An antibacterial peptide targeting staphylococcus aureus and having dual functions of inhibiting quorum sensing signal and antibacterial effect

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to an antibacterial peptide with the functions of targeting Staphylococcus aureus and inhibiting quorum sensing signal and antibacterial effect. The present application first synthesizes a non-sulfur lactone AIP-III variant CP7 with the functions of targeting and inhibiting QS signal pathway in Staphylococcus aureus cells, and synthesizes an FP13-2 antibacterial peptide with broad-spectrum antibacterial activity, and then successfully synthesizes a composite peptide containing the CP7 domain and the FP13-2 domain. The composite peptide of the present application can target and kill Staphylococcus aureus, and is also effective for methicillin-resistant Staphylococcus aureus. The composite peptide of the present application has high antibacterial activity in vivo and in vitro, and has moderate toxicity. In addition, the mechanism of the composite peptide is different from that of traditional antibiotics, and the composite peptide is not prone to drug resistance, thereby providing certain guidance value for treating Staphylococcus aureus infection and drug-resistant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antimicrobial peptide that targets Staphylococcus aureus and has dual functions of inhibiting quorum sensing signals and antibacterial activity. Background Technology

[0002] Staphylococcus aureus (S. aureus), a Gram-positive bacterium, is a common colonizing bacterium in human hosts. It is one of the most common pathogens causing clinical infections, and can cause systemic transmission in humans and animals, such as skin and soft tissue infections, urinary tract infections, pneumonia, osteomyelitis, and endocarditis. In recent years, antibiotic resistance has become increasingly prevalent. To address this pressing global infectious disease crisis, there is an urgent need to explore alternative treatments for Staphylococcus aureus infections using different mechanisms.

[0003] In addition to acquiring resistance genes, *Staphylococcus aureus* can synthesize several virulence factors and produce biofilms, which are closely related to bacterial resistance and pathogenicity. The formation of virulence factors and biofilms is strictly controlled by the helper gene regulator (agr) system, which is found in almost all staphylococci. The agr system is the master regulator of the *Staphylococcus aureus* quorum sensing (QS) system, controlling the expression of exogenous and surface proteins. This system consists of RNAII and RNAIII transcription units controlled by the P2 and P3 promoters, respectively. The P2 operon regulates a four-gene operon, agrBDCA, which biosynthesizes autoinducible peptides (AIPs) and processes AgrC and AgrA proteins. The P3 operon drives the expression of RNAIII units and is the main effector for *Staphylococcus aureus* virulence factor expression. *Staphylococcus aureus* detects extracellular autoinducible peptides through the AgrC system, thereby mediating the control of its virulence factors by the bacterial agr system. To date, four specific agr groups have been identified in Staphylococcus aureus based on the agr operon, each with distinct AIP (I-IV) sequences. Homologous AIPs bind to AgrC, activating the agr response and inducing virulence factors via RNAIII regulators, while non-homologous AIPs competitively bind to AgrC to suppress the agr response, still allowing bacterial growth. Therefore, variants of AIPs that inhibit the AgrC receptor and the production of associated Staphylococcus aureus toxins have attracted attention as novel strategies against Staphylococcus aureus and related infections.

[0004] Natural antimicrobial peptides (AMPs) are endogenous defense molecules in the innate immune system that protect the host against various pathogens. Furthermore, they are less prone to drug resistance. Therefore, developing methods for treating Staphylococcus aureus infections based on AIP variants and natural antimicrobial peptides has significant application value. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention designed an AIP-III variant called CP7, which binds to the extra-loop-II domain of the transmembrane protein AgrC in type I Staphylococcus aureus, and can reduce the expression of Staphylococcus aureus virulence factors in a non-biological killing manner. At the same time, FP (Fusogenic Peptides) with strong antibacterial activity were designed and identified. Finally, based on CP7 and FP, a CP7-FP13-2 complex peptide containing the CP7-targeting agr system domain and the FP13-2 broad-spectrum antibacterial domain was successfully constructed.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] The first aspect of this invention provides an antimicrobial peptide, CP7-FP13-2, whose amino acid sequence is shown below: (LLFA-Dab)-NIGGC-6MA-GIKNLWKKMIKLWY. The structural formula of CP7-FP13-2 is shown below:

[0008]

[0009] This invention first designed an AIP-III variant called CP7, which binds to the extra-loop-II domain of the transmembrane protein AgrC in type I Staphylococcus aureus, and can attenuate the expression of Staphylococcus aureus virulence factors in a non-biological killing manner. Simultaneously, based on previous research by our group, several potent AMPs were generated by replacing negatively charged or neutral FP residues with positively charged lysine residues (Wu, W.; Lin, D.; Shen, X. et al. New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides. PLoS One 2015, 10(9), e0138426. DOI:10.1371 / journal.pone.0138426). Based on these peptides, an antimicrobial peptide FP13-2 composed of 13 amino acids was designed. Although it has fewer amino acid residues, it still possesses potent antimicrobial activity, including anti-MRSA activity. Finally, the two N-termini of CP7 and FP13-2 were linked by -GGC6MA (6-maleimide hexanoic acid) G-, successfully constructing a CP7-FP13-2 complex peptide containing the CP7-targeting agr system domain and the FP13-2 broad-spectrum antibacterial domain.

[0010] The second aspect of this invention provides the use of the antimicrobial peptide described in the first aspect in the preparation of a medicament for inhibiting Staphylococcus aureus.

[0011] In a preferred embodiment of the present invention, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus (MRSA).

[0012] The third aspect of this invention provides the use of the antimicrobial peptide described in the first aspect in the preparation of a medicament for treating Staphylococcus aureus infections.

[0013] Studies have shown that CP7-FP13-2 not only retains the function of the CP7 domain by specifically inhibiting the Staphylococcus aureus QS signaling pathway, but also preserves the antibacterial function of the FP13-2 domain. In vivo studies have demonstrated that CP7-FP13-2 can improve the survival rate of Kunming mice infected with Staphylococcus aureus. In conclusion, the successful synthesis of CP7-FP13-2 provides a novel therapeutic strategy for treating Staphylococcus aureus infections, including MRSA.

[0014] Preferably, the antimicrobial peptide exerts its inhibitory effect on Staphylococcus aureus by inhibiting the formation of Staphylococcus aureus biofilm.

[0015] The antimicrobial peptide binds to the extra-loop-II domain of the AgrC-I transmembrane protein, thereby inhibiting Staphylococcus aureus.

[0016] Preferably, the antimicrobial peptide exerts its therapeutic effect on Staphylococcus aureus infectious diseases by downregulating the expression of Staphylococcus aureus RNAIII.

[0017] Preferably, the antimicrobial peptide exerts its therapeutic effect on Staphylococcus aureus infectious diseases by reducing the expression levels of HLA and PSM-α in Staphylococcus aureus.

[0018] Preferably, the antimicrobial peptide exerts its therapeutic effect on Staphylococcus aureus infectious diseases by inhibiting the hemolytic activity of Staphylococcus aureus.

[0019] Preferably, the drug further includes a pharmaceutically acceptable carrier. The carrier is a functional pharmaceutical excipient available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some novel pharmaceutical polymers, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0020] Preferably, the dosage form of the drug includes preparations such as injections, tablets, granules, capsules, pellets, sustained-release preparations, oral liquids, ointments, and patches.

[0021] Preferably, the drug is administered via injection, oral administration, or topical application. The drug formulation can be administered orally or via parenteral routes (e.g., intravenously, subcutaneously, intraperitoneally, or locally), as well as by topical application. If certain drugs are unstable under gastric conditions, they can be formulated as enteric-coated tablets.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention discloses a novel complex peptide CP7-FP13-2, which consists of a targeting peptide (CP7) that targets the agr system domain of Staphylococcus aureus and a broad-spectrum antimicrobial peptide (FP13-2). First, this invention synthesized a sulfur-free lactone AIP-III variant CP7 that exhibits intracellular targeting and QS signaling pathway inhibition in Staphylococcus aureus cells, and then synthesized the broad-spectrum antimicrobial peptide FP13-2. Then, a complex peptide containing both the CP7 and FP13-2 domains was successfully synthesized. The complex peptide of this invention can target and kill Staphylococcus aureus, and is also effective against methicillin-resistant Staphylococcus aureus. Both in vivo and in vitro studies demonstrate that the complex peptide of this invention has high antimicrobial activity and moderate toxicity. Furthermore, the mechanism of action of this complex peptide differs from that of traditional antibiotics, making it less likely to induce drug resistance, and it is effective against strains resistant to existing antibiotics. Therefore, it can effectively treat Staphylococcus aureus infections, providing valuable guidance for the treatment of Staphylococcus aureus infections and drug-resistant bacteria. Attached Figure Description

[0024] Figure 1 The images show the synthesis process, chemical structure, and HPLC chromatogram of the complex peptides. Image a shows the synthesis process of CP7-FP13-2: 1) 25% piperidine / DMF, 5+25 min; TBTU, HOBt, DIEA, Fmoc-protected amino acids, 4 h; 2) TFA, Tis, EDT, H2O, 3 h; 3) DIEA, DMF, 24 h; 4) TBTU, HOBt, DIEA, DMF, 24 h; 5) 25% piperidine / DMF, 5+25 min; TFA, Tis, EDT, H2O, 3 h. Image b shows the chemical structures of CP7, FP13-2, and CP7-FP13-2. Image c shows the HPLC chromatograms of CP7, FP13-2, and CP7-FP13-2.

[0025] Figure 2 This demonstrates the antimicrobial activity and cytotoxicity of antimicrobial peptides.

[0026] Figure 3CP7 and CP7-FP13-2 exhibited specific virulence factors against Staphylococcus aureus. After treatment with 10 nM, 100 μM, or 1 μM CP7 for 24 hours, RNAIII expression in (a) Staphylococcus aureus, (b) Staphylococcus epidermidis, and (c) Staphylococcus saprophyticus was detected by qRT-PCR. CP7 significantly inhibited RNAIII expression in Staphylococcus aureus in a concentration-dependent manner, but did not inhibit RNAIII expression in Staphylococcus epidermidis or Staphylococcus saprophyticus. CP7 and CP7-FP13-2 specifically inhibited RNAIII expression in Staphylococcus aureus at a concentration of 0.78 μM, but did not inhibit RNAIII expression in Staphylococcus epidermidis or Staphylococcus saprophyticus. Other peptides did not inhibit RNAIII expression in Staphylococcus aureus, Staphylococcus epidermidis, or Staphylococcus saprophyticus. Furthermore, CP7 and CP7-FP13-2 inhibited the virulence factors (g)hla and (h)psm-α of Staphylococcus aureus; CP7 and CP7-FP13-2 also inhibited the hemolytic toxicity (i) induced by Staphylococcus aureus. *P<0.05, **P<0.01, ***P<0.001.

[0027] Figure 4 The killing effect of a 6.25 μM peptide on (a) Staphylococcus aureus, (b) Staphylococcus epidermidis, (c) saprophytic tissue, (d) Streptococcus pneumoniae and (e) Escherichia coli within 120 minutes; the time-dependent killing effect of a 6.25 μM compound on Staphylococcus aureus after 10 minutes of pretreatment with 31.25 μM CCP7 (f).

[0028] Figure 5 This study investigated the inhibitory effect of peptides on Staphylococcus aureus biofilm formation.

[0029] Figure 6 To observe the biofilm structure of Staphylococcus aureus under a 400× fluorescence microscope.

[0030] Figure 7 To detect the tryptophan fluorescence emission spectra of FP13-2(a) and CP7-FP13-2(b) after incubation with 4 mg / mL Staphylococcus aureus membrane lipids, respectively, fluorescence spectroscopy was used.

[0031] Figure 8Figure (a) shows the cumulative survival rate of mice and (b) the change in body weight of mice within 7 days after Staphylococcus aureus infection. In Figure (a), mice in the normal control group were not infected with Staphylococcus aureus virus. Mice in the other groups were infected with Staphylococcus aureus virus and then injected intraperitoneally with the corresponding drug once 1 hour later. The survival rate of mice was calculated within 7 days after infection. In Figure (b), all mice in the normal control group survived and their body weight continued to increase; 3 days after Staphylococcus aureus infection, the body weight of infected mice continued to decrease, and surviving mice began to gain weight on day 5. Detailed Implementation

[0032] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0034] Example 1: Synthesis of antimicrobial peptides and their anti-Staphylococcus aureus activity.

[0035] 1. Materials and Methods

[0036] 1.1 Materials

[0037] Wang resin (100-200 mesh, substitution degree: 0.77 mmol·g) -1 Purchased from Xi'an Sunshine Resin New Materials Co., Ltd., China; amide-type MHBA resin (100-200 mesh, degree of substitution: 0.65 mmol·g) -1The (Fmoc) protected amino acids were purchased from Nankai Hecheng Sci. & Tech Co., Ltd. (Tianjin, China); the (Fmoc) protected amino acids were purchased from Wuxi Asia Peptide Biotechnology Co., Ltd. (Wuxi, China); the catalysts for 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) and 1-hydroxy-benzotriazole (HOBt) were purchased from GL Biochem. Ltd. (Shanghai, China); N,N-diisopropylethylamine (DIEA) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide (MTT), Dulbecco modified Eagle medium (DEME), and trypsin-TPCK were all purchased from Gibco (USA); fetal bovine serum (FBS) was purchased from Gibco (USA); dimethyl sulfoxide (DMSO) and N-dimethylformamide (DMF) were purchased from Tianjin Jingdong Tianzheng Precision Chemical Reagent Factory, China; ampicillin and vancomycin were purchased from Mym Biotechnology Co., Ltd. (USA); viability and virulence bacterial staining kit was purchased from Friendship Landi Biotechnology Co., Ltd. (Suzhou, China); propidium iodide (PI) was purchased from Yeasen (Shanghai, China); column chromatography was performed using Sephadex LH-20 (Amersham Pharmacia Biotech) and reversed-phase silica gel C18 (40-63 μm, Merck).

[0038] 1.2. Strains and Cell Lines

[0039] Staphylococcus aureus ATCC6538p, Staphylococcus aureus ATCC43300, Staphylococcus aureus ATCC12600, Staphylococcus epidermidis ATCC12228, Staphylococcus saprophyticus ATCC BAA-750, Streptococcus pneumoniae ATCC49619, Streptococcus mutans UA159, Streptococcus mutans ATCC25175, and Escherichia coli ATCC49619 were all stored at -80°C. Streptococcus mutans was cultured in brain-heart perfusion (BHI) broth under anaerobic conditions at 37°C, while the other strains were cultured in Mueller-hint (MH) broth under aerobic conditions at 37°C. Mouse monocyte-macrophages (RAW 264.7) were obtained from the American Type Culture Collection (ATCC), grown in DMEM, and supplemented with 10% fetal bovine serum (FBS).

[0040] 1.3 Peptide Design and Synthesis

[0041] The peptide sequences are shown in Table 1.

[0042] 1.3.1 Synthesis of FPs

[0043] Linear peptides were synthesized using a solid-phase synthesis method. Rink Amide MHBA resin was used for the synthesis of antimicrobial peptides (FPs), and Wang resin was used for the synthesis of targeting peptides. The specific solid-phase synthesis method is as follows:

[0044] (1) Activating the resin:

[0045] First, weigh 0.1 mmol of resin and add 1.5 mL of DCM to the reactor to swell and activate the resin. Shake for 5 minutes, repeating twice. Remove the DCM, add 2 mL of DMF, and wash twice, shaking for 10 minutes each time.

[0046] (2) Remove protection:

[0047] 1) Rink Amide MHBA resin: First add 2 mL of 25% piperidine / DMF, shake for 5 minutes and then remove it; then add the same 2 mL of 25% piperidine / DMF and activate for 25 minutes; then add 2 mL of DMF and wash 6 times to remove residual piperidine.

[0048] 2) Wang resin: No protection is required.

[0049] (3) Connecting amino acids:

[0050] Generally, 0.1 mmol of resin is used as a solid-phase support for peptide synthesis.

[0051] 1) The feeding ratio (molar ratio) of Rink Amide MHBA resin: Fmoc amino acid: TBTU: HOBt: DIEA is 1:3:3:3:6. Add an appropriate amount of DMF (1mL) as a solvent and shake to react for 4 hours.

[0052] 2) The molar ratio of Wang resin: Fmoc amino acid: DCC: HOBt: DIEA is 1:4:4:4; add an appropriate amount of DMF (1 mL) as a solvent and shake to react for 6 hours.

[0053] (4) Check the reaction:

[0054] 1) Rink Amide MHBA resin: First, weigh 0.3g of ninhydrin and dissolve it in 15mL of anhydrous ethanol to prepare a 2% ninhydrin solution; use a pipette to pipette a dozen resin particles into a glass tube, wash the resin resin twice with DMF and DCM in sequence, add 200uL of 2% ninhydrin indicator, shake to mix, heat for 3-5 minutes and observe the color change of the resin.

[0055] 2) Wang resin: After 6 hours, a white solid (urea formation) is observed, indicating that the reaction has occurred. Wash 4-6 times with 50% DCM / MeOH and 4 times with DMF (to remove residual MeOH). Squeeze out the reaction solution. Wash the resin in the reactor twice each with DMF and DCM (limited to the first amino acid linkage; for subsequent amino acid linkages, refer to Rink Amide MHBA resin).

[0056] (5) Complete the connection:

[0057] If the resin does not turn blue, the reaction is complete. Squeeze out the reaction liquid and wash the resin in the reactor twice each with DMF and DCM. Repeat steps (2)-(4) to connect the next amino acid in sequence.

[0058] (6) Cleavage of polypeptides:

[0059] After completing the amino acid synthesis, consider removing the Fmoc group on the last amino acid as needed. Wash the resin twice each with DMF, DCMM:MeOH (1:1, V / V), and DCM, and then air dry. Prepare a lysis buffer (prepared on ice and used immediately) using 5% EDT, 2.5% Tis, 87.5% TFA, and 5% ddH2O. Add 2 mL of the lysis buffer to the reactor and react for 4 hours. During this time, prepare a precipitant mixture of petroleum ether and methyl tert-butyl ether at a ratio of 1:1 (V / V). Add 10 mL of the precipitant to a separate test tube, squeeze the lysis buffer into the precipitant, mix thoroughly, centrifuge at 5000 rpm for 10 minutes, remove the supernatant, add another 5 mL of the precipitant, vortex to mix, and centrifuge again at 8000-9000 rpm. Remove the supernatant, and the resulting solid is the synthesized polypeptide. Air dry overnight.

[0060] (7) Dry and inspect

[0061] The following day, the peptide was dried using a rotary evaporator and then vacuum-dried for 24 hours. The compound was prepared to a 1 mM concentration using methanol solution, and its purity was checked by high-performance liquid chromatography (HPLC). The mobile phase consisted of solvent A (TFA: ddH2O = 0.075:1) and solvent B (TFA: chromatographic methanol: chromatographic acetonitrile = 0.075:0.5:0.5). The column was equilibrated with 15% B solution. After sample loading, separation was performed on the column at a flow rate of 0.8 mL / min and a gradient duration of 42 min. The elution gradient, according to the proportion of mobile phase solvent B, was as follows: 0–2 min, 15%–20%; 2–12 min, 20%–60%; 12–18 min, 60%–80%; 18–24 min, 80%–90%; 24–30 min, 90%; 30–30.5 min, 90%–70%; 30.5–32 min, 70%–40%; 32–34 min, 40%–15%; 34–40 min, 15%; 40–42 min, 15%. The molecular weight of the compounds was confirmed by mass spectrometry.

[0062] 1.3.2 Cycling of Target Peptides

[0063] Natural AIP-II consists of 7 amino acid residues (P7) and contains a 5-residue thiolactone macrocycle. CP7 was synthesized using natural AIP-III as a template, as detailed below:

[0064] (1) P7 was synthesized on Wang resin by solid-phase synthesis method;

[0065] (2) Remove the Fmoc of isoleucine with 25% piperidine, wash with DMF 6 times to remove piperidine;

[0066] (3) Add 10 times the volume of excess Ac2O and 10 times the volume of excess DIEA, with DMF (1 mL) as the solvent. Stir the reaction for 20 minutes to acetylate the amino group on isoleucine and test the reaction for completeness using the ninhydrin test.

[0067] (4) If the reaction is complete, squeeze out the reaction liquid, wash the resin in the reactor twice with DMF and twice with DCM, and then let it dry.

[0068] (5) The pyrolysis solution is used to pyrolyze the resin, and then the resin is dried.

[0069] (6) The next day, the peptide was dried by rotary evaporator and then dried by vacuum pump for 24 hours to obtain linear AC2O-P7.

[0070] (7) The molar ratio of AC2O-P7:Hobt:TBTU:DIEA is 1:1.5:1.5:3. It is fully dissolved in a round-bottom flask with DMF as solvent (1 mL) according to the ratio.

[0071] (8) The reaction was carried out under nitrogen protection for 12 hours. The carboxyl group on leucine and the amino group on Dab (2,3-diaminobutyric acid) were condensed into a ring in solution to obtain CP7, which was finally purified by gel column chromatography.

[0072] 1.3.3 Synthesis of Complex Peptides

[0073] Synthetic route for the complex peptide CP7-FP13-2 (see below) Figure 1 a) Specifically as follows:

[0074] (1) P7 was synthesized on Wang resin by solid-phase synthesis, and glycine (G), glycine (G), and cysteine ​​(C) were added sequentially to synthesize CGG-P7, retaining the Fmoc on the cysteine. 1 mL of lysis buffer was added, and the reaction was carried out for 3 hours. The residue was then cut off from the resin for later use.

[0075] (2) FP13-2 was synthesized in Rink Amide MHBA resin by solid-phase synthesis, and G and 6-maleimide hexanoic acid (6MA) were added in sequence.

[0076] (3) Add 2 times (twice the amount of resin material) of excess CGG-P7 (dissolved in DMF) and 10 times the amount of excess DIEA, and react at room temperature for 24 hours under nitrogen protection. This causes the carbon-carbon double bond on 6MA and the mercapto group on CGG-P7 to undergo an addition reaction on the resin.

[0077] (4) After the addition reaction is complete, squeeze out the reaction solution and wash with DMF 6 times to remove excess DIEA and CGG-P7.

[0078] (5) Add 3 times (3 times the amount of resin material) of excess TBTU, HOBt and 6 times the amount of excess DIEA in sequence, with DMF (1 mL) as solvent, and react at room temperature for 24 hours to esterify the amino group on Dab with the carboxyl group on leucine to form a ring.

[0079] (6) Add 2 mL of 25% piperidine / DMF, shake for 5 minutes and remove it, then add the same 2 mL of 25% piperidine / DMF, activate for 25 minutes to remove the Fmoc protecting group on cysteine, add 1 mL of lysis buffer, react for 3 hours, and lyse it from RinkAmide MHBA resin to complete the synthesis of complex peptide CP7-FP13-2.

[0080] (7) The synthesized complex peptide was dried by rotary evaporator and then dried by vacuum pump for 24 hours.

[0081] The synthetic route of CP7-FP13-2 is as follows: Figure 1As shown in a. As a control, random peptide (LGAKAGGG) and linear peptide P7 were also synthesized and coupled with FP13-2, and purified by gel column chromatography combined with preparative liquid chromatography. The purity of the peptides was identified by HPLC.

[0082] Table 1. Amino acid sequences of the synthesized peptides

[0083] Peptide name Sequence P7 IN-Dab-AFLL CP7 Ac-IN-(Dab-AFLL) FP13-2 IKNLWKKMIKLWY CP7-FP13-2 (LLFA-Dab)-NIGGC-6MA-GIKNLWKKMIKLWY P7-FP13-2 LLFA-Dab-NIGGGIKNLWKKMIKLWY Random-FP13-2 LGAKAGGGIKNLWKKMIKLWY

[0084] *The letters represent amino acid codes.

[0085] 1.4. MIC and MBC Measurement

[0086] Serially diluted (two-fold) complex peptides were added to 96-well plates at a concentration of 100 μL / well. The final peptide concentration was 0.78–100 μM. Bacteria in logarithmic growth phase were diluted to 2 × 10⁻⁶ in MH broth or BHI broth. 5 CFU / mL. Add 100 μL of bacterial suspension to the wells containing the peptide in a 96-well plate. Then, incubate the plates at 37°C for 18 to 24 hours. Next, determine the MIC (Minimum Inhibitory Concentration). The MIC is the lowest drug concentration at which no bacterial growth is observed. After MIC determination, take 50 μL of culture from the MIC, 2×MIC, and 4×MIC wells, respectively, and spread them evenly on BHI or MH agar plates. Incubate the agar plates at 37°C for 24 hours, and count the bacterial colonies on each plate. The minimum bactericidal concentration (MBC) is defined as the lowest drug concentration capable of killing >99.9% of the initial bacterial content (<100 CFU / mL). The experiment is repeated three times.

[0087] 1.5 In vitro cytotoxicity test

[0088] In a 96-well plate (4×10) 4 RAW 264.7 cells were cultured overnight in wells (cells / well) to allow for adhesion. The supernatant was then removed, washed three times with PBS, and fresh DMEM medium containing different concentrations (125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9, 0.9 μM) of complex peptides was added, and the plates were incubated at 37°C for 48 hours. Then, 100 μL of LTT solution (0.5 mg / mL) was added, and the plates were incubated at 37°C for 4 hours. The supernatant was then removed, and 150 μL of DMSO solution was added to the plates. The OD value at 570 nm was measured using a microplate reader.

[0089] 1.6 Bacterial Total RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

[0090] QRT-PCR was used to detect RNAIII expression, key factors in the Staphylococcus agr system, and related virulence factors hla and psm-α. After incubating bacteria with peptides for 18 hours, bacterial cells were collected by centrifugation (12,000 × g, room temperature, 5 min) and resuspended in PBS (pH 7.4). Lysozyme (100 μg / mL) and lysostaphylococcal lysin (100 μg / mL) were added, and the bacterial suspension was incubated at 37°C for 20 min. Total RNA was then isolated and purified using a bacterial RNA kit (GBCBIO, Guangdong, China) according to the manufacturer's instructions. RNA yield and purity were assessed spectrophotometrically. Subsequent reverse transcription was performed only on samples with a 260 / 280 nm ratio in the range of 1.8–2.0. cDNA was synthesized using PrimeScript RT MasterMix (Takara, Shiga Prefecture, Japan). QRT-PCR was performed using RR420A SYBR premix Ex Taq (Takara). By using 2 -ΔΔCt The method normalizes the data to housekeeping genes (16S rRNA) to determine relative gene expression profiles. All samples were analyzed three times. The QRT-PCR primer sequences are: 5'-AAACTAAAKGAATTGACGG-3' (16-S rRNA forward); 5'-CTACRCCGAGCTGAC-3' (16-S rRNA reverse); 5'-TTCACTGTGTCGATAATCCA-3' (RNA iii Staphylococcus aureus forward); 5'-TGATTTCAATGGCACAAGAT-3' (RNA iii Staphylococcus aureus reverse); 5'-TGAGTTGTTGAGCCATCCA-3' (Staphylococcus epidermidis RNA iii forward); 5'-ACCTAACACTGAGTCCAAGAAACTA-3' (Staphylococcus epidermidis RNA III reverse); 5'-ACGACCTTCACTTGTATCC-3' (Staphylococcus saprophytic RNA type III forward); 5'-GCTACGGCATCTTCTTCTA-3' (RNA iii RNA ... iii. Staphylococcus saprophyticus (reverse); 5'-ATGGATAGAAAAGCATCCAACA-3' (HLA forward); 5'-TTTCCAATTTGTTGAAGTCCAAT-3' (HLA reverse); 5'-TATCAAAAGCTTAATCGAACAATTC-3' (PSM-α forward); 5'-CCCCCCCCTCAAATAAGATGTTCATATC-3' (PSM-α reverse).

[0091] 1.7 Hemolysis Test

[0092] Five mL of venous blood was collected from the inferior vena cava of rats (Experimental Animal Center, Southern Medical University) and centrifuged at 1200 rpm for 10 minutes. The supernatant was then removed. The resulting red blood cells were washed three times with PBS buffer, diluted to 2% (v / v) with PBS, and then stored at 4°C. Staphylococcus aureus ATCC6538p (10 μL) was cultured at 37°C with or without compound (peptide) treatment. 5 After 20 hours of incubation (CFU / ml), the culture was centrifuged (12,000×g, 15 min), and the supernatant was filtered through a 0.22 μm microporous membrane for sterilization. 500 μL of the supernatant was added to a previously prepared 500 μL 2% erythrocyte suspension and incubated for 1 hour at 37°C using a constant-temperature shaker. The sample was then centrifuged (12,000×g, 10 min) to precipitate intact erythrocytes, and 150 μL of the supernatant was transferred to a 96-well plate for OD measurement at 570 nm. The hemolytic activity of the sterile culture medium was set to 0%, and the hemolytic activity of the untreated Staphylococcus aureus supernatant was set to 100%. The percentage of hemolytic inhibition of the control culture was calculated as: 1 - (OD value of the experimental group / OD value of the untreated Staphylococcus aureus supernatant).

[0093] 1.8 Bactericidal Kinetics Test

[0094] Dilute the bacteria in the logarithmic growth phase to 5 × 10⁻⁶. 5 CFU / mL, and treated with a compound (peptide) at a concentration of 6.25 μM (2×MIC). At different time intervals (5 s, 10 s, 30 s, 1 min, 5 min, 10 min, and 30 min), 50 μL of bacterial solution was taken and diluted to 1:10, 1:100, and 1:1000. Then, 20 μL of each dilution was plated onto MH or BHI agar plates. After incubation at 37°C under aerobic or anaerobic conditions for 24 hours, the bacterial colony count was determined. Additionally, 31.25 μM CP7 (100 μL) was mixed with 5×10⁻⁶ CFU / mL. 5 After pre-incubation with CFU / mL Staphylococcus aureus for 30 minutes, the bactericidal kinetics against Staphylococcus aureus were determined by plate count method. All results were obtained in three independent experiments.

[0095] 1.9 Biofilm formation inhibition test

[0096] The ability of the complex peptides to inhibit biofilm formation was assessed by crystal violet staining. Peptides at concentrations of 1 / 2×MIC, 1 / 4×MIC, 1 / 8×MIC, and 1 / 16×MIC were added to MH broth and Staphylococcus aureus suspension (1×10⁻⁶ ppm), respectively. 6The bacterial suspension was prepared in CFU / mL, with an untreated bacterial suspension used as a control. Each suspension was aliquoted into 96-well microtiter plates (200 μL / well) and incubated at 37°C for 24 hours. The supernatant was then removed, and the resulting biofilm was washed three times with PBS and fixed in 4% paraformaldehyde for 30 minutes. After air-drying, the biofilm was stained with 0.2% crystal violet for 30 minutes, rinsed with water, air-dried, and eluted with 33% acetic acid (v / v). The OD value was measured at 595 nm.

[0097] Simultaneously, bacterial biofilm formation was observed under a fluorescence microscope using a live / dead staining assay. Staphylococcus aureus was allowed to grow on sterile 8×8mm slides in 24-well plates at 37°C for 24 hours, with or without peptide treatment, to form a biofilm. The slides were then rinsed twice with 0.85% sodium chloride solution, stained with a bacterial viability / virulence assay kit (Bioscience, China), and observed under a fluorescence microscope (Axio Observer A1, ZEISS, Germany) at 400× magnification.

[0098] 1.10 Isothermal titration calorimetry experiment

[0099] The amino acid sequences of three AgrC-I extra-loop domains were synthesized using a solid-phase synthesis method, and their purity was confirmed by high-performance liquid chromatography (HPLC). The amino acid sequences of the three AgrC-I domains are: AgrC-I extra-loop-I: NH2-GIKYSKLDYF; AgrC-I extra-loop-II: NH2-ayitkidsi; AgrC-I extra-loop-III: NH2-SQINSDEAKVIRQ. These were then dissolved in 1% DMSO to a concentration of 100 μM. A peptide was also prepared in 1% DMSO at a concentration of 1 mM. After degassing for 10 min, the heat change of the peptide interacting with the three extra-loop domains of AgrC-I was determined by isothermal titration calorimetry (ITC) (MicroCal PEAQ-ITC, Malvern Panalytical, Malvern, UK). The ITC reaction conditions were: temperature: 25℃, reference power (μcal / s): 10, stirring speed (rpm): 750, initial delay (s): 60, number of injections: 19. Thermodynamic parameters were obtained by analyzing the data using MicroCal PEAQ-ITC software.

[0100] 1.11 Tryptophan Fluorescence Spectroscopy

[0101] Logarithmic-phase Staphylococcus aureus ATCC6538p cells were collected by centrifugation (4,000×g, 15 min), washed twice with PBS, and resuspended in 10 mL of PBS (pH 7.4). Then, 20 mL of chloroform-methanol (1:2, v / v) mixed solvent was added and stirred for 18 h, followed by the addition of 10 mL of chloroform-water (1:1, v / v), and further stirred for 30 min. The chloroform phase was collected using a separatory funnel; the solvent was removed by rotary evaporation and dried under vacuum for 24 h to obtain Staphylococcus aureus cell membrane lipids.

[0102] The prepared Staphylococcus aureus cell lipids were dissolved in 5% DMSO to a concentration of 4 mg / mL, and then treated with 100 μM CP7-FP13-2 or FP13-2 for 1 h. 100 μM CP7-FP13-2 and FP13-2 dissolved in PBS were used as controls, respectively. The fluorescence emission spectra of tryptophan were detected using fluorescence spectroscopy (FLS 980, Edinburgh, UK, excitation wavelength: 280 nm, scanning range: 300 to 400 nm), and the slit widths of the excitation and emission beams were 3 nm.

[0103] 1.12 In Vivo Experiments in Mice

[0104] Male Kunming mice at 4 weeks of age (16 - 20 g, specific pathogen free) were purchased from the Experimental Animal Center of Southern Medical University (license number: SYXK (Guangdong) 2016 - 0167, Guangzhou, China). During the experiment, all mice were fed standard laboratory food and given water ad libitum. This experiment was conducted in accordance with the standard operating procedures of the Institutional Animal Care and Use Committee of Southern Medical University and the Animal Welfare Act.

[0105] The mice were randomly divided into 5 groups with 10 mice in each group. Mice not infected with Staphylococcus aureus virus were used as the normal control group, and the remaining groups of mice were intraperitoneally injected with <0.5 mL (1.0×10 8 CFU / mL) of Staphylococcus aureus ATCC6538p to establish a Staphylococcus aureus-infected mouse model. One hour later, the normal group and the Staphylococcus aureus-infected control group mice were intraperitoneally injected with 0.9% normal saline, the low-dose group was injected with 15 mg / kg of CP7-FP13-2, the high-dose group was injected with 45 mg / kg of CP7-FP13-2, and the positive group was injected with 5 mg / kg of vancomycin. All mice were injected only once for 7 consecutive days, and the survival time and body weight changes of the mice were recorded. Survival rate analysis was performed using log pad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA), and the log-rank (Mantel-Cox) test was used. A p value < 0.05 was defined as having a significant difference.

[0106] 1.13 Statistical Analysis

[0107] Each experiment was independently repeated at least three times, and results are expressed as mean SD. All data were analyzed using unpaired Student's t-tests or one-way ANOVA, followed by Duncan's test using SPSS 25.0 software. Graphs were plotted using GraphPad Prism 8 software. Statistical significance was defined as... * P<0.05, ** P<0.01, *** P<0.001.

[0108] 2. Experimental Results

[0109] 2.1 Peptide Design and Synthesis

[0110] All Staphylococcus aureus AIP signals share two main structural features: 1) a macrocyclic thioester formed by a conserved cysteine ​​residue and a C-terminal carboxylic acid, and ii) an N-terminal external ring tail. In this example, natural AIP-III was used as a lead compound for a proof-of-concept study. An AIP-III variant, designated CP7, was constructed by replacing Asp with Ala and Cys with Dab to enhance its stability, water solubility, and reduce synthetic difficulty. Simultaneously, an antimicrobial peptide, FP-13-2, composed of 13 amino acids, was constructed. Next, the N-termini of the two peptide chains of CP7 and FPs were linked using the bifunctional molecule 6MA (6-maleimide hexanoic acid) via a Gly-Gly-Cys-6MA-Gly linker. Cyclation was then performed on resin, forming a lactam ring through the amino group of the Dab residue and the carboxyl group of the CP7 domain, yielding the complex peptide CP7-FP13-2. Figure 1 a and 1b show the synthetic process and chemical structure of these peptides, and their purity was determined by HPLC. Figure 1 c).

[0111] 2.2. Complex peptides have potent antibacterial activity.

[0112] The antibacterial activity of these peptides against a group of bacterial strains (including Gram-positive Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, and Streptococcus mutans, and Gram-negative Escherichia coli) was detected using MIC and MBC methods. Figure 2 It can be seen that FP13-2 has broad-spectrum antibacterial activity, while CP7 has no activity within the experimental range. After CP7-FPs are coupled with CP7, they have the same MIC and MBCs as FPs, indicating that they have stronger antibacterial activity in the antibacterial domain.

[0113] 2.3 The complex peptide showed moderate cytotoxicity.

[0114] The cytotoxicity of AMPs to RAW 264.7 cells was assessed using the MTT assay. Figure 2 As shown, CP7 and FP13-2 showed no cytotoxicity at concentrations up to 125 μM, and the CC of CP7-FP13-2 was [not specified]. 50 The concentration was 71.13 ± 2.13 μM. These results indicate that CP7-FP13-2 should be safe within the concentration range required for effective treatment of Staphylococcus aureus.

[0115] 2.4. CP7 can specifically inhibit the QS signaling pathway in Staphylococcus aureus.

[0116] The inhibitory effect of CP7 on the QS signaling pathway in Staphylococcus aureus was assessed by measuring the RNAIII transcription level of Staphylococcus aureus ATCC6538p 24 hours after CP7 treatment. Figure 3 As shown in a, CP7 downregulated Staphylococcus aureus RNAIII expression in a concentration-dependent manner. Therefore, the downregulation of Staphylococcus aureus RNAIII is due to a non-biological killing mechanism, as CP7 has no antibacterial activity. Figure 2 To verify whether CP7 inhibits QS signaling against other staphylococci, its effect on Staphylococcus epidermidis and Staphylococcus saprophyticus was measured. Figure 3 As shown in b and 3c, at the same concentration, CP7 did not inhibit RNAIII expression in Staphylococcus epidermidis and Staphylococcus saprophyticus, indicating that CP7 has a specific inhibitory effect on the QS signaling pathway in Staphylococcus aureus.

[0117] 2.5. CP7-FP13-2 specifically inhibits the QS signaling system of Staphylococcus aureus at non-bactericidal concentrations.

[0118] Based on the MIC value of CP7-FP13-2, the inhibitory effect of CP7-FP13-2 on RNAIII expression was evaluated using a non-bacterial concentration of 0.78 μM (1 / 4 × MIC). Figure 3 As shown in df, CP7-FP13-2 significantly downregulated the expression of Staphylococcus aureus RNAIII, but had no inhibitory effect on the expression of Staphylococcus epidermidis RNAIII or Staphylococcus saprophyticus RNAIII, indicating that CP7-FP13-2 retains the function of the CP7 domain in the targeted inhibition of the Staphylococcus aureus QS signaling system.

[0119] 2.6, CP7, and CP7-FP13-2 inhibit the expression of virulence factors.

[0120] The antiviral activity of peptides was assessed using qRT-PCR to determine the levels of virulence factors after peptide treatment with Staphylococcus aureus. For example... Figure 3As shown in the diagram, CP7 and CP7-FP13-2 significantly reduced the expression levels of Staphylococcus aureus RNAIII, as well as hla and psm-α. In contrast, FP13-2 and Random-FP13-2 did not downregulate the expression of these factors, suggesting that CP7-FP13-2 has antiviral activity against Staphylococcus aureus virus due to the presence of the CP7 domain.

[0121] 2.7, CP7, and CP7-FP13-2 can inhibit the hemolytic activity of Staphylococcus aureus.

[0122] Hemolysin, encoded by the hla gene, is one of the most important virulence factors in Staphylococcus aureus. Hemolysin produced by Staphylococcus aureus can be assessed using the erythrocyte bacterial hemolysis assay. Figure 3 i showed that CP7 and CP7-FP13-2 had a significant inhibitory effect on hemolysis of suspended red blood cells caused by Staphylococcus aureus, while FP13-2 and Random-FP13-2 had no inhibitory effect.

[0123] 2.8. CP7-FP13-2 accelerated the killing kinetics against Staphylococcus aureus.

[0124] The kinetics of peptide killing different bacterial strains were determined within 30 minutes. Figure 4 As shown in Figure ae, at a concentration of 6.25 μM, the CP7-FP13-2 compound exhibited faster killing kinetics against Staphylococcus aureus compared to FP13-2 within 20 minutes, while showing no killing tendency against Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, or Escherichia coli. The killing kinetics of CP7-FP13-2 against Staphylococcus aureus were also determined over 120 minutes after bacteria were pretreated with CP7 at a concentration of 31.25 μM for 10 minutes. Figure 4 As shown in f, CP7 had no bactericidal effect during the experiment, while CP7-FP13-2 exhibited similar killing kinetics to FP13-2, indicating that CP7 reduced the bactericidal and killing rate of CP7-FP13-2 against Staphylococcus aureus.

[0125] 2.9. Peptides inhibit the formation of Staphylococcus aureus biofilm.

[0126] Biofilm formation is a significant factor in the development of drug resistance in Staphylococcus aureus. For example... Figure 5 As shown, at concentrations of 1.56 and 0.78 μM, all peptides inhibited the formation of Staphylococcus aureus biofilm; at a concentration of 0.39 μM, both CP7 and CP7-FP13-2 inhibited the formation of Staphylococcus aureus biofilm. The formation of Staphylococcus aureus biofilm was also observed under a fluorescence microscope. Figure 6As shown, in the control group, Staphylococcus aureus formed a uniform and dense biofilm containing a small number of dead bacteria. In the peptide-treated group, the dense structure of the biofilm was disrupted, and scattered biofilm defects were visible. Furthermore, the number of red fluorescent cells, representing dead bacterial cells within the biofilm, was significantly increased.

[0127] 2.10, CP7, and CP7-FP13-2 may bind to the Extra-loop-II protein AgrC-I in Staphylococcus aureus.

[0128] Next, to explore the possible binding domains of the AgrC-I protein in Staphylococcus aureus, the thermodynamic parameters of the interactions between CP7, CP7-FP13-2, and the three exocyclic peptides were measured using an ITC instrument. The ITC isotherms clearly show that CP7 and CP7-FP13-2 bind more efficiently to extra-loop-II, extra-loop-I, or extra-loop-III (Table 2), indicating that P7 and FP13-2 interact with extra-loop-I, extra-loop-II, and extra-loop-III. As shown in Table 2, the binding affinity Kd between CP7 and extra-loop-II was calculated to be 2.32 × 10⁻⁶. - The binding affinity Kd between 3M, CP7-FP13-2, and extra-loop-II is 3.78 × 10⁻⁶. -3 M indicates that CP7 and CP7-FP13-2 may bind to the extra-loop-II domain of the AgrC-I transmembrane protein and may target the extra-loop-II domain of the Staphylococcus aureus AgrC-I transmembrane protein.

[0129] Table 2. Thermodynamic parameters of the interaction between peptides and AgrC- in extra-loop-II and extra-loop-III.

[0130]

[0131]

[0132] 2.11. The interaction between CP7-FP13-2 and Staphylococcus aureus biofilm resulted in a blue shift in the tryptophan spectrum.

[0133] Tryptophan fluorescence spectroscopy is used to investigate potential antibacterial mechanisms and to verify the interaction between the peptide and the bacterial cell membrane. FP13-2 and CP7-FP13-2 contain tryptophan residues and exhibit a maximum absorption peak at an emission wavelength of 300-400 nm under excitation at 280 nm. Figure 7As shown in ab, the emission wavelength of tryptophan residues in FP13-2 changed from 365.83±2.25nm to 355.17±2.75nm, a blue shift of 10.50±1.00nm; the emission wavelength of CP7-FP13-2 changed from 368.83±1.04nm to 358.50±0.50nm, a blue shift of 10.33±1.57nm.

[0134] 2.12. CP7-FP13-2 exhibits antibacterial activity in vivo.

[0135] The in vivo effects of CP7-FP13-2 were evaluated using male Kunming mice (16-20g) aged 4 to 6 weeks. Figure 8 As shown in Figure a, all mice in the normal control group survived and continued to gain weight, while those mice in the Staphylococcus aureus infection group died in less than 3 days. Approximately 20% and 50% of mice in the low-dose and high-dose groups survived, respectively. Approximately 90% of mice in the vancomycin group survived. All Staphylococcus aureus-infected mice continued to lose weight within 3 days post-infection, and surviving mice began to gain weight on day 5. Figure 8 b). It prolonged the survival time of mice and improved their survival rate.

[0136] In summary, this invention uses Staphylococcus aureus AIP-III as a lead compound, in which Asp is replaced by Ala, and Cys in the macrocycle is replaced by Dab, and then acylated to form a ring, to obtain a novel Staphylococcus aureus AIP-III variant, named CP7. QRT-PCR experiments confirmed that CP7 inhibits the expression of Staphylococcus aureus RNAIII in the range of 10 nM to 1 μM, without inhibiting the expression of Staphylococcus epidermidis RNAIII or Staphylococcus saprophyticus RNAIII. Biofilm inhibition experiments showed that CP7 also inhibits the formation of Staphylococcus aureus biofilms. Therefore, CP7 inhibits the lethal virulence of Staphylococcus aureus, providing a new therapeutic strategy for the treatment of Staphylococcus aureus infections. Simultaneously, a 13-amino acid residue FP was designed and identified, exhibiting potent antibacterial activity. MIC / MBC assays showed that FP13-2 possesses potent broad-spectrum antibacterial activity and high antibacterial efficacy against Staphylococcus aureus (including MRSA). Therefore, FP13-2 was selected for subsequent research. Finally, CP7 was coupled with FP13-2 to construct a dual-function complex peptide with antiviral factors and anti-Staphylococcus aureus properties. First, -GGC-P7 and -6MAG FPs were synthesized separately. Then, the N-termini of the two peptides were linked by an addition reaction between a carbon-carbon double bond on 6MA and a thiol group on Cys. Acylation of the amino group on Dab and the carboxyl group on Leu formed a ring in the P7 domain, allowing 6MA to successfully link the N-termini of CP7 and FPs. Subsequently, the inhibitory effect of CP7-FP13-2 on QS signaling was detected by qRT-PCR. The results showed that CP7-FP13-2 inhibited the expression of Staphylococcus aureus RNAIII at a concentration of 1 / 4×MIC, consistent with CP7. Furthermore, CP7 and CP7-FP13-2 significantly downregulated the expression of RNAIII, hla, and psm-α, while FP13-2 did not. Hemolysin is an important virulence factor of Staphylococcus aureus, encoded by the hla gene. Studies have shown that CP7 and CP7-FP13-2 inhibit Staphylococcus aureus hemolysis. These results indicate that the CP7 domain in CP7-FP13-2 retains the function of an anti-Staphylococcus aureus virulence factor. Furthermore, CP7-FP13-2 exhibits strong binding affinity to the extra-loop II of the AgrC-I protein, but weaker binding affinity to extra-loop I and extra-loop III.

[0137] MIC assays showed that FP13-2 and CP7-FP13-2 exhibited similar anti-Staphylococcus aureus activity. At a concentration of 6.25 μM, CP7-FP13-2 showed a faster bactericidal kinetic against Staphylococcus aureus than FP13-2. However, no similar trend was observed in Staphylococcus epidermidis, Staphylococcus saprophyticus, or Escherichia coli. Furthermore, CP7 reduced the bactericidal rate of CP7-FP13-2 against Staphylococcus aureus. All these results indicate that the FP13-2 antibacterial domain in CP7-FP13-2 retains its antibacterial activity.

[0138] The main antibacterial mechanism of AMPs is to disrupt bacterial membrane stability, leading to membrane rupture, which is achieved through annular pores, barrel walls, and carpet models. To further evaluate the therapeutic effect of CP7-FP13-2, an in vivo study was conducted to determine its in vivo anti-Staphylococcus aureus activity. The results showed that CP7-FP13-2 effectively improved the survival rate of Kunming mice infected with Staphylococcus aureus. These results indicate that the composite peptide synthesized in this invention is an antimicrobial peptide that targets Staphylococcus aureus and possesses dual functions of inhibiting quorum sensing signals and antibacterial activity, providing certain guiding value for the treatment of Staphylococcus aureus and drug-resistant bacteria.

[0139] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide is CP7-FP13-2, and its amino acid sequence is as follows: (LLFA-Dab)-NIGGC-6MA-GIKNLWKKMIKLWY.

2. The use of the antimicrobial peptide according to claim 1 in the preparation of a drug for inhibiting Staphylococcus aureus.

3. The use of the antimicrobial peptide according to claim 1 in the preparation of a medicament for treating Staphylococcus aureus infections.

4. The application according to claim 2 or 3, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 2 or 3, characterized in that, The dosage forms of the drugs include injections, tablets, granules, capsules, pills, sustained-release preparations, oral liquids, ointments, and patches.

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