: benzyl sulfide: phenol: triisopropylsilane: water = 82.5: 7.5: 5: 3: 2). Stir slowly and react at low temperature for 0.5 hours, then at room temperature for 2 hours. Filter to obtain the lysis buffer. Slowly add the lysis buffer to anhydrous ice-cold ether and stir. Filter to separate the crude polypeptide. Wash with ice-cold ether 3 times to obtain the crude peptide.

[0045] (5) Use mass spectrometry to detect whether the molecular weight of the crude product is correct. If it is correct, use high performance liquid chromatography to purify and separate it, freeze dry it, and then obtain pure polypeptide.

[0046] The antimicrobial peptide obtained in Example 1 was tested:

[0047] (1) Its three-dimensional structure was predicted using AlphaFold2, and the results are as follows: Figure 2 As shown.

[0048] (2) Determination of the antimicrobial peptide spectrum: The previously stored pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella enteritidis, Salmonella typhimurium, Listeria monocytogenes) were taken out from the -80℃ freezer and a small amount of bacterial solution was taken by the inoculation loop and streaked in three zones on LB solid medium (commercially available). The culture was then incubated in a 37℃ incubator for 16-24 h.

[0049] After single colonies have grown on the plate, use a sterilized inoculation loop to pick a single colony and inoculate it into 5 mL of LB liquid medium. Place the medium in a 37°C constant temperature shaker for 16-24 h to activate it.

[0050] 1 mL of the activated pathogenic bacteria culture was transferred to 100 mL of LB liquid medium (commercially available) and incubated at 37°C and 200 rpm for 16-24 hours. Biomass was then measured using a microplate reader to determine the final biomass OD. 600 It reaches between 1.0 and 1.2.

[0051] Take 40 μL of the cultured bacterial solution and spread it evenly on LB solid medium using a spreader. Place 32 sterilized filter paper discs (6 mm in diameter) at equal intervals on a plate covered with pathogenic bacteria. Take 20 μL of a 400 μM antimicrobial peptide solution (dispersed in sterilized water) and spot it on the filter paper disc. After spotting, incubate the LB solid medium at 37 °C for 16-24 h.

[0052] Take the LB solid culture medium after cultivation, observe and measure the inhibition zones formed. This experiment was repeated three times for each test strain, with three replicates each time. The average value of each inhibition zone was taken, and the average diameter of all measured inhibition zones was used to represent the final inhibition zone diameter of the antimicrobial peptide against that strain. Results showed that the antimicrobial peptide of this invention formed inhibition zones against Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii. Figure 3 As shown.

[0053] (3) Determination of the minimum inhibitory concentration (MIC) of antimicrobial peptides:

[0054] Prepare an LB medium solution containing antimicrobial peptides at a concentration of 400 μM.

[0055] Take 500 μL of the above-mentioned LB medium solution containing antimicrobial peptides and add it to 500 μL of LB medium to prepare a 200 μM medium solution containing antimicrobial peptides.

[0056] Similarly, the culture medium solutions containing antimicrobial peptides were successively diluted to 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, and 1.5625μM, and 6 mL of each concentration of the culture medium solution containing antimicrobial peptides was prepared.

[0057] Similarly, prepare LB medium solutions containing the natural antimicrobial peptide nisin at a concentration of 400 μM. Following the steps described above, sequentially dilute the natural antimicrobial peptide nisin to prepare medium solutions containing the antimicrobial peptide at concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM.

[0058] The previously stored pathogenic bacteria Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii were taken out of the -80℃ freezer and activated and cultured on LB solid medium, and incubated at 37℃ for 16-24h.

[0059] After single colonies have grown on the plate, use a sterilized inoculation loop to pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C in a shaker for 16-24 h to activate the culture. Measure the biomass using a microplate reader to determine the final biomass OD. 600 Once the concentration reaches between 1.0 and 1.2, dilute the activated bacterial solution 100 times and transfer it into a 2mL sterile centrifuge tube for later use.

[0060] 200 μL of culture medium solutions containing different concentrations of antimicrobial peptides were placed in sterile 96-well plates. Three parallel experiments were conducted 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, respectively. The culture medium solutions containing antimicrobial peptides without pathogenic bacteria were used as blank control groups.

[0061] Following the method described above, a control for Nisin was prepared using the same method.

[0062] The 96-well plates were incubated at 37°C and 200 rpm for 16-24 hours. The biomass was measured using a microplate reader, and the minimum inhibitory concentration (MIC) was determined.

[0063] (4) Application of antimicrobial peptides in chicken preservation:

[0064] Cut the raw chicken into 1cm pieces 3 The raw chicken meat was first sterilized by soaking in 0.5% sodium hypochlorite for 1 minute, then washed 3 times with PBS (phosphate buffer), then sterilized by soaking in 75% ethanol for 1 minute, washed 3 times with PBS, and then uniformly irradiated with ultraviolet light for 1.2 hours to make the raw chicken sterile.

[0065] Taking Staphylococcus aureus as an example, the previously stored Staphylococcus aureus was taken out of the -80℃ freezer and activated and cultured on LB solid medium, and cultured at 37℃ for 16-24 hours.

[0066] After single colonies have grown on the plate, use a sterilized inoculation loop to pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C in a shaker for 16-24 h to activate the culture. Measure the biomass using a microplate reader to determine the final biomass OD. 600 It reaches between 1.0 and 1.2.

[0067] Dilute the activated bacterial solution to a concentration of 5 × 10⁻⁶. 3The diluted bacterial solution was evenly spread onto the surface of chicken cubes using a sterile cotton swab at a concentration of CFU / mL, and then incubated in a 25°C incubator for 2 hours to infect the chicken.

[0068] A 3.125 μM antimicrobial peptide was evenly applied to the surface of chicken cubes using a sterile cotton swab and incubated in a 25°C incubator for 32 h. PBS buffer solution was used as a PBS control.

[0069] The chicken pieces were homogenized and centrifuged, and the supernatant was then diluted and spread on the appropriate selective medium to count the remaining viable bacteria.

[0070] PBS buffer solution (pH 7.2) formulation: Take 100 mL of 0.2 mol / L potassium dihydrogen phosphate solution and 70 mL of 0.2 mol / L sodium hydroxide solution, dilute with freshly boiled and cooled water to 400 mL, and adjust the pH to between 7.2 and 7.4.

[0071] (5) Application of antimicrobial peptides in milk preservation:

[0072] Divide 30mL of commercially available fresh milk into 50mL heat-resistant Erlenmeyer flasks and seal the flasks with sterile film. Sterilize the fresh milk in an autoclave at 115℃ for 30 minutes. After sterilization, slowly cool the milk and immediately store it in a 4℃ refrigerator.

[0073] Taking Staphylococcus aureus as an example, the previously stored Staphylococcus aureus was taken out of the -80℃ freezer and activated and cultured on LB solid medium, and cultured at 37℃ for 16-24 hours.

[0074] After single colonies have grown on the plate, use a sterilized inoculation loop to pick a single colony and inoculate it into 5 mL of LB liquid medium. Incubate at 37°C in a shaker for 16-24 h to activate the culture. Measure the biomass using a microplate reader to determine the final biomass OD. 600 It reaches between 1.0 and 1.2.

[0075] Dilute the activated bacterial solution to a concentration of 5 × 10⁻⁶. 3 The diluted bacterial solution (CFU / mL) was added to the conical flask containing sterilized milk and incubated at 25°C for 2 hours to infect the milk.

[0076] The 3.125 μM antimicrobial peptide was added to the conical flask containing the bacterial culture milk, and the flask was incubated at 25°C for 32 h. Sterile water was used as a blank control.

[0077] The milk samples were diluted and spread on the corresponding selective culture media to count the remaining viable bacteria.

[0078] Results and Analysis:

[0079] exist Figure 2 The antimicrobial peptide of this invention exhibits a typical α-helix structure. Antimicrobial peptide 746 (KVYVIKAPRFIKKLLSVFIKK) is a peptide with potential antimicrobial activity. Results show that this peptide has high structural confidence (pLDDT=74.8) and good matching with the experimental structure (pTM=0.121). Its electrostatic charge is 7, indicating that the peptide carries a positive charge under physiological conditions, which is beneficial for interaction with negatively charged bacterial membranes. The normalized hydrophobic moment is 1.1, and the amphiphilicity index is 1.41, indicating that the peptide has certain hydrophobicity and amphiphilicity, enabling it to interact with the lipid bilayer of bacterial membranes. The disordered structure tendency is 0.05, indicating that the peptide has a relatively stable secondary structure under certain conditions. The isoelectric point penetration depth is 13, further demonstrating its strong penetration ability near the isoelectric point. This peptide has a typical α-helix structure with 3 helices and a tendency to form triple helices of 1.03. These structural features help it bind to and insert into bacterial membranes, thereby exerting its antibacterial effect.

[0080] Figure 3 It can be found that the antimicrobial peptides obtained by this invention can inhibit Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii. Figure 3 (a) It may be invented that the diameter of the inhibition zone of Staphylococcus aureus is 14 mm, the diameter of the inhibition zone of Klebsiella pneumoniae is 9 mm, and the diameter of the inhibition zone of Acinetobacter baumannii is 12 mm.

[0081] Figure 4 It can be found that the antimicrobial peptide obtained by this invention has a mic value of 3.125 μM against Staphylococcus aureus, and its inhibitory effect on Staphylococcus aureus is significantly better than that of Nisin in the prior art. Furthermore, the antimicrobial peptide of this invention has a mic value of 50 μM against Klebsiella pneumoniae and 6.25 μM against Acinetobacter baumannii, while Nisin in the prior art has no inhibitory effect on Klebsiella pneumoniae and Acinetobacter baumannii. Therefore, the antimicrobial peptide of this invention has significant advantages in inhibiting Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

[0082] Figure 5 It can be found that the antimicrobial peptides obtained by the present invention can be used to inhibit Staphylococcus aureus in chicken, and its inhibitory effect on Staphylococcus aureus is significant within 32 hours.

[0083] Figure 6 It can be found that the antimicrobial peptides obtained by the present invention can be used to inhibit Staphylococcus aureus in milk, and its inhibitory effect on Staphylococcus aureus is significant within 32 hours.

[0084] In summary, the antimicrobial peptides obtained by this invention can inhibit Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii, which is significantly superior to the prior art.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A novel antibacterial peptide of Calorimonas adulescens, characterized in that, The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO:

1.

2. An antibacterial composition, characterized by, The antibacterial composition comprises the antibacterial peptide with the amino acid sequence of SEQ ID NO:

1.

3. A gene encoding a gene, characterized in that, The encoding gene is used for encoding the antibacterial peptide of claim 1.

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

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

6. Use of the antibacterial peptide of claim 1 in preparing a drug for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

7. Use of the antibacterial composition of claim 2 in preparing a drug for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

8. Use of the antibacterial peptide according to claim 1 for the preparation of an additive, characterized in that, The additive is any one of feed or food additives.

9. Use according to claim 5, characterized in that, The minimum inhibitory concentration of the antibacterial peptide for inhibiting Staphylococcus aureus is 3.125 μM, the minimum inhibitory concentration for inhibiting Klebsiella pneumoniae is 50 μM and the minimum inhibitory concentration for inhibiting Acinetobacter baumannii is 6.25 μM.

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