Antibacterial peptide pa targeting multi-drug resistant gram-negative bacteria and application thereof

Abstract

This invention discloses an antimicrobial peptide PA targeting multidrug-resistant Gram-negative bacteria and its application. The amino acid sequence of the antimicrobial peptide PA is shown in SEQ ID NO.1. The antimicrobial peptide PA provided by this invention has a significant antibacterial effect against multidrug-resistant Gram-negative bacteria, and has a rapid bactericidal onset, no hemolysis or cytotoxicity, does not cause nephrotoxicity after treatment, and has good biosafety. It can be used for the treatment of bacterial infections and can also be applied to other scenarios that require bactericidal or antibacterial growth inhibition.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antimicrobial peptide PA targeting multidrug-resistant Gram-negative bacteria and its applications. Background Technology

[0002] The growing problem of multidrug resistance (MDR) among various pathogens poses a threat to human health, making the development of new and potent antimicrobial drugs increasingly urgent. Various strategies have been employed to find promising new antimicrobial compounds. Among all known producers of natural products, microorganisms are an important source of bioactive metabolites with broad application value. Antimicrobial peptides (AMPs) offer numerous potential advantages as therapeutic agents, including a broad antimicrobial spectrum, rapid biocide action, and the ability to counteract existing antibiotic resistance mechanisms.

[0003] With the emergence of "superbugs," many traditional antibiotics are gradually becoming ineffective. Novel drugs (or their analogues) developed based on antimicrobial peptide mechanisms hold promise as "weapons" against drug-resistant bacterial infections. Their mechanisms of action make it difficult for bacteria to develop high levels of resistance through a single mutation. Through mechanistic studies, we can modify and optimize natural antimicrobial peptides. We can design broad-spectrum antimicrobial peptides for treating infections with unknown pathogens or for combination therapy, or we can design narrow-spectrum, precisely targeted antimicrobial peptides (such as killing only Pseudomonas aeruginosa without affecting the gut microbiota), thus avoiding side effects such as dysbiosis. Derivatives developed based on antimicrobial peptide mechanisms can be used in food preservation, medical device sterilization, and cosmetic preservation, replacing some chemical preservatives and offering greater safety and reducing the likelihood of drug resistance. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antimicrobial peptide PA that targets multidrug-resistant Gram-negative bacteria and its application. The antimicrobial peptide PA of this invention has the characteristics of strong antimicrobial activity and low toxicity.

[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0006] This invention provides an antimicrobial peptide PA targeting multidrug-resistant Gram-negative bacteria, the amino acid sequence of which is shown in SEQ ID NO.1:

[0007] MATEVLQTQVIQKAWEDASFREKLMADPKSAIRDVLGVVIPDHQIKTVEETSDQFYLVIPPNPSGVLATSQKPRSMW.

[0008] The present invention also provides the application of the above-mentioned antimicrobial peptide PA in inhibiting and / or killing drug-resistant Gram-negative bacteria.

[0009] The present invention also provides the use of the above-mentioned antimicrobial peptide PA in the preparation of a drug for inhibiting and / or killing drug-resistant Gram-negative bacteria.

[0010] Furthermore, the Gram-negative bacteria are any one of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0011] Furthermore, the *Escherichia coli* is *Escherichia coli* 25922, and the *Acinetobacter baumannii* is *Acinetobacter baumannii* A11.

[0012] The present invention also provides an agent for inhibiting and / or killing drug-resistant Gram-negative bacteria, the agent containing the above-mentioned antimicrobial peptide PA.

[0013] Furthermore, the content of the antimicrobial peptide PA in the pharmaceutical preparation is 0.5 μg / mL.

[0014] The present invention also provides the application of the above-mentioned antimicrobial peptide PA in the preparation of a drug for preventing and treating diseases caused by Gram-negative bacterial infections.

[0015] The present invention also provides an formulation for treating sepsis caused by Gram-negative bacterial infection, the formulation containing antimicrobial peptide PA, wherein the content of antimicrobial peptide PA in the formulation is 1.28 mg / mL.

[0016] The beneficial effects of this invention are:

[0017] 1. Antibacterial activity: The antimicrobial peptide PA of this invention has a significant inhibitory effect on multidrug-resistant Gram-negative bacteria, and has a good inhibitory effect on Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In particular, the MIC against Acinetobacter baumannii is 0.25~0.5μg / mL, which is comparable to colistin and significantly better than traditional antibiotics such as cephalosporins and florfenicol. At high concentrations (4×MIC and 8×MIC), bacteria can be completely killed in only 20~60 minutes, and the bactericidal effect has a clear concentration dependence.

[0018] 2. Stability: The antimicrobial peptide PA of the present invention can maintain more than 99% antibacterial activity at 100℃, maintain stable activity in the pH range of 2.0-12.0, and show strong tolerance to various proteases such as papain and pepsin. Moreover, its activity is not affected by common organic reagents and surfactants.

[0019] 3. Safety: The antimicrobial peptide PA of this invention does not cause hemolysis at concentrations below 20 μg / mL, and even at a concentration of 320 μg / mL, the hemolysis rate is only 15.2%; it has no significant toxic effect on human renal tubular epithelial cells: even at a high concentration of 512 μg / mL, no statistically significant change was observed compared with the blank control group. P All values ​​were greater than 0.05, and the IC50 value was greater than 512 μg / mL; treatment did not cause nephrotoxicity, and animal experiments showed good safety and tolerability;

[0020] 4. Therapeutic effects: In a mouse sepsis model, the biological agent of this invention achieved a 100% survival rate in the high-dose group (50 mg / kg), which was significantly better than the colistin control group; the bacterial load in all tissues was significantly reduced, and the high-dose group achieved complete clearance of bacteria in the liver; the liver morphology basically returned to normal after treatment, and the effect was better than traditional antibiotics.

[0021] In summary, the antimicrobial peptide PA provided by this invention has a significant antibacterial effect against multidrug-resistant Gram-negative bacteria, and its bactericidal effect is rapid, without hemolysis or cytotoxicity. It does not cause nephrotoxicity after treatment, has good biosafety, and can be used to treat bacterial infections. It can also be applied to other scenarios that require sterilization or inhibition of bacterial growth, and has great potential to replace traditional antibiotics. Attached Figure Description

[0022] Figure 1 The graph shows the effect of different conditions on the stability of the antimicrobial peptide PA.

[0023] In the figure, a is a graph showing the effect of antimicrobial peptide PA's stability under different temperature conditions;

[0024] b is a graph showing the stability of antimicrobial peptide PA under different enzyme conditions. The main enzymes are pepsin, trypsin, papain, creatine kinase, catalase and α-chymotrypsin.

[0025] c is a graph showing the stability of the antimicrobial peptide PA under different pH conditions;

[0026] d shows the effect of different organic reagents and surfactants on the stability of antimicrobial peptide PA;

[0027] Figure 2 A schematic diagram showing the time-killing curves of different concentrations of antimicrobial peptide PA on Escherichia coli ATCC25922 and Acinetobacter baumannii A11;

[0028] In the figure, A is a schematic diagram of the time-killing curves of different concentrations of antimicrobial peptide PA, colistin (COL), and florfenicol (FFC) against Escherichia coli ATCC25922.

[0029] B is a schematic diagram of the time-killing curves of different concentrations of antimicrobial peptide PA on Acinetobacter baumannii A11;

[0030] Figure 3 The diagram shows the bactericidal effect of different concentrations of antimicrobial peptide PA.

[0031] In the figure, A shows the bactericidal effect of different concentrations of antimicrobial peptide PA on Escherichia coli ATCC25922;

[0032] B shows the bactericidal effect of different concentrations of antimicrobial peptide PA on Acinetobacter baumannii A11.

[0033] Wherein, 0, 1, 2, and 4 represent concentrations of 0, 1, 2, and 4 μg / mL of antimicrobial peptide PA concentrate, respectively;

[0034] Figure 4 A schematic diagram illustrating the in vitro and in vivo safety evaluation of antimicrobial peptide PA at different concentrations;

[0035] In the figure, A is a schematic diagram of the hemolysis rate of defibrinated sheep blood by different contents of antimicrobial peptide PA.

[0036] B is a schematic diagram of the interaction between antimicrobial peptide PA and human renal tubular epithelial cells HK-2 for 0.5 hours;

[0037] C is a schematic diagram of the interaction between antimicrobial peptide PA and human renal tubular epithelial cells HK-2 for 1 hour;

[0038] D is a schematic diagram showing the survival rates of mice given different concentrations of antimicrobial peptide PA (25, 50, 100 mg / kg);

[0039] Where ns indicates: no significant difference;

[0040] Figure 5 A schematic diagram comparing the nephrotoxicity indicators of antimicrobial peptide PA and colistin (COL);

[0041] In the figure, A represents the content of the detection index SPP-1;

[0042] B is a graph showing the content of the detection index TIMP-1;

[0043] C represents the content of the detection index KIM-1;

[0044] Where ns represents: no significant difference, This indicates that p < 0.05. This indicates that p < 0.01. This means: p < 0.0001;

[0045] Figure 6A schematic diagram illustrating the dosing regimens of biological agents and control substances in mice is shown.

[0046] In the diagram, A is a schematic diagram of the drug administration method for mice.

[0047] B is a schematic diagram of organ damage in the liver and spleen of a mouse.

[0048] C is a schematic diagram of organ bacterial load (abdominal cavity, thigh muscles, liver, and spleen).

[0049] D is a schematic diagram showing the survival rate of mice after 7 days of treatment with biological agents and control substances.

[0050] Where ns represents: no significant difference, This indicates that p < 0.05. This means: p < 0.01, This means that P < 0.001. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0052] Example 1: Synthesis and Antibacterial Evaluation of Antimicrobial Peptide PA

[0053] 1. Synthesis of antimicrobial peptide PA

[0054] The antimicrobial peptide PA targeting multidrug-resistant Gram-negative bacteria was synthesized by Nanjing Peptide Research Biotechnology Co., Ltd. The amino acid sequence of antimicrobial peptide PA is shown in SEQ ID NO.1.

[0055] MATEVLQTQVIQKAWEDASFREKLMADPKSAIRDVLGVVIPDHQIKTVEETSDQFYLVIPPNPSGVLATSQKPRSMW.

[0056] 2. Drug susceptibility testing of antimicrobial peptide PA on bacterial strains.

[0057] (1) Strain

[0058] Standard strain: Escherichia coli ATCC25922.

[0059] Clinically resistant strains include: Extensive-drug resistant (XDR) Escherichia coli ST03, T28R, EPF42-4 and Klebsiella pneumoniae SW04, SW24; Multi-drug resistant (MDR) Acinetobacter baumannii A11, A12, A13; MDR Salmonella JS, 2a; Pseudomonas aeruginosa 091218, 091452; Actinobacillus pleuropneumoniae 1144; Riesella anatipestifer RA3; Haemophilus parasuis HPS-1; Streptococcus suis 1122S, 988S; Staphylococcus aureus P1-1; Micrococcus luteus 1-1; and Enterococcus faecium EF-1.

[0060] The drug sensitivity results are shown in Table 1: The broad-spectrum antibacterial activity of antimicrobial peptide PA was tested, and it was found that antimicrobial peptide PA had good antibacterial effects against drug-resistant Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae in clinical practice.

[0061] Table 1. Antimicrobial susceptibility results of clinical strains

[0062]

[0063] 3. Evaluation of the minimum inhibitory concentration (MIC) of the above-mentioned antimicrobial peptide PA

[0064] The minimum inhibitory concentration (MIC) was assessed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for MIC determination, using the microbroth dilution method. The specific steps are as follows:

[0065] a. First, dissolve the antimicrobial peptide PA in enzyme-free water at an initial concentration of 1280 μg / mL and store at 20°C.

[0066] b. When using, dilute the antimicrobial peptide to 128 μg / mL with LB broth, and then perform a 2-fold serial dilution in a 96-well plate.

[0067] c. Adjust the bacterial culture to 0.5 McFarland turbidity, then dilute it 100-fold with fresh LB medium. Add 100 μL of the diluted bacterial solution to 96-well plates containing different concentrations of the antimicrobial peptide PA, to achieve a final bacterial concentration of 5 × 10⁻⁶ in each well. 5 CFU / mL. After incubating the 96-well plate at 37℃ for 18 h, the lowest concentration of antimicrobial peptide PA corresponding to the wells with no obvious bacterial growth observed by the naked eye was determined as the minimum inhibitory concentration (MIC). The results are shown in Tables 2 and 3.

[0068] Table 2. Antimicrobial activity of antimicrobial peptide PA against Acinetobacter baumannii

[0069]

[0070] Notes: PA: Antimicrobial peptide in this study; CRO: Ceftriaxone sodium; COL: Colistin; LEV: Ciprofloxacin; FFC: Florfenicol; CEF: Ceftiofur;

[0071] Table 3. MIC of antimicrobial peptide PA against XDR-resistant bacteria

[0072]

[0073] Notes: PA: Antimicrobial peptide in this study; COL: Colistin; AZI: Azithromycin; GEN: Gentamicin; DOX: Doxycycline; FFC: Florfenicol; TLM: Tilmicosin; TIA: Tylosin; CEF: Cefotaxime; CEQ: Cefquinoxime; FOS: Fosfomycin; CIP: Ciprofloxacin; OLA: Quinolone.

[0074] ATCC: American Standard Microbial Bank.

[0075] As shown in Tables 2 and 3, the antimicrobial peptide PA exhibits good antibacterial effects against Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In particular, the MIC of antimicrobial peptide PA against Acinetobacter baumannii can reach 0.25–0.5 μg / mL, which is significantly better than first- and third-generation cephalosporins such as ceftriaxone sodium (8–256 μg / mL) and cefazolin sodium (512 μg / mL), as well as florfenicol (128–512 μg / mL) and levofloxacin (0.5–64 μg / mL). More importantly, its effect is even superior to colistin (0.25–2 μg / mL), a last-line treatment for Gram-negative bacteria (Table 2). In addition, minimum inhibitory concentration (MIC) testing of MDR bacteria revealed that the antimicrobial peptide PA exhibited good inhibitory effects against MDR Escherichia coli and Klebsiella pneumoniae, far exceeding the efficacy of most antibiotics, such as polypeptides (colistin), macrolides, tetracyclines, amyl alcohols, cephalosporins, ciprofloxacin, and quinoxaline antibiotics (Table 3). This indicates that the antimicrobial peptide possesses good antimicrobial efficacy and has great potential for development as an alternative to antibiotics.

[0076] Example 2: Stability test of antimicrobial peptide PA

[0077] The effects of temperature, pH, protease, and surfactant on the antimicrobial efficacy of antimicrobial peptide PA were measured to characterize the stability of antimicrobial peptide PA.

[0078] The results are as follows Figure 1 As shown: the antimicrobial peptide PA retains (99±0.5)% antibacterial activity at 100℃, exhibiting good thermal stability, but its activity is almost completely lost at 121℃. Figure 1a). Antimicrobial peptide PA exhibits strong resistance to various proteases (such as papain, pepsin, etc.), with its activity decreasing by approximately 40% only upon treatment with α-chymotrypsin. Figure 1 b). Its antibacterial activity remained stable within the pH range of 2.0–12.0, and even under strong acid and strong alkaline conditions (pH 2.0 and 12.0), the activity still reached (98±1.2)%. Figure 1 c). In addition to Tris, acetonitrile, and methanol having some effect on the antimicrobial peptide PA activity, common surfactants and organic reagents have no significant effect on its antibacterial activity. Figure 1 d).

[0079] Example 3 Time-based sterilization experiment of antimicrobial peptide PA

[0080] Single colonies of *Escherichia coli* 25922 and *Acinetobacter baumannii* A11 were inoculated into 5 mL of LB broth and incubated overnight at 37°C. The colonies were then transferred to fresh LB broth at a 1:100 ratio and incubated until the late logarithmic growth phase (6 h). The resulting cultures were then treated with the antimicrobial peptide PA at concentrations of 0.5×MIC (0.25 μg / mL), 2×MIC (1 μg / mL), 4×MIC (2 μg / mL), and 8×MIC (4 μg / mL), respectively. A growth control (CON) and a positive control (*E. coli* 25922 was treated with 4×MIC COL and 4×MIC FFC, while *Acinetobacter baumannii* A11 was not treated) were also established. The bacterial and drug suspensions were incubated on a shaker at 37°C and 220 rpm. Centrifuge 100 μL samples at 0, 10, 20, 40, 60, 120, 240, 360, 480, and 1400 min, resuspend in 0.9% sodium chloride, and perform serial 10-fold dilutions. Spread the dilutions evenly on LA agar plates, incubate overnight at 37°C, and count the colonies on LB agar. The experiment should be performed in at least two replicates.

[0081] The results are as follows Figure 2 As shown, the antimicrobial peptide PA mainly exhibits antibacterial activity at lower concentrations, while it shows bactericidal activity only at higher concentrations (≥4×MIC). Using *Escherichia coli* 25922 as an indicator bacterium, concentrations below 2×MIC were ineffective in inhibiting its growth; however, at concentrations of 4×MIC and 8×MIC, the antimicrobial peptide PA completely killed it within 60 min and 20 min, respectively, demonstrating superior efficacy compared to the control drug. Figure 2 A).

[0082] When Acinetobacter baumannii A11 was used as the indicator bacterium, the antimicrobial peptide PA at a concentration of 0.5 × MIC did not affect the bacterial growth. At a concentration of 2 × MIC, the antimicrobial peptide PA only produced a slight and transient inhibition of A11 growth within 60 min. When the concentration was increased to 4 × MIC and 8 × MIC, the antimicrobial peptide PA rapidly reduced the viable count of A11 to zero within 60 min. Figure 2 B).

[0083] Therefore, the antimicrobial peptide PA mainly exhibits antibacterial effects at low concentrations, but can quickly exert bactericidal effects when the concentration is increased to 4×MIC or 8×MIC. Moreover, this bactericidal effect is significantly concentration-dependent, and rapid sterilization can be achieved in just 20 minutes at high concentrations.

[0084] Example 4: High-efficiency bactericidal test of antimicrobial peptide PA

[0085] The Cell-Check™ Viability / Cytotoxicity Kit was used to calculate the number of surviving and dead Escherichia coli 25922 and Acinetobacter baumannii A11 cells after exposure to the antimicrobial peptide PA. The specific steps are as follows:

[0086] Indicator bacteria (Escherichia coli 25922 or Acinetobacter baumannii A11) were cultured to mid-logarithmic growth phase. Antimicrobial peptide PA was added to prepare concentrations of 2×MIC (1 μg / mL), 4×MIC (2 μg / mL), and 8×MIC (4 μg / mL), respectively. The same culture medium without PA served as a control (0 μg / mL). The culture was treated at 37°C for 2 hours. The procedure was performed according to the Cell-Check™ Viability / Cytotoxicity Kit, and the specific steps are as follows:

[0087] Mix 1.5 µL of SYTO9 stock solution and 1.5 µL of propidium iodide stock solution in a microcentrifuge tube, then add 7 µL of 0.85% NaCl solution and mix thoroughly to obtain a 100X dye mixture. For every 100 µL of bacterial sample and live / dead control suspension, add 1 µL of the 100X dye mixture. Mix thoroughly and incubate in the dark at room temperature for 15 min. Capture 5 µL of the stained bacterial suspension between a glass slide and an 18 mm square cap. Observe the sample on a slide under a fluorescence microscope at 200x magnification.

[0088] The results are as follows Figure 3 As shown: Whether it is Escherichia coli 25922 ( Figure 3 A) or Acinetobacter baumannii A11 ( Figure 3Similar results were observed in both groups (B). In the untreated control group, all bacteria exhibited green fluorescence (live bacteria). After treatment with the antimicrobial peptide PA at a concentration of 2×MIC, scattered red fluorescence (dead bacteria) began to appear in the samples. When the concentration was increased to 4×MIC, approximately 50% of the bacteria showed red fluorescence; with further increases to 8×MIC, approximately 95% of the bacteria showed red fluorescence, at which point green fluorescence was almost invisible.

[0089] This indicates that the bactericidal effect of antimicrobial peptide PA is concentration-dependent, and high concentrations can cause cell membrane damage and death in the vast majority of bacteria.

[0090] Example 5: Hemolysis and Cytotoxicity Test of Antimicrobial Peptide PA

[0091] 1. Antimicrobial peptide PA hemolysis test: Defibrinated sheep blood was centrifuged at 1000 r / min for 10 min. The supernatant was discarded, and the red blood cells were washed repeatedly with PBS until the supernatant was clear. The red blood cells were resuspended in 10 times their volume of PBS to prepare a red blood cell suspension. The antimicrobial peptide PA was serially diluted with PBS to concentrations of 320, 160, 80, 40, 20, 10, and 5 μg / mL. 10 μL of the peptide solution (containing antimicrobial peptide PA) was added to each well of a 96-well plate as the experimental group. 10 μL of PBS and 10% Triton X-100 were added as the negative control group and positive control group, respectively. 1×10⁻⁶ ppm of the solution was added to each well. 5 Red blood cell suspensions were prepared in 5 replicates per group. The cells were incubated in 5% CO2 at 37°C for 30 min, centrifuged at 1500 rpm for 10 min, and the supernatant was transferred to a new 96-well plate. The optical density was measured at 450 nm. D 450 [and calculate the hemolysis rate.]

[0092] Hemolysis rate = [experimental group] D 450 - Negative control group D 450 [Positive control group] D 450 - Negative control group D 450 ]×100%.

[0093] The results are as follows Figure 4 As shown in Figure A, the antimicrobial peptide PA does not cause hemolysis at concentrations below 20 μg / mL; when the concentration is increased to 320 μg / mL, the hemolysis rate is only 15.2%, with almost no hemolysis.

[0094] 2. Antimicrobial peptide PA cytotoxicity test

[0095] The toxicity of PA to human renal tubular epithelial cells HK-2 was determined using the CCK-8 assay. The specific steps are as follows:

[0096] HK-2 cells in logarithmic growth phase were subjected to 10 4 Cells were seeded into 96-well plates and grown overnight. After cell attachment, the plates were replaced with FBS-free basal medium and starved for 12 h. Antimicrobial peptide PA was diluted with serum-free basal medium at concentrations of 512, 128, 32, 8, and 2 μg / mL, and 100 μL was added to each well. Cells were incubated at 37°C and 5% CO2 for 24 h. Then, in the dark, 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37°C and in the dark for 0.5 h and 1 h, respectively. The absorbance was then measured at 450 nm using a microplate reader. The cytotoxicity of antimicrobial peptide PA to cells was calculated using the following formula: Cell viability (%) = [A...] 加药 -A 空白 ] / [A 不加药 -A 空白 ]×100%

[0097] In the formula, A 加药 : Absorbance of pores containing cells, CCK-8 solution, and drug solution;

[0098] A 空白 : Absorbance of pores containing culture medium and CCK-8 solution but without cells;

[0099] A 不加药 : Absorbance of pores containing cells and CCK-8 solution but no drug solution.

[0100] like Figure 4 As shown in B-C, the cytotoxicity assays indicated that even at a concentration of 512 μg / mL, the activity of HK-2 cells remained significantly different from that of the control group (0 μg / mL). Given that no significant cytotoxicity was observed at the highest tested concentration (512 μg / mL), the half-maximal inhibitory concentration (IC50) could not be fitted at this concentration; its IC50 value is presumed to be greater than 512 μg / mL.

[0101] Example 6: In vivo safety and therapeutic trials of antimicrobial peptide PA

[0102] The biological agent for treating sepsis caused by Gram-negative bacterial infection is prepared by suspending the antimicrobial peptide PA in ultrapure distilled water, wherein the concentration of the antimicrobial peptide PA in the biological agent is 1.28 mg / mL.

[0103] 1. In vivo safety of antimicrobial peptide PA

[0104] In this embodiment, ICR mice were used. After 7 days of quarantine and acclimatization, they were randomly divided into 4 groups of 5 mice each. The specific grouping and administration regimens were as follows: PBS control group (intraperitoneal injection of an equal volume of PBS), 25 mg / kg PA dose group, 50 mg / kg PA dose group, and 100 mg / kg PA dose group. All groups were administered the medication once daily via intraperitoneal injection for 7 consecutive days. During the administration period, the clinical symptoms of each group of animals were closely observed and recorded daily. 24 hours after the last administration, blood samples were collected from the mice and serum was separated. Subsequently, the animals were euthanized using ethical methods.

[0105] like Figure 4 As shown in D, during the 7-day continuous dosing experiment, compared with the PBS control group, the antimicrobial peptide PA treatment in all dose groups (25 mg / kg, 50 mg / kg, 100 mg / kg) did not result in a significant difference in the survival rate of experimental mice, and the survival rate of mice in all groups remained at 100%.

[0106] 2. Treatment trials of antimicrobial peptide PA

[0107] To establish a neutropenia model, reduce the immunity of mice, increase their susceptibility to Acinetobacter baumannii, and make them more prone to symptoms, thus improving the model's success, mice were intraperitoneally injected with cyclophosphamide (Sigma-Aldrich, catalog number C0768-5G) 150 mg / kg body weight 4 days before infection, and again injected with 100 mg / kg 1 day before infection. On the day of infection, 1×10⁻⁶ cytidine diphosphate lacryma-jobi (CPD) was administered via intramuscular injection in the thigh. 9 CFU of Acinetobacter baumannii strain A11 was administered, and mouse core body temperature and weight changes were continuously monitored (specific infection and administration methods can be found in [reference]). Figure 6 A).

[0108] Low-dose treatment group: The above-mentioned biological agent was administered intraperitoneally at 25 mg / kg at 1, 4, 8, and 20 hours post-infection.

[0109] High-dose treatment group: The above-mentioned biological agent was administered intraperitoneally at 50 mg / kg at 1, 4, 8 and 20 hours after infection.

[0110] PBS control group: The same volume of PBS as the high-dose group was injected.

[0111] COL control group: Colistin solution was injected intraperitoneally at 7.5 mg / kg at 1, 4, 8 and 20 hours after infection.

[0112] CON control group: Normal growth without bacterial infection.

[0113] After drug administration, mice were observed for 7 consecutive days, and their survival rate was recorded daily. At the end of the experimental period, blood samples were collected via ocular hemorrhage (PBS group, high-dose PA treatment group, and COL control group). Serum was separated and used to detect kidney injury-related indicators, such as osteopontin-1 (SPP-1), tissue inhibitor of metalloproteinases-1 (TIMP-1), and kidney injury molecule-1 (KIM-1), to assess the potential impact of the antimicrobial peptide PA on kidney function. Subsequently, the animals were euthanized using ethically sound methods. Under aseptic conditions, peritoneal contents (approximately 2 mL) and major organ samples were collected for subsequent Acinetobacter baumannii load counting. The collected organ samples were then fixed in 4% paraformaldehyde and subjected to pathological observation. This experimental protocol has been approved by the Experimental Animal Management and Ethics Review Executive Committee of Huazhong Agricultural University (HZAUMO-2025-0412).

[0114] like Figure 5 As shown, compared with the PBS group, treatment with the antimicrobial peptide PA at a dose of 50 mg / kg resulted in a statistically significant increase only in the level of the kidney injury biomarker KIM-1 (P<0.05), while the levels of SPP-1 and TIMP-1 showed no significant difference. Considering the slight fluctuations that KIM-1 may exhibit as an early sensitive indicator, PA at this dose hardly induced definite kidney injury. In stark contrast, in the positive control group treated with colistin (COL), the levels of all detected kidney injury biomarkers (SPP-1, TIMP-1, and KIM-1) were significantly elevated, exhibiting typical characteristics of severe nephrotoxicity.

[0115] like Figure 6 As shown, pathological observation of the organs first revealed that the liver and spleen of mice infected with Acinetobacter baumannii showed obvious blackening and hardening. The liver and spleen lesions were aggravated in the colistin treatment group, the pathological changes in the liver and spleen were slightly alleviated after treatment in the low-dose group, while the liver morphology in the high-dose treatment group basically returned to normal and was no different from that in the uninfected group. Figure 6B). In an ICR mouse sepsis model, tissue bacterial load analysis revealed that after Acinetobacter baumannii infection, the highest bacterial load was found in the thigh muscle, followed by the peritoneal cavity, with similar loads in the liver and spleen. Treatment with the biological agent PA (low-dose group 25 mg / kg and high-dose group 50 mg / kg) showed significant bactericidal effects in the peritoneal cavity, thigh muscle, and liver compared to the PBS control group. In the peritoneal cavity, PA's effect was slightly less than the positive control drug COL. However, in the thigh muscle and liver, PA achieved a bacterial load reduction comparable to COL. Notably, PA's antibacterial effect in the spleen exhibited a unique dose-dependent characteristic. Low-dose PA and COL treatments both led to an abnormal increase in spleen bacterial load; however, when the PA dose was increased to 50 mg / kg, complete clearance of spleen bacteria was achieved, with significantly better results than COL. Figure 6 C).

[0116] Compared with the PBS control group, the low-dose treatment group increased the survival rate of mice to 50%, and the high-dose treatment group achieved a survival rate of 100%, which was significantly better than the COL control group. Figure 6 D).

[0117] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide PA targeting multidrug-resistant Gram-negative bacteria, characterized in that: The amino acid sequence of the antimicrobial peptide PA is shown in SEQ ID NO.1: MATEVLQTQVIQKAWEDASFREKLMADPKSAIRDVLGVVIPDHQIKTVEETSDQFYLVIPPNPSGVLATSQKPRSMW.

2. The use of the antimicrobial peptide PA according to claim 1 in the preparation of a drug for killing drug-resistant Gram-negative bacteria, characterized in that: In the drug, the effective concentration of the antimicrobial peptide PA is not less than 4×MIC; the Gram-negative bacteria are any one of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

3. The application according to claim 2, characterized in that: The Escherichia coli is Escherichia coli 25922, and the Acinetobacter baumannii is Acinetobacter baumannii A11.

4. An agent for inhibiting and / or killing drug-resistant Gram-negative bacteria, characterized in that: The pharmaceutical preparation contains the antimicrobial peptide PA as described in claim 1.

5. The pharmaceutical preparation according to claim 4, characterized in that: The content of antimicrobial peptide PA in the drug is 1.28 mg / mL.

6. The use of the antimicrobial peptide PA according to claim 1 in the preparation of a medicament for preventing and treating diseases caused by infections with Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa, characterized in that: In the drug, the effective concentration of the antimicrobial peptide PA is not less than 4×MIC.

7. A preparation for treating sepsis caused by Gram-negative bacterial infection, characterized in that: The formulation contains the antimicrobial peptide PA according to claim 1, wherein the content of antimicrobial peptide PA in the formulation is 1.28 mg / mL.

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