Antibacterial peptide cAMP048 and application thereof

Abstract

The invention provides an antibacterial peptide cAMP048 sourced from microorganisms in a deep sea environment and application of the antibacterial peptide cAMP048, and belongs to the technical field of peptide inhibitors. The antibacterial peptide cAMP048 has an amino acid sequence as shown in SEQ ID NO: 1 (Sequence Identity Number 1). The antibacterial peptide can inhibit the growth of acinetobacter baumannii at 64 mu M, inhibit the growth of multi-drug-resistant acinetobacter baumannii and multi-drug-resistant enterococcus faecium at 128 mu M and inhibit the growth of escherichia coli, staphylococcus aureus and multi-drug-resistant klebsiella pneumoniae at 256 mu M. The antibacterial peptide can be used for preparing products with antibacterial and / or bactericidal performance, and can be applied to preparation of antibacterial and / or bactericidal products. Such as food, skin care products or medical supplies.

Description

Technical Field

[0001] The present application relates to the technical field of peptide inhibitors, and specifically, to an antibacterial peptide cAMP048 derived from deep-sea microorganisms and its applications. Background Art

[0002] Antibiotic resistance refers to the ability of microorganisms (including bacteria, fungi, and viruses) to resist antibiotics that could originally inhibit or kill them, resulting in a weakened or ineffective effect of these antibiotic drugs in treating microbial infections. Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis are common pathogenic bacteria in clinical practice, which can cause various infectious diseases, and the World Health Organization has identified them as key multidrug-resistant bacteria. Acinetobacter baumannii ( Acinetobacter baumannii,A. baumannii ), a Gram-negative bacterium, often causes pneumonia or bloodstream infections in critically ill patients. This bacterium is naturally resistant to many antibacterial drugs, and in recent years, its resistance to carbapenem antibiotics has increased significantly. Escherichia coli ( Escherichia coli , E. coli ) and Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae ) are Gram-negative bacteria, which often cause urinary tract infections, pneumonia, and bloodstream infections, etc. In recent years, the resistance of Escherichia coli and Klebsiella pneumoniae to key antibiotics such as carbapenems and third-generation cephalosporins has increased significantly. Enterococcus faecalis ( Enterococcus faecium , E. faecium ) is a Gram-positive bacterium, which often causes urinary tract infections and intra-abdominal infections. Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) is a Gram-positive bacterium and is the main pathogenic bacterium of suppurative infections of human skin and soft tissues. In recent years, the resistance of Enterococcus faecalis and Staphylococcus aureus to key antibiotics such as vancomycin and methicillin has increased significantly globally. The resistance of these key pathogenic bacteria has increased the difficulty of treating infections and brought huge challenges to clinical treatment.

[0003] Antibacterial peptides are a class of small peptides that can combat various microorganisms, including bacteria, fungi, viruses, and parasites. These peptides mainly bind to the cell membranes or cell walls of microorganisms, causing the leakage of intracellular substances and ultimately resulting in the death of microorganisms. Bacteria are difficult to resist by mutating to change the membrane structure, so antibacterial peptides are not likely to develop resistance and are expected to become effective alternatives to traditional antibiotics. Summary of the Invention

[0004] This application aims to solve at least one of the technical problems in the related art to some extent. For this purpose, an object of this application is to provide an antibacterial peptide cAMP048 and its application. This antibacterial peptide is derived from deep-sea environmental microorganisms and has broad-spectrum antibacterial activities against Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae. The antibacterial peptide cAMP048 is not prone to drug resistance and has low mammalian cell toxicity, low hemolytic toxicity, and high cell selectivity. It can be used to prepare antibacterial compositions for treating Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae. At the same time, it can also be used as a peptide inhibitor in the fields of food, skin care, and medicine.

[0005] Specifically, the technical solution of this application is as follows: In a first aspect, this application proposes an antibacterial peptide cAMP048. According to an embodiment of this application, this antibacterial peptide cAMP048 has an amino acid sequence as shown in SEQ ID NO: 1.

[0006] PGRRLSARERLGIYHRSYWA (SEQ ID NO: 1).

[0007] In some examples of this application, the antibacterial peptide cAMP048 consists of the amino acid sequence shown in SEQ ID NO: 1.

[0008] In a second aspect, this application proposes the application of the antibacterial peptide cAMP048 described in the first aspect in the preparation of a product with antibacterial and / or bactericidal properties.

[0009] It can be understood that the aforementioned products include but are not limited to antibacterial agents, hand sanitizers, feeds, etc.

[0010] In some examples of this application, the minimum inhibitory concentration of the antibacterial peptide cAMP048 against Acinetobacter baumannii is 64 μM.

[0011] In some examples of this application, the minimum inhibitory concentration of the antibacterial peptide cAMP048 against multidrug-resistant Acinetobacter baumannii or multidrug-resistant Enterococcus faecium is 128 μM.

[0012] In some examples of this application, the minimum inhibitory concentration of the antibacterial peptide cAMP048 against Escherichia coli or Staphylococcus aureus or multidrug-resistant Klebsiella pneumoniae is 256 μM.

[0013] In a third aspect, the present application provides the use of the antibacterial peptide cAMP048 described in the first aspect in the preparation of antibacterial drugs, wherein the bacteria are selected from at least one of Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae.

[0014] In a fourth aspect, the present application provides a composition. According to an embodiment of the present application, the active ingredient of the composition is the antibacterial peptide cAMP048 described in the first aspect.

[0015] In some examples of the present application, the aforementioned composition is selected from facial cleanser, hand sanitizer, body wash, shampoo, mouthwash, toothpaste, soap, cosmetics, feminine care lotion, laundry soap, laundry detergent, washing powder, dishwashing liquid, disinfectant, or toilet cleaner.

[0016] In a fifth aspect, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition includes: the antibacterial peptide cAMP048 described in the first aspect.

[0017] In a sixth aspect, the present application provides an antibacterial additive. According to an embodiment of the present application, the active ingredient of the antibacterial additive is the antibacterial peptide cAMP048 described in the first aspect.

[0018] It can be understood that "cAMP048" in the aforementioned antibacterial peptide cAMP048 only represents the number of the antibacterial peptide, and this number does not limit the composition and functional activity of the antibacterial peptide itself.

[0019] The aforementioned antibacterial peptide cAMP048 is derived from deep-sea environmental microorganisms and has the following beneficial technical effects: 1) It has been verified that the antibacterial peptide cAMP048 of the present application has broad-spectrum antibacterial activity against Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae.

[0020] 2) It has been verified that the antibacterial peptide cAMP048 of the present application is not prone to drug resistance, has low mammalian cell toxicity, low hemolytic toxicity, and high cell selectivity, and can be used to prepare antibacterial compositions for the treatment of infections caused by Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae; in addition, the antibacterial peptide cAMP048 can be used as a peptide inhibitor in the fields of food, skin care, and medicine.

[0021] Additional aspects and advantages of the present application will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0023] Figure 1 It is a schematic diagram of the HPLC detection result of the antibacterial peptide cAMP048 provided by an embodiment of the present application; Figure 2 It is a schematic diagram of the LCMS detection result of the antibacterial peptide cAMP048 provided by an embodiment of the present application; Figure 3 It is a schematic diagram of the minimum inhibitory concentration determination result of the antibacterial peptide cAMP048 provided by an embodiment of the present application; among them, (a) is Acinetobacter baumannii A. baumannii ATCC 19606; (b) is multi-drug resistant (MDR) Acinetobacter baumannii A. baumannii BAA-1605; (c) is Escherichia coli E. coli ATCC 25922; (d) is multi-drug resistant Klebsiella pneumoniae MDR K. pneumoniae ATCC 700603; (e) is multi-drug resistant Enterococcus faecium MDR E. faecium BM4105; (f) is Staphylococcus aureus S. aureus ATCC 29213; Figure 4 It is a schematic diagram of the mammalian cell toxicity determination result of the antibacterial peptide cAMP048 provided by an embodiment of the present application; Figure 5 It is a schematic diagram of the hemolytic toxicity determination result of the antibacterial peptide cAMP048 provided by an embodiment of the present application. Detailed implementation manners

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present application in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0025] It should be noted that the terms "first", "second", etc. in the description, claims and the above-mentioned drawings of this application are used to distinguish similar objects, and do not necessarily have to be used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of the present application described here can be implemented in an order other than those illustrated or described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or server that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0026] This application is based on the Global Extreme Environment Microbiome Catalogue (EEMC) constructed by BGI Research Sanya to mine and screen for novel antimicrobial peptides. First, the antiSMASH (v7.0) (Kai Blin, Simon Shaw, Hannah E Augustijn, et al., antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation, Nucleic Acids Research, Volume 51, Issue W1, 5 July 2023, Pages W46–W50) tool was used with the parameter set to --minlength 5000 to identify the biosynthesis gene clusters (BGCs) in extreme environment microbial genomes, and a deep learning model (RNN, LSTM) was used to predict the core peptide sequences in ribosomally synthesized and post-translationally modified peptides (RiPPs) - type BGCs. Further, three pre-trained protein language models (ESM2-3B, ESM3, PTRANS) were used to predict low-toxicity antimicrobial peptides for the obtained core peptide sequences, and the polypeptide sequences with a prediction score ranking in the top 50% for each model were screened as candidate low-toxicity antimicrobial peptides. The candidate antimicrobial peptides were compared with a self-built antimicrobial peptide database to remove the polypeptide sequences that were identical to the known database, and then novel candidate low-toxicity antimicrobial peptide sequences were obtained. These antimicrobial peptides were then experimentally verified. Finally, the antimicrobial peptide of the present invention derived from deep-sea environmental microorganisms was obtained and named cAMP048. The following are the processes and results of wet experiments to verify the antibacterial function and toxicity of this antimicrobial peptide.

[0027] In the following examples, for those without specific technologies or conditions indicated, the technologies or conditions described in the literature in this field or in accordance with the product specifications shall be followed. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through commercial purchase.

[0028] In the following examples A. baumannii represents Acinetobacter baumannii (ATCC), E. coli represents Escherichia coli (ATCC), S. aureus represents Staphylococcus aureus (ATCC), MDR A. baumannii represents multidrug-resistant Acinetobacter baumannii (BeiNuo Biotechnology), MDR E. faecium represents multidrug-resistant Enterococcus faecium (ATCC), MDR K. pneumoniae represents multidrug-resistant Klebsiella pneumoniae (BeiNuo Biotechnology).

[0029] Example 1: Synthesis and purification of antibacterial peptide cAMP048 The antibacterial peptide cAMP048 was chemically synthesized by solid-phase peptide synthesis method (commercial service provided by Shanghai Sangon Biotech Co., Ltd.). The purity of the antibacterial peptide was determined by high-performance liquid chromatography (HPLC), and the accurate molecular weight of cAMP048 was detected by mass spectrometry.

[0030] The model of the HPLC analysis column was SHIMADZU shim-pack GIST(4.6*250MM*5UM). Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was fixed at 1 mL / min, the detection wavelength was 214 nm. The sample for detection was 0.5 mg of antibacterial peptide dissolved in 0.5 mL with 20% acetonitrile and 80% water. The injection volume was 30 μL, and the detection method was to linearly increase the gradient of mobile phase B (20%~80%) from 0 minute to 20 minutes.

[0031] The sample for LCMS detection was 0.1 mg of antibacterial peptide dissolved in 0.5 mL with 50% acetonitrile and 50% water. The atomizing gas flow rate was 1.50 L / min, the CDL temperature was 250 °C, the CDL voltage was 0 v, the module temperature was 200 °C, the pre-rod deviation was +4.5 kv, the detector was -0.2 kv, the T.Flow was 0.2 mL / min, and the mobile phase was 50% water / 50% methanol.

[0032] The HPLC detection results are as Figure 1 shown. From this result, it can be known that the purity of the synthesized antibacterial peptide cAMP048 is greater than 95%. The amino acid sequence is PGRRLSARERLGIYHRSYWA, as shown in SEQ ID No. 1.

[0033] The LCMS detection results are as Figure 2 shown. From these results, it can be seen that the theoretical molecular weight of the antimicrobial peptide cAMP048 is 2444.777, and the synthesized cAMP048 is consistent with the theoretical molecular weight.

[0034] Example 2: Activity analysis of antimicrobial peptides (1)Determination of minimum inhibitory concentration (MIC) Six bacterial strains, including Acinetobacter baumannii A. baumannii , Escherichia coli E. coli , Staphylococcus aureus S. aureus , multi-drug resistant Acinetobacter baumannii MDR A. baumannii , multi-drug resistant Enterococcus faecium MDR E. faecium and multi-drug resistant Klebsiella pneumoniae MDR K. pneumoniae were streaked on Luriae–Bertani (LB) agar medium and incubated overnight at 37°C.

[0035] Single colonies of the above six bacterial strains were picked into Mueller-Hinton Broth (MHB) (ThermoFisher Scientific) liquid medium and shaken overnight at 37°C at 120 rpm. The culture was diluted 1:100 with fresh MHB and then cultured until the exponential phase (OD 600 was 0.4 - 0.6), and then the cell concentration of the bacterial suspension was adjusted to 1×10 6 cfu / mL. Subsequently, 100 μL of the diluted bacterial suspension was transferred to each well of a 96-well plate, and 100 μL of the MHB medium containing cAMP048 obtained by serial two-fold dilution was added, so that the final cAMP048 concentration range was 256 μM to 0.5 μM. The negative control group was 100 μL of MHB liquid medium plus 100 μL of the bacterial suspension, and the blank group was 200 μL of MHB medium.

[0036] Among them, the preparation process of the two-fold diluted cAMP048 is as follows: Prepare a stock solution of cAMP048 at 512 μM, and take equal volumes of MHB medium for serial dilutions in equal ratios. Add 100 μL of the highest concentration solution containing cAMP048 to the first well. Subsequently, take 100 μL of the solution from this well and add it to the second well, which already contains 100 μL of MHB medium. After mixing, the solution concentration is halved. Repeat the above operation, transfer 100 μL from the second well to the third well (containing 100 μL of MHB medium), mix well, and halve the concentration again. And so on until a concentration of 1 μM is reached. As described above, after mixing 100 μL of MHB medium containing cAMP048 at each concentration with 100 μL of the bacterial solution, the final cAMP048 concentration range is from 256 μM to 0.5 μM.

[0037] After incubation at 37 °C for 16 - 18 hours, the MIC value is the lowest concentration of the antimicrobial peptide at which no bacterial growth is visually observed. All experiments have 3 technical replicates. The experimental results are as Figure 3 shown.

[0038] Figure 3 The results of A. baumannii show that the MIC of the antimicrobial peptide cAMP048 against A. baumannii (a) is 64 μM, and the MIC against MDR E. faecium baumannii (b) or MDR E. faecium K. pneumoniae (e) is 128 μM, and the MIC against E. coli (c), MDR K. pneumoniae S. aureus (d) or S. aureus (f) is 256 μM.

[0039] (2) Mammalian cell cytotoxicity assay Set up a blank group (Blank), a control group (Control), and an antimicrobial peptide group (cAMP048) in the experiment. Prepare an antimicrobial peptide cAMP048 solution using PBS solution at a concentration of 60 μM. Use a culture medium containing 10% fetal bovine serum (04 - 001 - 1acs, Biologicalindustries, Israel) to prepare single - cell suspensions of L - 02 human hepatocytes (Beina Biotechnology, China) and 293T human embryonic kidney cells (Procell, China). Seed 90 μL of 5×10 4 / mL adherent cells and 9×10 4Suspension cells at / mL were pre-cultured for 24 hours under the conditions of 5% CO2 and 37°C. In the control group, 10 μL of cisplatin (D8810, Solarbio, Germany) was added to each well to make the final concentration of cisplatin 60 μM. In the antimicrobial peptide group, 10 μL of cAMP048 solution was added to each well and incubated in the incubator for 48 hours. The old culture medium and drug solution were aspirated out. 100 μL of CCK-8 solution (BS350A, White Shark Bio, China) diluted tenfold was added to each well of the 96-well plate. The CCK-8 reagent was diluted 1:10 with serum-free medium, and the final concentration was 10% v / v. It was continued to be cultured for 1 hour under the conditions of light avoidance, 5% CO2 and 37°C. The absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the original data results were recorded. The formula for calculating the inhibition rate of mammalian cells is (OD Control - OD cAMP048 ) / (OD Control - OD Blank ) × 100%. All experiments had 3 technical replicates. The experimental results were as Figure 4 shown.

[0040] Figure 4 The results showed that compared with the control group, the antimicrobial peptide cAMP048 group had a lower inhibition rate and significantly lower mammalian cell toxicity. After calculation, the inhibition rate of antimicrobial peptide cAMP048 on L-02 human hepatocytes was 0.45%, and the inhibition rate on 293T human embryonic kidney cells was 21.22%.

[0041] (3) Hemolytic toxicity assay The blank group (Blank), control group (Control) and antimicrobial peptide group (cAMP048) were set up in the experiment. Fresh sheep blood cells (defibrinated sheep blood, Bickman Bio, China) were centrifuged at 1500 rpm for 10 minutes to separate plasma and red blood cells. The red blood cells were washed 3 - 4 times repeatedly with PBS buffer until the supernatant was clear and transparent. The red blood cells were resuspended with PBS, and the final concentration of the red blood cell suspension was adjusted to 4%. 100 μL of the red blood cell suspension was inoculated into each well of the 96-well plate. In the control group, Triton X-100 (Sigma-Aldrich, Germany) was added to each well to a final concentration of 1%. In the antimicrobial peptide group, cAMP048 solution was added to each well to a final concentration of 60 μM. It was incubated in the incubator at 37°C for 1 hour. After the experiment, it was immediately centrifuged at 4°C and 1500 rpm for 10 minutes, and the supernatant was collected into a new 96-well plate. The absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the original data results were recorded. The formula for calculating the hemolysis rate is (OD cAMP048 - OD Blank ) / (OD Control - OD Blank) × 100%. All experiments had 4 technical replicates. The experimental results are as Figure 5 shown.

[0042] Figure 5 The results of show that, compared with the control group, the antimicrobial peptide cAMP048 group had a lower OD value and significantly lower hemolytic toxicity. After calculation, the hemolysis rate of the antimicrobial peptide cAMP048 was 0.79%.

[0043] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0044] Although the embodiments of this application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting this application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application without departing from the principles and purposes of this application.

Claims

1. An antibacterial peptide cAMP048, characterized in that, It has the amino acid sequence shown in SEQ ID NO:

1.

2. Use of the antimicrobial peptide cAMP048 according to claim 1 in the preparation of a product having bacteriostatic and / or bactericidal properties.

3. The application according to claim 2, characterized in that The minimum inhibitory concentration of the antimicrobial peptide cAMP048 against Acinetobacter baumannii is 64 μM.

4. The application according to claim 2, characterized in that The minimum inhibitory concentration of the antimicrobial peptide cAMP048 against multidrug-resistant Acinetobacter baumannii or multidrug-resistant Enterococcus faecium is 128 μM.

5. The application according to claim 2, wherein The minimum inhibitory concentration of the antimicrobial peptide cAMP048 against Escherichia coli or Staphylococcus aureus or multidrug-resistant Klebsiella pneumoniae is 256 μM.

6. Use of the antibacterial peptide cAMP048 according to claim 1 in the preparation of antibacterial drugs, wherein, The bacterium is selected from at least one of Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae.

7. A composition, characterized in that, The active ingredient of the composition is the antimicrobial peptide cAMP048 according to claim 1.

8. The composition according to claim 7, characterized in that, The composition is selected from facial cleanser, hand sanitizer, body wash, shampoo, mouthwash, toothpaste, soap, cosmetics, feminine care lotion, laundry soap, laundry detergent, washing powder, dishwashing liquid, disinfectant, or toilet cleaner.

9. A pharmaceutical composition, characterized in that, Comprising: The antimicrobial peptide cAMP048 according to claim 1.

10. An antibacterial additive, characterized in that, The active ingredient of the bacteriostatic additive is the antimicrobial peptide cAMP048 as claimed in claim 1.

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