Antimicrobial peptide cAMP048 and its application

Abstract

The present application proposes an antimicrobial peptide cAMP048 derived from deep-sea microorganisms and its application, which belongs to the technical field of peptide inhibitors. The aforementioned antimicrobial peptide cAMP048 has an amino acid sequence as shown in SEQ ID NO: 1. The aforementioned antimicrobial peptide can inhibit the growth of Acinetobacter baumannii at 64 μM, inhibit the growth of multidrug-resistant Acinetobacter baumannii and multidrug-resistant Enterococcus faecium at 128 μM, and inhibit the growth of Escherichia coli, Staphylococcus aureus and multidrug-resistant Klebsiella pneumoniae at 256 μM, and can be used to prepare products with antibacterial and / or bactericidal properties, 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 in particular, to an antimicrobial peptide cAMP048 derived from deep-sea microorganisms and its application. Background Art

[0002] Antibiotic resistance refers to the ability of microorganisms (including bacteria, fungi and viruses) to resist antibiotics that can inhibit or kill them, resulting in these antibiotics becoming less effective or ineffective in treating microbial infections. Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Enterococcus faecium are common clinical pathogens that can cause a variety of infectious diseases. The World Health Organization has identified them as key multidrug-resistant bacteria. Acinetobacter baumannii, A. baumannii ) is a Gram-negative bacterium that often causes pneumonia or bloodstream infections in critically ill patients. The bacterium is naturally resistant to many antimicrobial drugs, and its resistance to carbapenem antibiotics has increased significantly in recent years. Escherichia coli , E. coli ) and Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae ) is a Gram-negative bacterium that often causes urinary tract infections, pneumonia, and bloodstream infections. 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 faecium , E. faecium ) is a Gram-positive bacterium that often causes urinary tract infections and abdominal infections. Staphylococcus aureus , S. aureus ) is a Gram-positive bacterium and a major pathogen of purulent skin and soft tissue infections in humans. In recent years, resistance of Enterococcus faecium and Staphylococcus aureus to key antibiotics such as vancomycin and methicillin has increased significantly globally. This resistance among these key pathogens complicates the treatment of these infections and poses significant challenges to clinical practice.

[0003] Antimicrobial peptides are a class of small peptides that can fight a wide range of microorganisms, including bacteria, fungi, viruses, and parasites. These peptides primarily bind to the cell membrane or cell wall of microorganisms, causing leakage of intracellular substances and ultimately killing the microorganisms. Bacteria are less likely to resist by mutating their membranes, making antimicrobial peptides less likely to develop drug resistance, making them a promising alternative to traditional antibiotics. Summary of the Invention

[0004] The present application aims to solve one of the technical problems in the related art to at least a certain extent. To this end, one purpose of the present application is to provide an antimicrobial peptide cAMP048 and its application, which is derived from deep-sea environmental microorganisms and 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. The antimicrobial peptide cAMP048 is not easy to develop drug resistance, has low mammalian cell toxicity, low hemolytic toxicity, and high cell selectivity, and can be used to prepare antimicrobial compositions for treating Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium, and multidrug-resistant Klebsiella pneumoniae. It can also be used as a peptide inhibitor in food, skin care, and pharmaceutical fields.

[0005] Specifically, the technical solution of this application is as follows:

[0006] In a first aspect, the present application provides an antimicrobial peptide cAMP048. According to an embodiment of the present application, the antimicrobial peptide cAMP048 has an amino acid sequence as shown in SEQ ID NO: 1.

[0007] PGRRLSARERLGIYHRSYWA (SEQ ID NO: 1).

[0008] In some examples of the present application, the antimicrobial peptide cAMP048 consists of the amino acid sequence shown in SEQ ID NO: 1.

[0009] In a second aspect, the present application proposes the use of the antimicrobial peptide cAMP048 described in the first aspect in the preparation of products with antibacterial and / or bactericidal properties.

[0010] It is understandable that the aforementioned products include but are not limited to antibacterial agents, hand sanitizers, feed, etc.

[0011] In some examples of the present application, the minimum inhibitory concentration of the antimicrobial peptide cAMP048 against Acinetobacter baumannii is 64 μM.

[0012] In some examples of the present application, the minimum inhibitory concentration of the antimicrobial peptide cAMP048 against multidrug-resistant Acinetobacter baumannii or multidrug-resistant Enterococcus faecium is 128 μM.

[0013] In some examples of the present application, the minimum inhibitory concentration of the antimicrobial peptide cAMP048 against Escherichia coli, Staphylococcus aureus, or multidrug-resistant Klebsiella pneumoniae is 256 μM.

[0014] In a third aspect, the present application proposes the use of the antimicrobial 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.

[0015] 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 antimicrobial peptide cAMP048 described in the first aspect.

[0016] In some examples of the present application, the aforementioned composition is selected from facial cleanser, hand soap, shower gel, shampoo, mouthwash, toothpaste, soap, cosmetics, feminine care wash, laundry soap, laundry liquid, laundry powder, detergent, disinfectant or toilet cleaning liquid.

[0017] In a fifth aspect, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises: the antimicrobial peptide cAMP048 described in the first aspect.

[0018] 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 antimicrobial peptide cAMP048 described in the first aspect.

[0019] It is understandable that the “cAMP048” in the aforementioned antimicrobial peptide cAMP048 only represents the number of the antimicrobial peptide, and the number does not limit the composition and functional activity of the antimicrobial peptide itself.

[0020] The aforementioned antimicrobial peptide cAMP048 is derived from deep-sea microorganisms and has the following beneficial technical effects:

[0021] 1) It has been verified that the antimicrobial 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.

[0022] 2) It has been verified that the antimicrobial peptide cAMP048 of the present application is not easy to develop drug resistance, has low mammalian cytotoxicity, low hemolytic toxicity and high cell selectivity, and can be used to prepare antimicrobial compositions for infections of Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Enterococcus faecium and multidrug-resistant Klebsiella pneumoniae; in addition, the antimicrobial peptide cAMP048 can be used as a peptide inhibitor in the fields of food, skin care and medicine.

[0023] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 This is a schematic diagram of the HPLC detection results of the antimicrobial peptide cAMP048 provided in one embodiment of the present application;

[0026] Figure 2 This is a schematic diagram of the LCMS detection results of the antimicrobial peptide cAMP048 provided in one embodiment of the present application;

[0027] Figure 3 This is a schematic diagram of the minimum inhibitory concentration determination results of the antimicrobial peptide cAMP048 provided in an embodiment of the present application; wherein (a) is Acinetobacter baumannii A. baumannii ATCC 19606; (b) multi-drug resistant (MDR) Acinetobacter baumannii A. baumannii BAA-1605; (c) Escherichia coli E. coli ATCC 25922; (d) is multidrug-resistant Klebsiella pneumoniae MDR K. pneumoniae ATCC 700603; (e) is multidrug-resistant Enterococcus faecium MDR E. faecium BM4105; (f) Staphylococcus aureus S. aureus ATCC 29213;

[0028] Figure 4 This is a schematic diagram of the mammalian cell toxicity assay results of the antimicrobial peptide cAMP048 provided in one embodiment of the present application;

[0029] Figure 5 This is a schematic diagram of the hemolytic toxicity assay results of the antimicrobial peptide cAMP048 provided in one embodiment of the present application. DETAILED DESCRIPTION

[0030] The following will be combined with the accompanying drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0031] It should be noted that the terms "first", "second", etc. in the specification and claims of this application and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or server that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products, or devices.

[0032] This application is based on the global extreme environment microbiome database (The Extreme Environment Microbiome Catalogue, EEMC) constructed by Sanya BGI Life Sciences Institute to mine and screen new antimicrobial peptides. First, the antiSMASH (v7.0) tool (Kai Blin, Simon Shaw, Hannah E Augustijn, et al., antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualization, Nucleic Acids Research, Volume 51, Issue W1, 5 July 2023, Pages W46–W50) was used with the parameter --minlength 5000 to identify biosynthesis gene clusters (BGCs) in the genomes of extreme environment microorganisms. Deep learning models (RNN, LSTM) were used to predict the core peptide sequences in BGCs of ribosomal postsynthetic modified polypeptides (RiPPs). Furthermore, the obtained core peptide sequences were used to predict low-toxicity antimicrobial peptides using three pre-trained protein language models (ESM2-3B, ESM3, and PTRANS). Peptide sequences ranked in the top 50% by each model's prediction score were screened as candidate low-toxicity antimicrobial peptides. The candidate peptides were then compared with a self-built antimicrobial peptide database, and peptide sequences that matched known databases were removed. New candidate low-toxicity antimicrobial peptide sequences were then identified and experimentally validated. Ultimately, the antimicrobial peptide derived from deep-sea microorganisms of the present invention was obtained and named cAMP048. The following describes the process and results of wet-bed experiments to verify the antimicrobial activity and toxicity of this antimicrobial peptide.

[0033] If no specific techniques or conditions are specified in the following examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without manufacturer specified were commercially available conventional products.

[0034] In the following embodiments A. baumannii Indicates Acinetobacter baumannii (ATCC), E. coli Indicates Escherichia coli (ATCC), S. aureus Indicates Staphylococcus aureus (ATCC), MDR A. baumannii Indicates multidrug-resistant Acinetobacter baumannii (Beinuo Bio), MDR E. faecium Indicates multidrug-resistant Enterococcus faecium (ATCC), MDR K. pneumoniae Indicates multidrug-resistant Klebsiella pneumoniae (Beinuo Biotechnology).

[0035] Example 1: Synthesis and purification of antimicrobial peptide cAMP048

[0036] The antimicrobial peptide cAMP048 was chemically synthesized by solid-phase peptide synthesis (commercial service provided by Shanghai Sangon Biotechnology Co., Ltd.). The purity of the antimicrobial peptide was determined by high-performance liquid chromatography (HPLC), and the exact molecular weight of cAMP048 was determined by mass spectrometry.

[0037] The model of the HPLC analytical column is SHIMADZU shim-pack GIST (4.6*250MM*5UM), the mobile phase A is deionized water containing 0.1% trifluoroacetic acid, the mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid, the flow rate is fixed at 1 mL / min, the detection wavelength is 214 nm, the detection sample is 0.5 mg of antimicrobial peptide dissolved in 20% acetonitrile and 80% water to 0.5 mL, the injection volume is 30 μL, and the detection method is a linear increase in the mobile phase B gradient (20%~80%) from 0 minute to 20 minutes.

[0038] The LCMS detection sample was 0.1 mg of antimicrobial peptide dissolved in 50% acetonitrile and 50% water to 0.5 mL, the nebulizer 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 rod front 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.

[0039] HPLC test results are as follows Figure 1 As shown in the results, the purity of the synthesized antimicrobial peptide cAMP048 is greater than 95%. The amino acid sequence is PGRRLSARERLGIYHRSYWA, as shown in SEQ ID No. 1.

[0040] LCMS test results are as follows Figure 2 As shown in FIG, the results show that the theoretical molecular weight of the antimicrobial peptide cAMP048 is 2444.777, and the synthesized cAMP048 is consistent with the theoretical molecular weight.

[0041] Example 2: Activity analysis of antimicrobial peptides

[0042] (1) Minimum inhibitory concentration (MIC) determination

[0043] Six bacterial strains, including Acinetobacter baumannii A. baumannii , Escherichia coli E. coli , Staphylococcus aureus S. aureus , multidrug-resistant Acinetobacter baumannii MDR A. baumannii , multidrug-resistant Enterococcus faecium MDR E. faecium and multidrug-resistant Klebsiella pneumoniae MDR K. pneumoniae Streak onto Luriae–Bertani (LB) agar medium and incubate overnight at 37°C.

[0044] Single colonies of the six bacterial strains were picked into Mueller-Hinton Broth (MHB) (ThermoFisher Scientific) liquid medium and shaken at 120 rpm at 37°C overnight. The culture was diluted 1:100 with fresh MHB and then cultured to the exponential phase (OD 600 The cell concentration of the bacterial solution was then adjusted to 1×10 6 cfu / mL. Subsequently, 100 μL of the diluted bacterial solution was transferred to each well of a 96-well plate, and 100 μL of MHB medium containing cAMP048, obtained by serial two-fold dilution, was added to achieve a final cAMP048 concentration ranging from 256 μM to 0.5 μM. The negative control group consisted of 100 μL of bacterial solution added to 100 μL of MHB liquid medium, and the blank control group consisted of 200 μL of MHB medium.

[0045] The preparation process of two-fold diluted cAMP048 is as follows:

[0046] A 512 μM stock solution of cAMP048 was prepared and serially diluted in equal volumes of MHB medium. 100 μL of the highest cAMP048 concentration was added to the first well. 100 μL of this solution was then transferred from this well to a second well containing 100 μL of MHB medium, mixed, and the concentration reduced by half. Repeat this process, transferring 100 μL from the second well to a third well (containing 100 μL of MHB medium), mixing, and reducing the concentration by half again. This process was repeated until a 1 μM concentration was reached. As described above, 100 μL of each concentration of cAMP048 in MHB medium was mixed with 100 μL of the bacterial suspension, resulting in final cAMP048 concentrations ranging from 256 μM to 0.5 μM.

[0047] After incubation at 37°C for 16-18 hours, the MIC value was calculated as the lowest concentration of antimicrobial peptide at which no bacterial growth was observed. All experiments were repeated three times. Figure 3 shown.

[0048] Figure 3 The results showed that the antimicrobial peptide cAMP048 A. baumannii The MIC of (a) is 64 μM, and the MDR A. baumannii (b) or MDR E. faecium The MIC of (e) is 128 μM. E. coli (c) MDR K. pneumoniae (d) or S. aureus The MIC of (f) is 256 μM.

[0049] (2) Mammalian cell toxicity assay

[0050] The experiment was conducted using blank, control, and cAMP048 groups. The cAMP048 solution was prepared in PBS at a concentration of 60 μM. L-02 human hepatocytes (Beina Biotech, China) and 293T human embryonic kidney cells (Punosai, China) were cultured in medium containing 10% fetal bovine serum (04-001-1acs, Biological Industries, Israel) to form a single cell suspension. 5 × 10 cells were seeded into each well of a 96-well plate (90 μL). 4 / mL of adherent cells and 9×10 4 / mL of suspended cells were pre-cultured for 24 hours under 5% CO2 and 37°C. 10 μL of cisplatin (D8810, Solarbio, Germany) was added to each well of the control group to make the final concentration of cisplatin 60 μM. 10 μL of cAMP048 solution was added to each well of the antimicrobial peptide group and cultured in an incubator for 48 hours. The old culture medium and drug solution were aspirated, and 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 culture medium to a final concentration of 10% v / v. The culture was continued for 1 hour in the dark, 5% CO2, and 37°C. The absorbance at 450 nm was measured with an enzyme 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 were repeated three times. Figure 4 shown.

[0051] 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. The inhibition rate of the antimicrobial peptide cAMP048 on L-02 human hepatocytes was 0.45%, and the inhibition rate on 293T human embryonic kidney cells was 21.22%.

[0052] (3) Hemolytic toxicity assay

[0053] The experiment set up a blank group (Blank), a control group (Control), and an antimicrobial peptide group (cAMP048). Fresh sheep blood cells (defibrinated sheep blood, Beekman Bio, China) were centrifuged at 1500 rpm for 10 minutes to separate plasma and red blood cells. The red blood cells were repeatedly washed 3-4 times with PBS buffer until the supernatant was clear and transparent. The red blood cells were resuspended in 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 a 96-well plate. Triton X-100 (Sigma-Aldrich, Germany) was added to each well of the control group to a final concentration of 1%. cAMP048 solution was added to each well of the antimicrobial peptide group to a final concentration of 60 μM. The plates were incubated in an incubator at 37°C for 1 hour. After the experiment, the plates were immediately centrifuged at 4°C and 1500 rpm for 10 minutes. The supernatant was collected onto a new 96-well plate and the absorbance at 570 nm was measured using a microplate reader. The raw data results were recorded. The hemolysis rate was calculated as (OD cAMP048 -OD Blank ) / (OD Control -OD Blank) × 100%. All experiments were repeated 4 times. Figure 5 shown.

[0054] Figure 5 The results showed that compared with the control group, the antimicrobial peptide cAMP048 group had a lower OD value and significantly lower hemolytic toxicity. The hemolytic rate of the antimicrobial peptide cAMP048 was calculated to be 0.79%.

[0055] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0056] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application without departing from the principles and purpose of the present application.

Claims

1. An antimicrobial peptide cAMP048, characterized in that It consists of 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, wherein: The bacteria 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.

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

4. The use 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 use according to claim 2, characterized in that The minimum inhibitory concentration of the antimicrobial peptide cAMP048 against Escherichia coli, Staphylococcus aureus or multidrug-resistant Klebsiella pneumoniae is 256 μM.

6. Use of the antimicrobial peptide cAMP048 according to claim 1 in the preparation of antibacterial drugs, wherein: The bacteria 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 hand soap, body wash, shampoo, mouthwash, toothpaste, soap, cosmetics, feminine care wash, laundry soap, laundry liquid, laundry powder, detergent, disinfectant or toilet cleaning liquid.

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

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

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