Antibacterial peptide for inhibiting drug-resistant acinetobacter baumannii as well as preparation method and application of antibacterial peptide

Abstract

The invention relates to the technical field of antibacterial peptide application, in particular to an antibacterial peptide for inhibiting drug-resistant acinetobacter baumannii as well as a preparation method and application of the antibacterial peptide. The antibacterial peptide is cd77AHP-2, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1. The antibacterial peptide cd77 obtained through artificial chemical synthesis is modified, distribution of cationic amino acid is optimized, and the antibacterial activity of the antibacterial peptide is remarkably improved. The antibacterial peptide cd77AHP-2 has a remarkable antibacterial effect on acinetobacter baumannii which are gram-negative bacteria and multi-drug resistant acinetobacter baumannii, and the antibacterial peptide cd77AHP-2 has a remarkable antibacterial effect on the acinetobacter baumannii which are gram-negative bacteria. The antibacterial peptide cd77AHP-2 provided by the invention has relatively low cytotoxicity to human bronchial epithelial cells, and has the potential of preparing a drug for resisting drug-resistant acinetobacter baumannii. The invention provides a novel functional polypeptide for the field of antibacterial peptides, and the novel functional polypeptide has important practical application value.

Description

Technical Field

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

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

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

[0004] Antimicrobial peptides are a class of natural antimicrobial molecules composed of polypeptide chains. With their unique mechanism of action and broad-spectrum antimicrobial activity, they have become an important research direction for the treatment of drug-resistant bacterial infections. Unlike traditional antibiotics, antimicrobial peptides work by directly destroying bacterial cell membranes or inhibiting their metabolic functions, and are less likely to induce drug resistance. At the same time, they can synergize with the host immune system to enhance infection control. However, many antimicrobial peptides are currently limited in their clinical transformation due to problems such as insufficient environmental stability, low specificity, and high toxicity to host cells. Summary of the invention

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

[0006] The present invention adopts the following technical solution: 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, molecular weight is 2090.2.

[0007] The antimicrobial peptide cd77AHP-2 of the present invention specifically targets Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii, and exhibits significant antimicrobial activity. Studies have shown that the antimicrobial 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 antimicrobial peptide has low toxicity to human bronchial epithelial cells BEAS-2B, providing a good safety basis for its development of drugs for lung infections.

[0008] The antimicrobial peptide cd77AHP-2 of the present invention has a minimum inhibitory concentration of 16 μg / mL for both Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii, showing strong antibacterial potential. The antimicrobial peptide has excellent performance and wide application value, and provides new research ideas and technical support for solving the major clinical challenge of drug-resistant pathogen infection.

[0009] Secondly, the present invention provides a method for preparing the antimicrobial peptide cd77AHP-2, by optimizing and modifying the cationic amino acid distribution of the antimicrobial peptide cd77, adjusting the cationic amino acids from a clustered distribution to a dispersed distribution, thereby obtaining the antimicrobial peptide cd77AHP-2. After optimization, the minimum inhibitory concentration of the antimicrobial 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.

[0010] Finally, the present invention provides the use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products, and the antimicrobial products are used to inhibit Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

[0011] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0012] Specifically, the antimicrobial peptide cd77AHP-2 can be used as a specific antimicrobial drug for Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii, and is used to treat pneumonia and other related diseases caused by infection thereof, especially showing excellent potential in the treatment of ventilator-associated pneumonia (VAP). The antibacterial agent can be used in the development of anti-infective preparations in the medical and health field. In addition, combined with the characteristics of antimicrobial peptides, its products can also be widely used in the preparation of disinfectants and biological preservatives.

[0013] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide cd77AHP-2 against Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii was 16 μg / mL.

[0014] Furthermore, the antibacterial agent is obtained by dissolving the antimicrobial peptide cd77AHP-2 in sterile water.

[0015] Furthermore, the concentration of the antimicrobial peptide cd77AHP-2 in the antibacterial agent is 16 μg / mL to 128 μg / mL.

[0016] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0017] Furthermore, the auxiliary material is any one or a combination of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0018] Compared with the prior art, the present invention has the following beneficial effects: Improved efficiency: By optimizing the distribution of cationic amino acids through molecular design, the antibacterial activity of cd77AHP-2 was significantly improved, and the minimum inhibitory concentration against multidrug-resistant Acinetobacter baumannii was reduced from 32μg / mL of cd77 to 16μg / mL.

[0019] 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.

[0020] Low cytotoxicity: Toxicity experiments showed that the antimicrobial peptide cd77AHP-2 has low toxicity to bronchial epithelial cells, providing a solid guarantee for its safety.

[0021] Broad application prospects: The antimicrobial peptide cd77AHP-2 is not only suitable for the development of anti-infection drugs, but also can be used in the development of other antimicrobial products in the fields of medical care and environmental disinfection.

[0022] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a structural comparison of the wheel diagrams of the antimicrobial peptide cd77 and the antimicrobial peptide cd77AHP-2. A is the wheel diagram of the antimicrobial peptide cd77, and B is the wheel diagram of the antimicrobial peptide cd77AHP-2.

[0024] Figure 2 are the amino acid composition and structure of the antimicrobial peptide cd77AHP-2, A is the amino acid composition of the antimicrobial peptide cd77AHP-2, and B is the structure of cd77AHP-2.

[0025] Figure 3These are the MIC test results of the antimicrobial peptide cd77AHP-2 against Acinetobacter baumannii 1 and Acinetobacter baumannii 2. In the figure, bauman 1 is Acinetobacter baumannii 1, and bauman 2 is Acinetobacter baumannii 2.

[0026] Figure 4 To determine the bacterial growth when the MIC of the antimicrobial peptide cd77AHP-2 was used to detect Acinetobacter baumannii 1 and Acinetobacter baumannii 2, rows a to c were three replicates of the MIC of the antimicrobial peptide cd77AHP-2 to Acinetobacter baumannii 1, rows d to f were three replicates of the MIC of the antimicrobial peptide cd77AHP-2 to Acinetobacter baumannii 2, columns 1 to 6 corresponded to the concentrations of the antimicrobial peptide cd77AHP-2 of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL, respectively; rows g, columns 1 to 3 were Acinetobacter baumannii 1 + sterile water; rows h, columns 1 to 3 were Acinetobacter baumannii 2 + sterile water; rows g, columns 4 to 6 were antimicrobial peptide cd77AHP-2 + LB liquid culture medium; rows h, columns 4 to 6 were sterile water + LB liquid culture medium.

[0027] Figure 5 These are the results of the stability experiment of the antimicrobial peptide cd77AHP-2.

[0028] Figure 6 These are the results of the cytotoxicity experiment of the antimicrobial peptide cd77AHP-2.

[0029] Figure 7 These are photos of the MTT staining plate from the cytotoxicity experiment of the antimicrobial peptide cd77AHP-2. Columns 1 to 4 are human bronchial epithelial cells BEAS-2B treated with the antimicrobial peptide cd77AHP-2 at concentrations of 0 μg / mL, 64 μg / mL, 96 μg / mL, and 128 μg / mL, respectively. Rows a to c are repeated three times. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] The pathogenic bacteria used in the following examples are from: Acinetobacter baumannii 1: Acinetobacter baumannii ATCC19606, purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0032] Acinetobacter baumannii 2: Acinetobacter baumannii bio-53272, a multidrug-resistant strain, was purchased from Beijing Bio-Bowei Biological Co., Ltd.

[0033] The present invention provides a broad-spectrum antimicrobial peptide cd77AHP-2 for multidrug-resistant bacteria, and its amino acid sequence is shown in SEQID NO.1. The antimicrobial peptide cd77AHP-2 exhibits high-efficiency antimicrobial activity under the conditions of pH=2, pH=7.4 and pH=8.4, respectively, and exhibits significant antibacterial effects on Gram-negative bacteria Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii, with the minimum inhibitory concentration value MIC being 16 μg / mL. The antimicrobial peptide cd77AHP-2 has extremely high antibacterial value as a medical drug, and can provide an alternative medicinal resource for clinical treatment of multidrug-resistant pathogens.

[0034] Example 1: An antimicrobial peptide cd77AHP-2 and a preparation method thereof.

[0035] The amino acid sequence of the antimicrobial peptide cd77AHP-2 is shown in SEQ ID NO. 1. The antimicrobial peptide cd77AHP-2 consists of 16 amino acids. Its molecular formula is C 93 H 159 N 33 O 18 S2, molecular weight is 2090.20Da. The sequence of the antimicrobial peptide cd77 before modification is shown in SEQ ID NO.2. In order to modify its antimicrobial activity, the online tool HeliQuest was used to predict its wheel structure, and it was found that its cationic antimicrobial peptide distribution was relatively concentrated, and the hydrophobic amino acid distribution was relatively dispersed due to the concentrated cationic amino acids. By exchanging the positions of the R at position 15 and the C at position 4 of the original antimicrobial peptide, cd77AHP-2 was obtained. The cationic distribution in its wheel diagram was more dispersed, and the hydrophobic amino acids were more concentrated, as shown in Figure 2. Figure 1 As shown, the concentrated hydrophobic amino acids contributed to the overall amphipathicity of the antimicrobial peptide, reducing the minimum inhibitory concentration of cd77AHP-2 against multidrug-resistant Acinetobacter baumannii from 32 μg / mL before the modification to 16 μg / mL, and doubling its activity.

[0036] SEQ ID NO. 1: MKKRRRKVLFGLWRCN.

[0037] SEQ ID NO. 2: MKKCRRKVLFGLWRRN.

[0038] The amino acid structure and related schematics are from the peptide website: https: / / www.allpeptide.com / jiegoutu.html. The structure of cd77AHP-2 was predicted using the online tool AlphaFold3. The prediction results showed that the peptide was a typical α-helical peptide, such as Figure 2As shown, it has amphiphilic characteristics, which helps it interact with bacterial membranes. The website of AlphaFold3 is: https: / / golgi.sandbox.google.com / about. Preparation method: The antimicrobial peptide cd77AHP-2 was prepared by Sangon Biotechnology Co., Ltd. using solid phase chemical synthesis, with a purity of >95%.

[0039] Example 2: MIC determination of antimicrobial peptide cd77AHP-2.

[0040] The MIC of antimicrobial peptides was determined by broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991. The above pathogens were inoculated into sterile LB medium and cultured at 37°C overnight with shaking. Each pathogen was inoculated into fresh LB medium at a 1% inoculum and cultured until the exponential phase, and the cell concentration was adjusted to 1×10 5 cfu / mL to obtain the bacterial solution. Then transfer 180μL of the bacterial solution to a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted to a 2-fold serial dilution of the antimicrobial peptide solution. 20μL was added to the bacterial solution in the 96-well plate to make the antimicrobial peptide concentration in the bacterial solution range from 0.5 to 256μg / mL. After incubating the 96-well plate at 37°C for 24 hours, the bacterial growth was detected by an enzyme reader. MIC was defined as the minimum antimicrobial peptide concentration for detecting bacterial growth. The experiment was set up with 3 replicates.

[0041] The results are as follows Figure 3 and Figure 4 As shown, the MIC of the antimicrobial peptide cd77AHP-2 against Acinetobacter baumannii 1 and Acinetobacter baumannii 2 was tested to be 16 μg / mL.

[0042] Example 3: Determination of the stability of the antimicrobial peptide cd77AHP-2.

[0043] Sterile PBS solution was added with 1 mol / L HCl and NaOH solution 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. Then the MIC changes of the antimicrobial peptide under different pH values ​​were determined according to the method described in Example 2. The results showed that Figure 5As shown, pH has no effect on the action of antimicrobial peptides. At pH = 2, pH = 7.4 and pH = 8.4, the MIC of the antimicrobial peptide cd77AHP-2 did not change. The antimicrobial peptide cd77AHP-2 can maintain stability and exert its function under different pH environments.

[0044] Example 4: Toxicity determination of antimicrobial peptide cd77AHP-2.

[0045] Human bronchial epithelial cells BEAS-2B were used to conduct cytotoxicity experiments on the antimicrobial peptide cd77AHP-2. BEAS-2B cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin, and cultured in a cell culture incubator at 37°C with 5% CO2. Cell subculture: When the cells grow to 80%-90%, remove the medium and add PBS to wash 1-2 times. After removing PBS, add 1ml of trypsin to digest for 1-3min, add 3ml of complete medium to neutralize the trypsin to terminate the digestion, transfer the digested cells to a 15ml centrifuge tube, and centrifuge at 1000rpm for 5min. After discarding the supernatant, add 3ml of medium to resuspend the cells, and subculture them in a culture dish at a ratio of 1:3. Digest the BEAS-2B cells that have grown to 80% to 90% of the culture dish with trypsin, centrifuge at 1000rpm, remove the supernatant, add complete culture medium to resuspend the cells, and plant the cells in 96-well plates, with 5000 cells in each well. After the cells adhere to the wall, add drugs at concentrations of 0μg / mL, 64μg / mL, 96μg / mL, and 128μg / mL, mix gently, add to the incubator and cultivate for 24h, add 10μl of MTT at a concentration of 5mg / ml, and continue to culture in the cell culture incubator for about 3h. Remove the culture medium, add 150μl of DMSO to each well, shake evenly, and measure the absorbance with an enzyme plate reader. Use an enzyme plate reader to read the OD value of each well at a wavelength of 570nm, and calculate the cell survival rate. The formula is as follows: Cell survival rate (%) = (OD sample group / OD blank control group) × 100.

[0046] The cytotoxicity of the antimicrobial peptide cd77AHP-2 was tested. Figure 6 and Figure 7 As shown, when the concentration of antimicrobial 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 cytotoxicity of antimicrobial peptide cd77AHP-2 was extremely low and had great medical therapeutic potential.

[0047] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and 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 method for preparing the antimicrobial peptide cd77AHP-2 according to claim 1, characterized in that: The cationic amino acid distribution of the antimicrobial peptide cd77 is optimized and modified, and the cationic amino acids are adjusted from aggregated distribution to dispersed distribution, thereby obtaining the antimicrobial peptide cd77AHP-2. The amino acid sequence of the antimicrobial peptide cd77 is shown in SEQ ID NO.

2.

3. 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 for inhibiting Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

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

5. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 3, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide cd77AHP-2 against Acinetobacter baumannii is 16 μg / mL.

6. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 3, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide cd77AHP-2 against multidrug-resistant Acinetobacter baumannii is 16 μg / mL.

7. 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.

8. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 7, characterized in that: The concentration of the antimicrobial peptide cd77AHP-2 in the antibacterial agent is 16 μg / mL to 128 μg / mL.

9. The use of the antimicrobial peptide cd77AHP-2 in the preparation of antimicrobial products according to claim 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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