Polypeptides and uses thereof

Abstract

The application discloses a polypeptide derived from deep-sea microorganisms and belongs to the technical field of peptide inhibitors. The polypeptide has an amino acid sequence as shown in SEQ ID NO:1; the polypeptide has the functions of inhibiting bacteria and / or killing bacteria. The polypeptide can inhibit the growth of Acinetobacter baumannii at 8 muM, and can inhibit the growth of Escherichia coli and multi-drug resistant Acinetobacter baumannii at 16 muM, and can be used for preparing products with the functions of inhibiting bacteria and / or killing bacteria, such as food, daily necessities or medical products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of peptide inhibitors, in particular, to polypeptides and applications thereof. More particularly, to polypeptides, compositions containing polypeptides, methods for inhibiting and / or killing bacteria, use of polypeptides in the preparation of products having inhibitory and / or killing properties, pharmaceutical compositions and bacteriostatic additives. BACKGROUND

[0002] Antibiotic resistance refers to the ability of microorganisms (including bacteria, fungi and viruses) to resist antibiotics that can otherwise inhibit or kill their growth by acquiring certain physiological or genetic mechanisms, resulting in a decrease in therapeutic effect or even complete failure. Currently, antibiotic resistance is occurring worldwide and is rising to a dangerous level. New drug resistance mechanisms are emerging and spreading globally, threatening the ability to treat common infectious diseases. In the clinic, Acinetobacter baumannii and Escherichia coli are common pathogens that cause infections, and are identified by the World Health Organization as key multi-drug resistant bacteria due to their extensive resistance to multiple antibiotics. Acinetobacter baumannii (A. baumannii) Acinetobacter baumannii, A. baumannii ) is a gram-negative bacterium commonly found in intensive care patients and can cause severe infections such as pneumonia and bacteremia. This bacterium is naturally resistant to multiple classes of antibacterial drugs, and in recent years its resistance to carbapenem antibiotics has increased significantly. Escherichia coli (E. coli) Escherichia coli , E. E. coli ) is a gram-negative bacterium that is a common pathogen of urinary tract infections, lung infections and bloodstream infections. The resistance of this strain to third-generation cephalosporins and carbapenem antibiotics is increasing. The widespread presence and spread of the above drug-resistant pathogens pose an unprecedented challenge to the treatment of infectious diseases in the clinic, and there is an urgent need to develop new antibacterial strategies and alternative treatment methods.

[0003] Antibacterial peptides are a class of short peptide molecules with broad-spectrum antimicrobial activity, which can effectively inhibit or kill a variety of pathogens including bacteria, fungi, viruses and parasites. Its main mechanism of action is to interact with the cell membrane or cell wall of microorganisms, disrupt the integrity of the membrane, and thus cause the contents of the cell to leak and trigger microbial death. Since it is difficult for microorganisms to significantly change the membrane structure through genetic mutation, the use of antibacterial peptides is less likely to induce the development of drug resistance, and therefore they are considered as potential alternatives to traditional antibiotics. Although antibacterial peptides show good prospects in the field of anti-infection, there are still limited products on the market with high antibacterial activity and high safety, so it is still necessary to continuously explore and optimize new antibacterial peptide resources. SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems existing in the prior art. To this end, one object of the present application is to provide a polypeptide and its application, which has broad-spectrum bacteriostatic activity against Acinetobacter baumannii, Escherichia coli and multi-drug resistant Acinetobacter baumannii. The polypeptide is not prone to drug resistance, has low mammalian cell toxicity, low hemolytic toxicity and high cell selectivity, and can be used for preparing an antibacterial composition for treating Acinetobacter baumannii, Escherichia coli and multi-drug resistant Acinetobacter baumannii, and can also be used as a peptide inhibitor in food, daily necessities or medical products, etc.

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

[0006] In a first aspect, the present application provides a polypeptide. According to an embodiment of the present application, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1; the polypeptide has a bacteriostatic and / or bactericidal function.

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

[0008] In a second aspect, the present application provides a method for bacteriostatic and / or bactericidal purposes other than treatment. According to an embodiment of the present application, the method contacts the polypeptide of the first aspect of the present application with a non-living sample to be treated.

[0009] According to an embodiment of the present application, the bacteria include at least one of Acinetobacter baumannii, Escherichia coli and multi-drug resistant Acinetobacter baumannii.

[0010] According to an embodiment of the present application, the bacteria include Acinetobacter baumannii, and the minimum concentration of the polypeptide in the contact system is 8 μM.

[0011] According to an embodiment of the present application, the bacteria include at least one of multi-drug resistant Acinetobacter baumannii and Escherichia coli, and the minimum concentration of the polypeptide in the contact system is 16 μM.

[0012] In a third aspect, the present application provides the use of the polypeptide of the first aspect in the preparation of a product having bacteriostatic and / or bactericidal properties.

[0013] It can be understood that the aforementioned product includes but is not limited to bacteriostatic agents, hand sanitizers, feed, etc.

[0014] According to an embodiment of the present application, the bacteria include at least one of Acinetobacter baumannii, Escherichia coli and multi-drug resistant Acinetobacter baumannii.

[0015] According to an embodiment of the present application, the minimum bacteriostatic concentration of the polypeptide against Acinetobacter baumannii is 8 μM.

[0016] According to embodiments of the present application, the polypeptide has a minimum inhibitory concentration of 16 μM against multi-drug resistant Acinetobacter baumannii and Escherichia coli.

[0017] In a fourth aspect, the present application provides a composition. According to embodiments of the present application, the active ingredient of the composition comprises the polypeptide of the first aspect of the present application.

[0018] It can be understood that as long as the composition comprises the aforementioned polypeptide, and the aforementioned polypeptide is the active ingredient of the composition, the specific type of the composition is not limited, for example, the composition can be a daily necessity, a medicine, a food additive, etc.

[0019] According to embodiments of the present application, the aforementioned composition is selected from at least one of face wash, hand sanitizer, shower gel, shampoo, mouthwash, toothpaste, soap, cosmetic, care lotion, laundry soap, laundry detergent, laundry powder, washing liquid, spray, ointment, gel base, disinfectant, and toilet cleaner.

[0020] In a fifth aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the polypeptide of the first aspect of the present application. It can be understood that the aforementioned polypeptide is the active ingredient of the pharmaceutical composition.

[0021] According to embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as a thickening agent, a dispersing agent, an emulsifying agent, a preservative, etc.

[0022] In a sixth aspect, the present application provides an antibacterial additive. According to embodiments of the present application, the antibacterial additive comprises the polypeptide of the first aspect of the present application. It can be understood that the aforementioned polypeptide is the active ingredient of the antibacterial additive.

[0023] The polypeptide described in the present application has the following beneficial technical effects:

[0024] 1) It has been verified that the polypeptide of the present application has broad-spectrum antibacterial activity against Acinetobacter baumannii, Escherichia coli and multi-drug resistant Acinetobacter baumannii;

[0025] 2) It has been verified that the polypeptide 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 for preparing antibacterial compositions for Acinetobacter baumannii, Escherichia coli and multi-drug resistant Acinetobacter baumannii infections; in addition, the polypeptide can be used as a peptide inhibitor in the fields of food, daily necessities or medicine.

[0026] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0027] The above and / or additional aspects and advantages of the present application will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

[0028] Figure 1 is a schematic diagram of HPLC detection results of the antibacterial peptide cAMP081 provided in an embodiment of the present application;

[0029] Figure 2 is a schematic diagram of LCMS detection results of the antibacterial peptide cAMP081 provided in an embodiment of the present application;

[0030] Figure 3 is a schematic diagram of minimum inhibitory concentration determination results of the antibacterial peptide cAMP081 provided in an embodiment of the present application; wherein (a) is Acinetobacter baumannii ATCC 19606; (b) is multi-drug resistant (MDR) Acinetobacter baumannii BAA-1605; and (c) is Escherichia coli ATCC 25922. A. baumannii A. baumannii E. coli

[0031] Figure 4 is a schematic diagram of mammalian cell toxicity determination results of the antibacterial peptide cAMP081 provided in an embodiment of the present application;

[0032] Figure 5 is a schematic diagram of hemolytic toxicity determination results of the antibacterial peptide cAMP081 provided in an embodiment of the present application. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative effort fall within the scope of protection of the present application.

[0034] ​​​It has to be noted that, as used herein, the terms "first", "second", etc. merely serve for differentiating between two entities or steps whose names might otherwise be identical, but do not necessarily indicate a specific order or chronology. It is to be understood that data thus designated can be interchanged, where appropriate, so that embodiments of the application described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising", "containing", "including", and "having" and any variations thereof, are intended to cover a non-exclusive inclusion such that a process, method, system, product, or server that comprises, contains, includes or has a list of steps or elements, but not only those recited as method, product, or device, but also any other step or element of structural inherent to such process, method, product, or device.

[0035] In the present context, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing into association active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active antibody or antigen-binding fragment with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0036] In the present context, the term "pharmaceutically acceptable excipient" can include any solvent, solid or liquid excipient, diluent, or other liquid vehicle, and the like, suitable for the particular target dosage form. The use of any particular excipient is contemplated to be within the scope of the application except to the extent that any conventional excipient is incompatible with the antibody or antigen-binding fragment according to the application, for example, as it produces any adverse biological effect or any other interaction with any other component of the pharmaceutically acceptable composition in a deleterious manner.

[0037] The present application is based on the global extreme environment microbiome database (The Extreme Environment Microbiome Catalogue, EEMC) constructed by Sanya Huada Life Science Research Institute to mine and screen new antibacterial 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 is used, and the parameter is set to --minlength 5000 to identify the biosynthesis gene clusters (BGCs) of the extreme environment microorganism genome. The deep learning model (RNN, LSTM) is used to predict the core peptide sequence in the RiPPs type BGCs after ribosome synthesis. Further, three pre-trained protein language models (ESM2-3B, ESM3, PTRANS) are used to predict low-toxicity polypeptides for the obtained core peptide sequence. The polypeptide sequence ranked in the top 50% of each model prediction score is screened as a candidate low-toxicity antibacterial peptide. The candidate antibacterial peptide is compared with the known antibacterial peptide database, and the polypeptide sequence identical to the known database is removed to obtain a new candidate low-toxicity antibacterial peptide sequence, and the next step of experimental verification is carried out. Through the above sequence mining and experimental verification steps, an antibacterial peptide is obtained, which is derived from a deep-sea microbial genome and is named cAMP081, i.e., antibacterial peptide cAMP081. The following is the process of antibacterial function and toxicity verification of the polypeptide through wet experiments.

[0038] The technology of the present application is described below through specific examples. If the specific technology or condition is not specified in the examples, it is performed according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0039] In the following examples, A. baumannii indicates Acinetobacter baumannii (ATCC 19606), E. coli indicates Escherichia coli (ATCC 25922), MDR A. baumannii indicates multi-drug resistant Acinetobacter baumannii (North Star Biological BAA-1605).

[0040] Example 1: Synthesis and purification of antibacterial peptide cAMP081

[0041] The antibacterial peptide cAMP081 was synthesized by Shanghai Generay Biotech Co., Ltd. The purity of the antibacterial peptide was determined by high performance liquid chromatography (HPLC), and the molecular weight of the antibacterial peptide was detected by liquid chromatography-mass spectrometry (LC-MS).

[0042] (1) High performance liquid chromatography detection

[0043] HPLC analysis column type: SHIMADZU shim-pack GIST (4.6*250MM*5UM);

[0044] Mobile phase A: deionized water containing 0.1% trifluoroacetic acid;

[0045] Mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid;

[0046] Detection sample: 0.5 mg of antibacterial peptide was dissolved with 20% acetonitrile and 80% water, and the final volume was 0.5 mL.

[0047] Detection method: The sample injection volume was 30 μL, the total flow rate of the mobile phase was fixed at 1 mL / min, the mobile phase B gradient was linearly increased (20%~80%) from 0 min to 20 min, and the mobile phase A gradient was linearly decreased (80%~20%), and the detection signal was recorded. The results are shown in Figure 1 .

[0048] (2) Mass spectrometry detection

[0049] Mass spectrometer: Shimadzu LC-MS-2020;

[0050] Mobile phase: 50% water / 50% methanol;

[0051] Detection sample: 0.1 mg of antibacterial peptide was dissolved with 50% acetonitrile and 50% water, and the final volume was 0.5 mL.

[0052] The atomization gas flow rate was set to 1.50 L / min, the CDL temperature was 250℃, the CDL voltage was 0 v, the module temperature was 200℃, the pre-rod deviation was +4.5 kv, the detector was -0.2 kv, T. Flow was 0.2 mL / min, and the detection signal was recorded. The results are shown in Figure 2 .

[0053] Figure 1The results shown indicate that the purity of the synthetic antibacterial peptide cAMP081 is greater than 95%. The amino acid sequence is GHQDLHRTLAGLRRRIRDPRTLARLAKRVS, as shown in SEQ ID NO: 1.

[0054] According to the amino acid sequence estimation, the theoretical molecular weight of the antibacterial peptide cAMP081 is 3520.104, Figure 2 The results shown indicate that the molecular weight of the synthetic antibacterial peptide cAMP081 is consistent with the theoretical molecular weight.

[0055] Example 2: Activity analysis of antibacterial peptide cAMP081

[0056] (1) Minimum inhibitory concentration (MIC) determination

[0057] Strains: Acinetobacter baumannii A. baumannii , Escherichia coli E. coli , and multi-drug resistant Acinetobacter baumannii MDR A. baumannii.

[0058] The above three bacterial strains were streaked on Luriae-Bertani (LB) agar medium and incubated at 37°C overnight.

[0059] After overnight culture, single colonies were picked into Mueller-Hinton Broth (MHB) liquid medium and cultured at 37°C with 120 rpm shaking overnight. Fresh MHB was used to dilute the culture by 1:100, and then cultured to the exponential phase (OD 600 0.4-0.6), and then the cell concentration was adjusted to 1×10 6 cfu / mL.

[0060] cAMP081 was serially diluted by two-fold to obtain a concentration range from 512 μM to 1 μM. The specific preparation process is as follows: first, prepare a cAMP081 stock solution with a concentration of 1024 μM. In the first well, add 100 μL of the prepared cAMP081 stock solution, and add an equal volume of MHB medium, mix well, and the cAMP081 concentration is diluted to 512 μM. Take 100 μL of the solution from the first well and add it to the second well, and add an equal volume of MHB medium, mix well, and the cAMP081 concentration is diluted to 256 μM. Take 100 μL of the solution from the second well and add it to the third well, and add an equal volume of MHB medium, mix well, and the cAMP081 concentration is halved again. Repeat the above operation until the cAMP081 concentration is diluted to 1 μM.

[0061] Subsequently, 100 μL of the diluted bacterial solution was transferred to a 96-well plate containing 100 μL of a two-fold serially diluted cAMP081-MHB medium, with a final cAMP081 concentration of 256 μM to 0.5 μM. The negative control group was 100 μL of MHB liquid medium added with 100 μL of the bacterial solution, and the blank group was 200 μL of MHB medium. After incubation at 37°C for 16-18 hours, the MIC value was taken as the lowest concentration of the antimicrobial peptide at which no bacterial growth was observed. All experiments were repeated three times, and the results are shown in Table 1. Figure 3

[0062] Figure 3 The results shown in Table 1 indicate that the antimicrobial peptide cAMP081 has an MIC of 8 μM against A. baumannii (a), an MIC of 16 μM against MDR A. baumannii (b), or E. coli (c).

[0063] (2) Mammalian cell toxicity assay

[0064] A blank group (Blank), a negative control group (Control), a positive control group (cisplatin), and an antimicrobial peptide group (cAMP081) were set up. In the blank group, only 10% fetal bovine serum (04-001-1acs, Biological industries, Israel) was added to the MHB culture solution in each well of the 96-well plate. L-02 human liver cells (BeNa'Biotech) and 293T human embryonic kidney cells (Promocell) were prepared into a single cell suspension with MHB culture solution containing 10% fetal bovine serum. In each well of the 96-well plate, 90 μL of 5×10 4 / mL of adherent cells were inoculated, except for the blank group. The cells were pre-cultured for 24 hours under the condition of 5% CO2 and 37°C. In the negative control group, 10 μL of PBS solution was added to each well. A 600 μM cisplatin (D8810, Solarbio, Germany) solution was prepared using PBS solution, and 10 μL of cisplatin was added to each well of the positive control group, resulting in a final cisplatin concentration of 60 μM. A 600 μM antimicrobial peptide cAMP081 solution was prepared using PBS solution, and 10 μL of antimicrobial peptide cAMP081 solution was added to each well of the antimicrobial peptide group, resulting in a final antimicrobial peptide cAMP081 concentration of 60 μM. The plate was incubated in an incubator for 48 hours. The old culture medium and drug solution were aspirated, and 100 μL of CCK-8 solution (BS350A, Boster Biological) diluted ten times with serum-free medium to a final concentration of 10% v / v was added to each well of the 96-well plate. The plate was further incubated for 1 hour under the conditions of light avoidance, 5% CO2, and 37°C. The absorbance at 450 nm was measured using a microplate reader, and the raw data results were recorded. The mammalian cell inhibition rate was calculated using the formula: (OD Control ​-OD cAMP081 / 顺铂 ) / (OD Control -OD Blank ) × 100%. All experiments were repeated 3 times, and the results are shown in Figure 4 shown.

[0065] Figure 4 The results showed that the antimicrobial peptide cAMP081 had an inhibition rate of 3.10% on L-02 human hepatocytes and 19.76% on 293T human embryonic kidney cells. Furthermore, compared to the positive control group (cisplatin), the antimicrobial peptide cAMP081 group had a lower inhibition rate and significantly lower mammalian cell toxicity.

[0066] (3) Hemolytic toxicity assay

[0067] The experiment set up a blank group (Blank), a control group (Control), and an antimicrobial peptide group (cAMP081). Fresh sheep blood cells (defibrinated sheep blood, Beekman Bio) 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 red blood cell suspension was added to each well of the control group, antimicrobial peptide group, and blank group on a 96-well plate. Triton X-100 was added to each well of the control group to a final concentration of 1%. cAMP081 solution was added to each well of the antimicrobial peptide group to a final concentration of 60 μM. The plates were incubated 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, and the raw data results were recorded. The hemolysis rate was calculated as (OD cAMP081 -OD Blank ) / (OD Control -OD Blank ) × 100%. All experiments were repeated 3 times, and the results are shown in Figure 5 shown.

[0068] Figure 5 The results showed that the cAMP081 group had a lower OD value and significantly lower hemolytic toxicity compared to the control group. The hemolytic rate of the cAMP081 group was calculated to be 1.55%.

[0069] In the description of the specification, the description using the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the particular feature, structure, material or characteristic being described is included in at least one embodiment or example of the present application. The illustrative appearances of the above-mentioned terms in various places in the specification are not necessarily referred to the same embodiment or example. Moreover, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. Furthermore, the terms "comprise", "comprising", "include", "including", "contain", "containing" or variations thereof are used inclusively and do not exclude the additional inclusion of unrecited features, structures, materials, or characteristics. In addition, it is understood that where the description indicates that certain features, structures, materials, or characteristics are present in "some" embodiments or examples, it is understood that these features, structures, materials, or characteristics are not necessarily present in all embodiments or examples.

[0070] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary, and are not to be interpreted as limiting the present application, and that the ordinary skilled person in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1; The polypeptide has a bacteriostatic and / or bactericidal function.

2. A non-therapeutic bacteriostasis and / or sterilization method, characterized in that: The polypeptide of claim 1 is contacted with a non-living sample to be treated; The bacteria are Acinetobacter baumannii or Escherichia coli.

3. The method of claim 2, wherein, The Acinetobacter baumannii includes multi-drug resistant Acinetobacter baumannii.

4. The method of claim 2, wherein, The bacteria include Acinetobacter baumannii, and the polypeptide has a minimum concentration of 8 μM in the contacting system.

5. The method of claim 3, wherein, The bacteria include at least one of multi-drug resistant Acinetobacter baumannii and Escherichia coli, and the polypeptide has a minimum concentration of 16 μM in the contacting system.

6. Use of the polypeptide of claim 1 in the preparation of a product having a bacteriostatic and / or bactericidal property. The bacteria are Acinetobacter baumannii or Escherichia coli.

7. Use according to claim 6, characterized in that, The Acinetobacter baumannii includes multi-drug resistant Acinetobacter baumannii.

8. A composition characterized in that, The active ingredient of the composition includes the polypeptide of claim 1.

9. The composition of claim 8, wherein, The composition is selected from a cosmetic product.

10. The composition of claim 8, wherein, The composition is selected from a soap.

11. The composition of claim 8, wherein, The composition is selected from at least one of a facial cleanser, a hand sanitizer, a body wash, a shampoo, a mouthwash, a toothpaste, a skin care lotion, a laundry detergent, a laundry soap, a laundry powder, a dishwashing liquid, a spray, a cream, a gel base, a disinfectant, and a toilet bowl cleaner.

12. A pharmaceutical composition, characterized by, The polypeptide of claim 1 is included.

13. A bacteriostatic additive characterized in that, The polypeptide of claim 1 is included.

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