Tryptophan-rich antimicrobial peptides with self-assembling properties and their applications

Abstract

The present invention aims to provide potential drug candidates for multidrug-resistant bacterial infections. [Solution] We provide short-chain antimicrobial peptides that are rich in tryptophan and have self-assembly properties. These antimicrobial peptides consist of tryptophan (W) and a positively charged amino acid and have the ability to self-assemble. As broad-spectrum antimicrobial peptides, these antimicrobial peptides can be used to treat microbial infections, such as mixed infections caused by Pseudomonas aeruginosa and Staphylococcus aureus.

Description

[Technical Field]

[0001] The present invention falls in the field of biotechnology and relates to novel antimicrobial peptides and their applications, especially antimicrobial peptides for bacterial infections. [Background technology]

[0002] Multidrug-resistant (MDR) bacterial infections are a serious global health problem. According to a recent Global Burden of Disease study, 4.95 million deaths were associated with antibiotic-resistant bacterial infections in 2019, and 1.27 million deaths were directly attributable to antibiotic-resistant bacterial infections (Non-Patent Document 1). MDR bacterial infections are one of the leading causes of death worldwide. MDR bacteria often exist in the form of biofilms, which are complex three-dimensional bacterial aggregates surrounded by water and extracellular polymers. Biofilms promote the development of antibiotic resistance by altering the microenvironment for cell replication, slowing the penetration of antibiotics into cells, and inducing highly resistant phenotypes (Non-Patent Document 2). Compared to biofilms composed of a single bacterium, biofilms containing multiple bacteria promote the transfer of drug resistance genes between bacterial species, further promoting biofilm resistance to antibiotics. For example, Staphylococcus aureus enhanced the resistance of Pseudomonas aeruginosa to tobramycin (Non-Patent Document 3), while Pseudomonas aeruginosa enhanced the resistance of S. aureus to vancomycin (Non-Patent Document 4). In patients with chronic infections or cystic fibrosis, P. aeruginosa and S. aureus often coexist, and such mixed infections lead to more severe illness and higher medical costs (Non-Patent Documents 5-7). Therefore, an ideal antibacterial formulation must be effective not only against planktonic bacteria but also against bacteria in multi-species complex biofilms.

[0003] Antimicrobial peptides (AMPs), also known as antimicrobial polypeptides, are one of the most promising alternatives to conventional antibiotics and have shown great potential in the treatment of multidrug-resistant bacterial infections. However, their high production costs remain a significant constraint in their clinical application. Antimicrobial peptides generally contain 10–100 amino acids. While the synthesis cost of short peptides is relatively low, their antimicrobial activity is also relatively low (Non-Patent Document 8). In an antimicrobial peptide library containing 3,324 sequences, only two of the 72 sequences effective against biofilms were short peptides of less than 10 amino acids (Non-Patent Document 9). Therefore, improving the antimicrobial activity of short peptides is an urgent challenge in the current stage of antimicrobial peptide development.

[0004] Previous studies have shown that the self-assembly of antimicrobial peptides into nanostructures can extend their half-life and enhance their antimicrobial activity by increasing their local concentration. Other studies have shown that structures known as "tryptophan zippers" or "π-π stacking bonds" can be formed between tryptophan groups, promoting intermolecular self-assembly. Therefore, the present invention provides short-chain antimicrobial peptides that are rich in tryptophan and have self-assembling activity, thereby offering potential drug candidates for multidrug-resistant bacterial infections. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] C. Antimicrobial Resistance, Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis, Lancet 399(10325) (2022) 629-655. [Non-patent document 2] PS Stewart, JW Costerton, Antibiotic resistance of bacteria in biofilms, Lancet 358(9276) (2001) 135-8.

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[0006] The present invention aims to provide potential drug candidates for multidrug-resistant bacterial infections. [Means for solving the problem]

[0007] To solve the above problems, the present invention provides short antimicrobial peptides that are rich in tryptophan and have self-assembling properties. [Effects of the Invention]

[0008] This application has designed short-chain polypeptides rich in tryptophan and possessing self-assembly properties. These polypeptides, particularly Z4, exhibit broad-spectrum antibacterial properties. Polypeptide Z4 has a minimum inhibitory concentration (MIC) of 4 μM against a mixed bacterial solution of Pseudomonas aeruginosa and Staphylococcus aureus. Compared to gentamicin, polypeptide Z4 exhibits a faster killing rate against P. aeruginosa, but no significant difference in killing rate against Staphylococcus aureus. Furthermore, polypeptide Z4 can remove more than 50% of biofilms at concentrations up to 8 μM, significantly better than the 32 μM required for gentamicin. This indicates that polypeptide Z4 has good direct antibiofilm activity.

[0009] In order to more clearly explain the technical idea of ​​the present invention, a brief description is given below in conjunction with the drawings. Obviously, these drawings are merely some specific embodiments described in this application. The present invention includes, but is not limited to, these drawings. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the minimum inhibitory concentrations (MICs) of the antimicrobial peptides of the present invention against multiple species of bacteria. [Figure 2]FIG. 2 shows the minimum inhibitory concentrations (MIC) of the antimicrobial peptides of the present invention against a mixed bacterial solution of Pseudomonas aeruginosa and Staphylococcus aureus. [Figure 3] FIG. 3 shows the results of spectral analysis of polypeptides Z1 to Z4. [Figure 4] FIG. 4 shows SEM images (A) and micelle size distribution (B) of polypeptide Z4 at MIC. [Figure 5] 5 shows the killing curves of the polypeptide Z4 of the present invention against P. aeruginosa (A) and S. aureus (B) at 1×MIC and against P. aeruginosa (C) and S. aureus (D) at 2×MIC, respectively, where FIG. 5A shows the viable counts (CFU / mL) of P. aeruginosa after treatment with Z4 or gentamicin at 1×MIC, FIG. 5B shows the viable counts (CFU / mL) of S. aureus after treatment with Z4 or gentamicin at 1×MIC, FIG. 5C shows the viable counts (CFU / mL) of P. aeruginosa after treatment with Z4 or gentamicin at 2×MIC, and FIG. 5D shows the viable counts (CFU / mL) of S. aureus after treatment with Z4 or gentamicin at 2×MIC. [Figure 6] FIG. 6 shows the removal effect (%) of the polypeptide Z4 of the present invention or gentamicin on formed biofilms. [Figure 7] Figure 7 shows the in vivo antibacterial activity of the polypeptide Z4 of the present invention, where Figure 7C shows the bactericidal activity (CFU) of Z4 or PBS against P. aeruginosa and S. aureus in catheter biofilms, Figure 7D shows the IL-6 content (pg / g) in skin tissue from the infected site of mice, and Figure 7F shows the number of inflammatory cells in skin tissue sections from the infected site of mice. [Figure 8] FIG. 8 shows that the dextrorotatory isomer Z4-D of polypeptide Z4 is highly resistant to protease digestion. DETAILED DESCRIPTION OF THE INVENTION

[0011] As shown in the present invention, the antimicrobial peptide is a broad-spectrum antimicrobial peptide.

[0012] According to the present invention, the antimicrobial peptide is a centrosymmetric nonapeptide (which may contain either a left-handed or a right-handed amino acid) consisting of tryptophan (W) and a positively charged amino acid, and the number of the positively charged amino acids is 3 to 6, for example, 3, 4, 5, or 6, preferably 4 or 6, and more preferably 4.

[0013] According to the present invention, the proportion of hydrophobic amino acids in the antimicrobial peptide is 30 to 60%, preferably 50% or more, and more preferably 55% or more, and / or the hydrophobicity is -0.1 to 0.8, preferably 0.6 or more, and / or the hydrophobic moment is 0.01 to 1.8, preferably 0.5 or more, more preferably 1.2 or more, and more preferably 1.7 or more.

[0014] According to the invention, the positively charged amino acid is selected from arginine (R) and lysine (K).

[0015] According to the present invention, the antimicrobial peptides contain a single tryptophan (W) motif, a double tryptophan motif (WW), a triple tryptophan motif (WWW) or a combination thereof, which are uniformly separated by positively charged amino acids (+).

[0016] According to the present invention, the antimicrobial peptide has a primary symmetric structure with a triple tryptophan (WWW) motif at its center, or a primary symmetric structure with a tryptophan motif at its center and double tryptophan (WW) motifs at both ends. Preferably, the antimicrobial peptide has a double tryptophan motif (WW) located at both ends.

[0017] According to the present invention, antimicrobial peptides include, but are not limited to, the following general formula: a single W motif, e.g., W+++W+++W, +W++W++W+, +++W+W+W++; combinations with a WW motif or a single W motif, for example WW+++++WW, WW+W+W+WW, +WW+++WW+, +++WW+WW++, WW++W++WW (e.g., Z3:WWRRWRRWW (SEQ ID NO: 3) and Z4:WWKKWKKWW (SEQ ID NO: 4) of the present invention), +WW+W+WW+, WWW motif or a single W motif, a combination with a WW motif, for example +++WWW+++ (e.g., Z2:RRRWWWRRR (SEQ ID NO: 2) of the present invention), +W+WWW+W+ (e.g., Z1: KWKWWWKWK (SEQ ID NO: 1) of the present invention), WWW+W+WWW, WWW+++WWW, WW+WWW+WW, +WWW+WWW+. Here, "W" represents tryptophan and "+" represents a positively charged amino acid, such as arginine (R) or lysine (K). Preferably, "+" in the same general formula are the same positively charged amino acid.

[0018] In a preferred embodiment, the antimicrobial peptide is selected from any one of SEQ ID NOs: 1 to 4 and variants thereof containing a dextrorotatory amino acid, preferably SEQ ID NO: 4 (Z4) or Z4-D.

[0019] The present invention further provides a pharmaceutical composition comprising the antimicrobial peptide.

[0020] The present invention further provides use of the antimicrobial peptide in antimicrobial mixed infections, such as anti-Pseudomonas aeruginosa-Staphylococcus aureus mixed infections, or use of the antimicrobial peptide for manufacturing an antimicrobial mixed infection medicament.

[0021] The present invention further provides use of the antimicrobial peptide for treating infection caused by multidrug-resistant bacteria, or use of the antimicrobial peptide for producing a medicament for treating infection caused by multidrug-resistant bacteria.

[0022] In order to better understand the present invention, the following describes the preferred embodiments of the present invention in conjunction with examples, which are only intended to illustrate the features and advantages of the technical invention of the present invention, and are not intended to limit the protection scope of the present invention. [Example]

[0023] Example 1 Preparation of polypeptides Z1 to Z4 Polypeptides Z1 to Z4 can be ordered from a polypeptide synthesis company or prepared according to the following conventional method.

[0024] 1. Using a polypeptide synthesizer, the antimicrobial peptides were synthesized stepwise from the C-terminus to the N-terminus according to their amino acid sequences. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) was attached to the AAM / MBHA resin, and then the Fmoc group was removed to obtain the X-AAM / MBHA resin. Next, Fmoc-Y-Trt-OH (9-fluorenylmethyloxycarbonyl-trityl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide) was attached. Following this procedure, the peptides were synthesized sequentially from the C-terminus to the N-terminus until the synthesis was complete, yielding peptide resins with the side chain protection by the Fmoc group removed.

[0025] 2. A cleavage reagent was added to the peptide resin obtained above, and the reaction was carried out at 20°C for 2 hours in the dark, followed by filtration. The precipitate was washed with TFA (trifluoroacetic acid), and the wash solution was combined with the filtrate and concentrated on a rotary evaporator. After that, approximately 10 volumes of pre-cooled anhydrous ether was added and the mixture was allowed to precipitate at -20°C for 3 hours, resulting in the deposition of a white powder. The mixture was centrifuged at 2500 g for 10 minutes, and the precipitate was collected, washed again with anhydrous ether, and dried in vacuo to obtain the polypeptide. The cleavage reagent was a mixture of TFA, water, and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5.

[0026] The column was equilibrated with 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid) for 30 min. The polypeptide was dissolved in 90% aqueous acetonitrile and filtered. A C18 reversed-phase atmospheric pressure column was used for gradient elution (eluent: methanol and aqueous sodium sulfate in a volume ratio of 30:70 to 70:30) at a flow rate of 1 mL / min and a detection wavelength of 220 nm. The main peak was collected and lyophilized. Further purification was performed using a reversed-phase C18 column. Eluent A was a 0.1% TFA / aqueous solution, and eluent B was a 0.1% TFA / acetonitrile solution. The elution concentrations were 25% B to 40% B and 75% A to 60% A. The elution time was 12 min and the flow rate was 1 mL / min. The main peak was collected and lyophilized in the same manner as above.

[0027] 4. Identification of antimicrobial peptides: The physicochemical parameters of the antimicrobial peptides are shown in Table 1.

[0028] [Table 1]

[0029] Example 2 Measurement of the bacteriostatic activity of polypeptides Z1-Z4: A. For use, the antimicrobial peptide was prepared into a fixed stock solution. A dilution series of the antimicrobial peptide solution was prepared by two-fold dilution using 0.01% acetic acid (containing 0.2% BSA) as the diluent.

[0030] B. 100 μL of the above antimicrobial peptide stock solution was taken and placed in a 96-well cell culture plate. An equal volume of test bacterial solution (approximately 10 5A positive control (containing bacterial solution but no antimicrobial peptide) and a negative control (containing neither bacterial solution nor peptide) were set up. The test bacterial species included Pseudomonas aeruginosa (PL1, SP1), Staphylococcus aureus (544, 103), Escherichia coli (729, 112), Klebsiella pneumoniae (106, 727), Streptococcus pneumoniae (383), Acinetobacter baumannii (946, 780) (Figure 1), or Pseudomonas aeruginosa (PAO1), Staphylococcus aureus (780, 946) and Staphylococcus aureus (780) (Figure 1). SP1), and a mixed solution of the above two bacterial species (Figure 2).

[0031] C. The culture plate was incubated at 37°C for 20 hours. The minimum inhibitory concentration was determined when no turbidity was observed with the naked eye at the bottom of the well.

[0032] The results in Figure 1 show that all of the antimicrobial peptides of the present invention have a certain degree of broad-spectrum antimicrobial activity, and that polypeptides Z1 and Z4 in particular have good broad-spectrum antimicrobial activity. The results in Figure 2 show that polypeptide Z4 has the lowest minimum inhibitory concentration (MIC) against a mixed solution of Pseudomonas aeruginosa and Staphylococcus aureus (4 μM, much lower than that of indolicidin (ILPWKWPWWPWRR, SEQ ID NO: 5), a well-studied antimicrobial peptide).

[0033] Example 3 Measurement of the self-assembly activity of polypeptides Z1-Z4: A. Measurement of the secondary structure of antimicrobial peptides by circular dichroism spectroscopy: The absorption spectra of polypeptides Z1–Z4 were measured at 25°C using a quartz colorimetric tube (path length 1.0 mm) on a Chirascan-plus spectrometer (Applied Photophysical, London, England). Spectra were recorded from 195–250 nm and scanned three times at a scan rate of 10 nm / min. The obtained CD spectra were used to calculate the mean residue molar ellipticity according to the formula θ = (θ × 1000) / (c × l × n). θ is the residue molar ellipticity ((deg cm) 2 ) dmol -1 ), where θ is the actually measured buffer-corrected ellipticity (mdeg), c is the antimicrobial peptide concentration (mM), l is the optical path length (mm), and n is the number of amino acids.

[0034] As shown in Figure 3, all peptides have two positive absorption peaks located at 200 nm and 220 nm, respectively (indicated by arrows), suggesting that the polypeptides may form supramolecular structures with self-assembly activity.

[0035] Without being bound by any theory, it is speculated that the reason why WW++W++WW has superior self-assembly activity and MIC compared to +++WWW+++ is due to the WW at both ends of the polypeptide, rather than the number of positively charged amino acids (K and / or R).

[0036] B. Observation of polypeptide Z4 by scanning electron microscopy (SEM): To prepare SEM samples for observing polypeptide self-assembly, 10 μL of the polypeptide was placed on tin foil that had been sonicated for 30 minutes, dried, and then metal-coated. All samples were observed using a Gimini SEM 500.

[0037] As shown in FIG. 4, polypeptide Z4 can form a nanomicelle structure with an average particle size of 174.62 nm by self-assembly in phosphate buffered saline (PBS) (pH=7.2).

[0038] Example 4 Measurement of the bactericidal and antibiofilm activities of polypeptide Z4 A. To measure the bactericidal activity of polypeptide Z4, Pseudomonas aeruginosa and Staphylococcus aureus were suspended in PBS to an OD600 nm of 0.4, diluted 1000-fold with fresh PBS, and mixed in equal volumes. Polypeptide Z4 was added to the bacterial suspension at concentrations of 1x and 2x the MIC. The bacterial suspension was diluted 10-fold, 100-fold, and 1000-fold at 0, 5, 15, 30, 60, 120, and 180 minutes, respectively, and 10 μl of each solution was spot-inoculated onto LB medium. After 18 hours of incubation at 37°C, single colonies of the microorganisms were counted. The experiment was repeated three times, and the average was calculated.

[0039] As shown in the results, at 1×MIC (4 μM), polypeptide Z4 completely killed P. aeruginosa and S. aureus within 60 and 360 minutes, respectively (Figure 5A-B and Tables 2-3). At 2×MIC (8 μM), polypeptide Z4 completely killed P. aeruginosa and S. aureus within 30 and 360 minutes, respectively (Figure 5C-D and Tables 4-5). These results indicate that, compared with gentamicin, polypeptide Z4 has a faster killing rate against P. aeruginosa, but no significant difference was observed in the killing rate against S. aureus.

[0040] [Table 2]

[0041] [Table 3]

[0042] [Table 4]

[0043] [Table 5]

[0044] B. To examine the antibiofilm activity of polypeptide Z4, P. aeruginosa and S. aureus were cultured overnight in tryptone soy broth medium at 37°C until they reached the logarithmic phase, and then 1 × 10 7 CFU / mL and 1 × 10 6 The bacteria were diluted to CFU / mL and mixed in equal amounts to prepare a mixed bacterial solution. 100 μL of the mixed bacterial solution was then incubated at 37°C for 24 hours to obtain a mixed biofilm. Antimicrobial peptides were then added to a 96-well plate at final concentrations of 2–32 μM. After incubation at 37°C for 3 hours, the medium was discarded and the 96-well plate was washed three times with PBS (pH 7.2). After drying at room temperature, 200 μL of 0.1% crystal violet staining solution was added to the plate at 200 μL / well. After incubation at 37°C for 30 minutes, the plate was washed three times with PBS. After air-drying at room temperature, 200 μL of 95% ethanol was added to the plate at 200 μL / well, and the plate was incubated at 37°C for 30 minutes. The absorbance was measured at 595 nm. A total of three experiments were performed, with each experiment repeated three times.

[0045] As shown in Figure 6 and Table 6, polypeptide Z4 was able to remove more than 50% of biofilms at a concentration of 8 μM, which is significantly better than the 32 μM required for gentamicin. This indicates that polypeptide Z4 has good direct antibiofilm activity.

[0046] [Table 6]

[0047] Example 5 Measurement of the in vivo antibacterial activity of polypeptide Z4 As shown in Figure 7, the recovery of skin tissue in the Z4 polypeptide-treated group was similar to that of the uninfected control catheter (MOCK), whereas mice in the control group (treated with PBS only) showed abundant pus and tissue damage consistent with ongoing infection (Figure 7B). Furthermore, Z4 treatment significantly reduced the number of P. aeruginosa and S. aureus colonizing the catheter (Figure 7C and Table 7). Z4 treatment also reduced IL-6 levels in the skin tissue (Figure 7D and Table 8) and the number of infiltrating inflammatory cells (Table 9, and black arrows in Figures 7E and 7F). Collectively, these data demonstrate that polypeptide Z4 kills bacteria within biofilms and reduces inflammatory responses in mice.

[0048] [Table 7]

[0049] [Table 8]

[0050] [Table 9]

[0051] Example 6 Measurement of the bacteriostatic and antiprotease hydrolytic activity of the dextrorotatory isomer (Z4-D) of polypeptide Z4 A. Polypeptide Z4, which consists only of dextrorotatory amino acids, is called the dextrorotatory isomer of polypeptide Z4. For use, polypeptide Z4-D was prepared into a fixed stock solution, and serial dilutions of the antimicrobial peptide solution were prepared by two-fold dilution using 0.01% acetic acid (containing 0.2% BSA) as the diluent. B. 100 μL of the above antimicrobial peptide stock solution was taken and added to a 96-well cell culture plate, and then an equal volume of test bacterial solution (approximately 10 5Each sample was added at a concentration of 1000 cells / mL. A positive control (containing bacterial solution but no antimicrobial peptide) and a negative control (containing neither bacterial solution nor peptide) were also included. The tested bacterial species included Pseudomonas aeruginosa (Pseudomonas aeruginosa PL1, Pseudomonas aeruginosa SP1), Staphylococcus aureus (Staphylococcus aureus 544, Staphylococcus aureus 103), Escherichia coli (Escherichia coli 729, Escherichia coli 112), Klebsiella pneumoniae (Klebsiella pneumoniae 106, Klebsiella pneumoniae 727), Streptococcus pneumoniae 383, and Acinetobacter baumannii (Acinetobacter baumannii 946, Acinetobacter baumannii 780). C. The culture plate was incubated at 37°C for 20 hours. The minimum inhibitory concentration was determined when no turbidity was observed with the naked eye at the bottom of the well.

[0052] The results in Table 10 indicate that Z4-D is similar to the polypeptide Z4 consisting only of levorotatory amino acids and has good broad-spectrum antibacterial activity.

[0053] [Table 10]

[0054] Example 7 Measurement of antiprotease hydrolysis ability of dextrorotatory isomer (Z4-D) of polypeptide Z4 Polypeptides Z4 and Z4-D were sonicated for 30 minutes and then allowed to stand at room temperature for 2 hours to promote self-assembly. Proteinase K (0.2 μg / mL) was then added to the polypeptide solution (640 μM final concentration) and incubated at 37°C for 0, 30, 60, 90, or 120 minutes, respectively. The MICs of the polypeptides against a mixed bacterial solution of P. aeruginosa PAO1 and S. aureus SP1 under different treatment conditions were then measured according to the method described in Example 2.

[0055] As a result, as shown in Figure 8, the MIC of polypeptide Z4-D remained unchanged throughout protease K treatment from 0 to 120 minutes, remaining at 4 μM. The MIC of polypeptide Z4 increased from 4 μM to 8 μM at 90 minutes, indicating that the antibacterial activity of polypeptide Z4 was reduced by 50% due to hydrolysis by protease K, whereas polypeptide Z4-D exhibited the ability to resist hydrolysis by protease K. This suggests that polypeptide Z4-D has a longer half-life in vivo than polypeptide Z4 and therefore has long-lasting bacteriostatic activity.

[0056] The above description of the specific embodiments is only used to help understand the core idea of ​​the present invention. Those skilled in the art may further make some improvements and refinements to the technical concept of the present invention without departing from the principles of the present invention, and it should be noted that these improvements and refinements also fall within the scope of the claims of the present invention.

Claims

1. A short-chain antimicrobial peptide rich in tryptophan and possessing self-assembling activity.

2. The antimicrobial peptide of claim 1, which is a centrosymmetric 9-peptide (which may contain either levorotatory or dextrorotatory amino acids), preferably consisting of tryptophan (W) and positively charged amino acids, and the number of positively charged amino acids is 3 to 6, for example 4 or 6.

3. The antimicrobial peptide of claim 2, wherein the positively charged amino acid is selected from arginine (R) and lysine (K).

4. The antimicrobial peptide according to claim 1, which has a primary symmetric structure with a triple tryptophan (WWW) at its center or with a tryptophan at its center and double tryptophan (WW) at both ends.

5. The antimicrobial peptide of claim 1, which is a broad-spectrum antimicrobial peptide.

6. The antimicrobial peptide according to claim 1 or 2, comprising a single tryptophan (W) motif, a double tryptophan motif (WW), a triple tryptophan motif (WWW) or a combination thereof, which are evenly spaced by positively charged amino acids (+), preferably comprising double tryptophan motifs (WW) spaced at both ends.

7. 7. The antimicrobial peptide of claim 6, selected from the following general formulas: W+++W+++W, +W++W++W+, ++W+W+W++; WW+++++WW, WW+W+W+WW, +WW+++WW+, ++WW+WW++, WW++W++WW, +WW+W+WW+; +++WWW+++, +W+WWW+W+, WWW+W+WWW, WWW+++WWW, WW+WWW+WW, +WWW+WWW+; Here, "W" represents tryptophan, and "+" represents a positively charged amino acid, such as arginine (R) or lysine (K), and preferably, "+" in the same sequence are the same positively charged amino acid.

8. The antimicrobial peptide according to any one of claims 1 to 7, selected from any one of SEQ ID NOs: 1 to 4 and variants thereof containing dextrorotatory amino acids, preferably SEQ ID NO: 4 (Z4) or Z4-D.

9. A pharmaceutical composition comprising the antimicrobial peptide according to any one of claims 1 to 8.

10. Use of the antimicrobial peptide according to any one of claims 1 to 8 for the manufacture of an antimicrobial mixed infection medicament.

11. 11. The use according to claim 10, wherein the antimicrobial mixed infection is an anti-Pseudomonas aeruginosa-Staphylococcus aureus mixed infection.

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