An antibacterial peptide for inhibiting drug-resistant Acinetobacter baumannii, its preparation method and application

Abstract

The present invention relates to the technical field of the application of antimicrobial peptides, and particularly relates to an antimicrobial peptide for inhibiting drug-resistant Acinetobacter baumannii, a preparation method thereof, and an application thereof. The antimicrobial peptide is cd77AHP-2, and the amino acid sequence is as shown in SEQ ID NO.1. The present invention modifies the artificially chemically synthesized antimicrobial peptide cd77, optimizes the distribution of cationic amino acids, and significantly improves the antibacterial activity of the antimicrobial peptide. The antimicrobial peptide cd77AHP-2 shows a significant antibacterial effect against Gram-negative bacterium Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii. The antimicrobial peptide cd77AHP-2 provided by the present invention has low cytotoxicity to human bronchial epithelial cells and has the potential to prepare drugs against drug-resistant Acinetobacter baumannii. The present invention provides a new functional polypeptide in the field of antimicrobial peptides and has important practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of the application of antimicrobial peptides, and particularly relates to an antimicrobial peptide for inhibiting drug-resistant Acinetobacter baumannii, a preparation method thereof, and an application thereof. Background Art

[0002] Acinetobacter baumannii is an important opportunistic pathogen. Especially in patients in the intensive care unit, hospital-acquired pneumonia and ventilator-associated pneumonia caused by this bacterium are the most common pathogenic factors. These infections are often accompanied by high morbidity and mortality.

[0003] Multidrug-resistant Acinetobacter baumannii forms a complex drug resistance mechanism by carrying multiple drug resistance genes and shows significant drug resistance to a variety of traditional antibiotics, such as β-lactams, aminoglycosides, and quinolones, making the clinical treatment options extremely limited. In addition, currently commonly used anti-infective drugs, such as polymyxin, although can effectively control the infection to a certain extent, its significant nephrotoxicity and neurotoxicity limit its wide application. With the continuous increase of drug-resistant strains, the development of new anti-infective drugs against multidrug-resistant Acinetobacter baumannii has become an urgent need in the global public health field.

[0004] Antimicrobial peptides are a class of natural antimicrobial molecules composed of polypeptide chains and have become an important research direction for the treatment of drug-resistant bacterial infections with their unique action mechanisms and broad-spectrum antimicrobial activities. Different from traditional antibiotics, antimicrobial peptides act by directly destroying the bacterial cell membrane or inhibiting its metabolic function, are not easily induced to produce drug resistance, and can act synergistically with the host immune system to enhance the control of infection. However, at present, many antimicrobial peptides are limited in their clinical transformation due to problems such as insufficient environmental stability, low specificity, and high toxicity to host cells in actual applications. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an antimicrobial peptide for inhibiting drug-resistant Acinetobacter baumannii, a preparation method thereof, and an application thereof.

[0006] The present invention adopts the following technical solutions:

[0007] First, the present invention provides an antimicrobial peptide cd77AHP-2 for inhibiting drug-resistant Acinetobacter baumannii. The amino acid sequence of the antimicrobial peptide cd77AHP-2 is shown in SEQ ID NO.1. The molecular formula of the antimicrobial peptide cd77AHP-2 is C 93 H 159 N 33 O 18 S2, and the molecular weight is 2090.2.

[0008] The antibacterial peptide cd77AHP-2 of the present invention specifically targets Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii, showing significant antibacterial activity. Research shows that the antibacterial peptide cd77AHP-2 maintains high stability under the conditions of pH = 2, pH = 7.4, and pH = 8.4. In addition, in the toxicity assessment, the antibacterial peptide has low toxicity to human bronchial epithelial cells BEAS-2B, providing a good safety basis for the development of drugs against lung infections.

[0009] The minimum inhibitory concentration of the antibacterial peptide cd77AHP-2 of the present invention against Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii is 16 μg / mL, demonstrating strong antibacterial potential. This antibacterial peptide has excellent performance and broad application value, providing new research ideas and technical support for solving the major clinical challenge of drug-resistant pathogen infections.

[0010] Secondly, the present invention provides a preparation method of the antibacterial peptide cd77AHP-2. By optimizing and transforming the distribution of cationic amino acids in antibacterial peptide cd77, the cationic amino acids are adjusted from aggregated distribution to dispersed distribution, thereby obtaining antibacterial peptide cd77AHP-2. After optimization, the minimum inhibitory concentration of this antibacterial peptide against multidrug-resistant Acinetobacter baumannii is reduced from 32 μg / mL of cd77 to 16 μg / mL, and the antibacterial activity is doubled, significantly enhancing its anti-infection potential.

[0011] Finally, the present invention provides the application of the antibacterial peptide cd77AHP-2 in the preparation of antibacterial products, and the antibacterial products are used to inhibit Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

[0012] Furthermore, the antibacterial product is a drug or a bacteriostatic agent.

[0013] Specifically, the antibacterial peptide cd77AHP-2 can be used as a specific antibacterial drug against Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii, for the treatment of pneumonia and other related diseases caused by their infections, especially showing excellent potential in the treatment of ventilator-associated pneumonia VAP. The bacteriostatic agent can be used for the development of anti-infection preparations in the medical and health field. In addition, combined with the characteristics of the antibacterial peptide, its products can also be widely used in the preparation of disinfectants and biological preservatives.

[0014] Furthermore, the minimum inhibitory concentration of the antibacterial peptide cd77AHP-2 against Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii is 16 μg / mL.

[0015] Furthermore, the bacteriostatic agent is obtained by dissolving the antibacterial peptide cd77AHP-2 in sterile water.

[0016] Further, the concentration of the antimicrobial peptide cd77AHP-2 in the bacteriostatic agent is 16 μg / mL to 128 μg / mL.

[0017] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0018] Further, the excipient is any one or a combination of several of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] Improved efficiency: By optimizing the distribution of cationic amino acids through molecular design, the antibacterial activity of cd77AHP-2 is significantly improved, and the minimum inhibitory concentration against multidrug-resistant Acinetobacter baumannii is reduced from 32 μg / mL of cd77 to 16 μg / mL.

[0021] Environmental stability: It can maintain good activity under the conditions of pH = 2, pH = 7.4, and pH = 8.4, which is significantly better than existing antimicrobial peptides.

[0022] Low cytotoxicity: Toxicity experiments show that the antimicrobial peptide cd77AHP-2 has low toxicity to bronchial epithelial cells, providing a solid guarantee for its safety.

[0023] Broad application prospects: The antimicrobial peptide cd77AHP-2 is not only suitable for the development of anti-infective drugs, but also can be used for the development of other antimicrobial products in the fields of medical and health and environmental disinfection.

[0024] In summary, the present invention provides important technical support and potential resources for the treatment of multidrug-resistant bacterial infections and the development of antibacterial preparations, and provides a new direction for solving the global problem of antibiotic resistance. Description of the Drawings

[0025] Figure 1 It is a comparison of the rotavirus-like structures of antimicrobial peptide cd77 and antimicrobial peptide cd77AHP-2. A is the rotavirus-like diagram of antimicrobial peptide cd77, and B is the rotavirus-like diagram of antimicrobial peptide cd77AHP-2.

[0026] Figure 2 It is the amino acid composition and structure of antimicrobial peptide cd77AHP-2. A is the amino acid composition of antimicrobial peptide cd77AHP-2, and B is the structure of cd77AHP-2.

[0027] Figure 3The MIC determination results of antibacterial peptide cd77AHP-2 against Acinetobacter baumannii 1 and Acinetobacter baumannii 2 are shown in the figure. In the figure, Baumann 1 refers to Acinetobacter baumannii 1, and Baumann 2 refers to Acinetobacter baumannii 2.

[0028] Figure 4 The bacterial growth conditions when determining the MIC of antibacterial peptide cd77AHP-2 against Acinetobacter baumannii 1 and Acinetobacter baumannii 2 are as follows. Among them, rows a to c are three replicates for determining the MIC of antibacterial peptide cd77AHP-2 against Acinetobacter baumannii 1, and rows d to f are three replicates for determining the MIC of antibacterial peptide cd77AHP-2 against Acinetobacter baumannii 2. Columns 1 to 6 correspond to the concentrations of antibacterial peptide cd77AHP-2 being 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL in sequence; columns 1 to 3 in row g are Acinetobacter baumannii 1 + sterilized water; columns 1 to 3 in row h are Acinetobacter baumannii 2 + sterilized water; columns 4 to 6 in row g are antibacterial peptide cd77AHP-2 + LB liquid medium; columns 4 to 6 in row h are sterilized water + LB liquid medium.

[0029] Figure 5 The results of the stability experiment of antibacterial peptide cd77AHP-2.

[0030] Figure 6 The results of the cytotoxicity experiment of antibacterial peptide cd77AHP-2.

[0031] Figure 7 The photographed MTT staining plate of the cytotoxicity experiment of antibacterial peptide cd77AHP-2. Columns 1 to 4 are human bronchial epithelial cells BEAS-2B treated with antibacterial peptide cd77AHP-2 at concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL in sequence, and rows a to c are three replicates. Detailed implementation manners

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] The sources of the pathogenic bacteria used in the following examples are as follows:

[0034] Acinetobacter baumannii 1: Acinetobacter baumannii ATCC19606, purchased from Beijing NaChuangLian Biotechnology Co., Ltd.

[0035] Acinetobacter baumannii 2: Acinetobacter baumannii bio-53272, a multi-drug resistant strain, purchased from Beijing BioWin Biotech Co., Ltd.

[0036] The present invention provides a broad-spectrum antibacterial peptide cd77AHP-2 against multi-drug resistant bacteria, and its amino acid sequence is shown in SEQ ID NO.1. The antibacterial peptide cd77AHP-2 exhibits high antibacterial activity under the conditions of pH = 2, pH = 7.4 and pH = 8.4 respectively, and shows significant antibacterial effects against Gram-negative bacteria Acinetobacter baumannii and multi-drug resistant Acinetobacter baumannii, and the minimum inhibitory concentration value MIC is 16 μg / mL for both. The antibacterial peptide cd77AHP-2 has extremely high antibacterial value for medical drugs and can provide alternative medicinal resources for clinical treatment of multi-drug resistant pathogenic bacteria.

[0037] Example 1: An antibacterial peptide cd77AHP-2 and its preparation method.

[0038] The amino acid sequence of the antibacterial peptide cd77AHP-2 is shown in SEQ ID NO.1. The antibacterial peptide cd77AHP-2 is composed of 16 amino acids. Its molecular formula is C 93 H 159 N 33 O 18 S2, with a molecular weight of 2090.20 Da. The sequence of the antibacterial peptide cd77 before modification is shown in SEQ ID NO.2. To modify its antibacterial activity, the online tool HeliQuest was used to predict its wheel diagram structure, and it was found that the distribution of its cationic antibacterial peptides was relatively concentrated, and the distribution of hydrophobic amino acids was more dispersed due to the concentrated cationic amino acids. By swapping the positions of the R at the 15th position and the C amino acid at the 4th position of the original antibacterial peptide, cd77AHP-2 was obtained. In its wheel diagram, the cation distribution is more dispersed, and at the same time, the hydrophobic amino acids are more concentrated. As Figure 1 shown, the concentrated hydrophobic amino acids contribute to the amphiphilicity of the whole antibacterial peptide, making the minimum inhibitory concentration value of cd77AHP-2 against multi-drug resistant Acinetobacter baumannii decrease from 32 μg / mL before modification to 16 μg / mL, and the activity is doubled.

[0039] SEQ ID NO.1: MKKRRRKVLFGLWRCN.

[0040] SEQ ID NO.2: MKKCRRKVLFGLWRRN.

[0041] The amino acid structure and related schematic diagrams are from the special peptide website: https: / / www.allpeptide.com / jiegoutu.html. The structure of cd77AHP-2 was predicted using the online tool AlphaFold3, and the prediction results show that this peptide is a typical α-helical peptide. As Figure 2As shown, it has amphiphilic characteristics, which contribute to its interaction with bacterial membranes. The AlphaFold3 website is: https: / / golgi.sandbox.google.com / about. Preparation method: The antimicrobial peptide cd77AHP-2 was prepared by Sangon Biotech Co., Ltd. using solid-phase chemical synthesis method, and the purity reached >95%.

[0042] Example 2: Determination of the MIC of the antimicrobial peptide cd77AHP-2.

[0043] The MIC of the antimicrobial peptide was determined by the broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, P.A. Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991. The above-mentioned pathogenic bacteria were inoculated into sterile LB medium and cultured overnight with shaking at 37°C. Each pathogenic bacterium was inoculated into fresh LB medium at an inoculum size of 1%, cultured until the exponential phase, and the cell concentration was adjusted to 1×10 5 cfu / mL to obtain the bacterial suspension. Then, 180 μL of the bacterial suspension was transferred to a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted to a two-fold serial dilution of the antimicrobial peptide solution. 20 μL was taken and added to the bacterial suspension in the 96-well plate to make the antimicrobial peptide concentration range in the bacterial suspension 0.5 - 256 μg / mL. After incubating the 96-well plate at 37°C for 24 hours, the growth of bacteria was detected by a microplate reader. The MIC was defined as the minimum antimicrobial peptide concentration at which bacterial growth was detected. The experiment was set up with 3 replicates.

[0044] The results are as Figure 3 and Figure 4 shown. The MICs of the antimicrobial peptide cd77AHP-2 against Acinetobacter baumannii 1 and Acinetobacter baumannii 2 were both detected to be 16 μg / mL.

[0045] Example 3: Determination of the stability of the antimicrobial peptide cd77AHP-2.

[0046] Sterile PBS solution was added to 1 mol / L HCl and NaOH solutions to prepare PBS solutions with pH = 2, pH = 7.4, and pH = 8.4. Then, the antimicrobial peptide was dissolved in PBS solutions with different pH values and incubated at 37°C for 1 h. After that, the change in the MIC of the antimicrobial peptide under different pH treatments was determined according to the method described in Example 2. The results showed that, as Figure 5As shown, pH has no effect on the antibacterial peptide. At pH = 2, pH = 7.4, and pH = 8.4, the MIC of the antibacterial peptide cd77AHP-2 remains unchanged, indicating that the antibacterial peptide cd77AHP-2 can maintain stability and perform its function in different pH environments.

[0047] Example 4: Toxicity determination of the antibacterial peptide cd77AHP-2.

[0048] Cytotoxicity experiments of the antibacterial peptide cd77AHP-2 were carried out using human bronchial epithelial cells BEAS-2B. The cells BEAS-2B were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured in a cell incubator at 37°C with 5% CO2. Cell passage culture: When the cells grew to 80% - 90%, the medium was removed, and the cells were washed 1 - 2 times with PBS. After removing the PBS, 1 ml of trypsin was added to digest the cells for 1 - 3 minutes, and then 3 ml of complete medium was added to neutralize the trypsin and terminate the digestion. The digested cells were transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After pouring off the supernatant, 3 ml of medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:3 into culture dishes for culture. The BEAS-2B cells that grew to 80% - 90% in the culture dish were digested with trypsin, centrifuged at 1000 rpm, and the supernatant was removed. The cells were resuspended with complete medium and seeded into 96-well plates, with 5000 cells seeded in each well. After the cells adhered, drugs with concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL were added respectively. After gently mixing, the plates were placed in the incubator for 24 h. Then 10 μl of MTT with a concentration of 5 mg / ml was added, and the cells were cultured in the cell incubator for about 3 h. The medium was removed, 150 μl of DMSO was added to each well, and the plates were shaken evenly. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The OD values of each well were read at a wavelength of 570 nm using the ELISA reader, and the cell survival rate was calculated using the following formula:

[0049] Cell survival rate (%) = (OD of the sample group / OD of the blank control group) × 100.

[0050] After detection, the cytotoxicity results of the antibacterial peptide cd77AHP-2 are as Figure 6 and Figure 7 shown. When the concentration of the antibacterial peptide cd77AHP-2 was as high as 128 μg / ml, the survival rate of human bronchial epithelial cells BEAS-2B was still greater than 90%, indicating that the antibacterial peptide cd77AHP-2 has extremely low cytotoxicity and great potential for medical treatment.

[0051] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic inventive concept. Therefore, the appended claims are intended to be construed to cover the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

Claims

1. An antimicrobial peptide cd77AHP-2 for inhibiting drug-resistant Acinetobacter baumannii, characterized in that: The amino acid sequence of the antimicrobial peptide cd77AHP-2 is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide cd77AHP-2 according to claim 1 in the preparation of antimicrobial products, characterized in that: The antibacterial product is used to inhibit Acinetobacter baumannii.

3. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 2, characterized in that: The antibacterial product is a medicine.

4. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 3, characterized in that: The drug is a bacteriostatic agent.

5. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 4, characterized in that: The antibacterial agent is obtained by dissolving the antimicrobial peptide cd77AHP-2 in sterile water.

6. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 5, characterized in that: The concentration of the antimicrobial peptide cd77AHP-2 in the antibacterial agent is 16 μg / mL to 128 μg / mL.

7. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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