Modified antibacterial peptides

Abstract

The invention provides a modified antibacterial peptide, and belongs to the technical field of antibacterial peptides. The invention provides a modified antibacterial peptide. The modified antibacterial peptide comprises at least one of a first antibacterial peptide, a second antibacterial peptide, a third antibacterial peptide, a fourth antibacterial peptide, a fifth antibacterial peptide and a sixth antibacterial peptide, the amino acid sequences of the first antibacterial peptide, the second antibacterial peptide, the third antibacterial peptide, the fourth antibacterial peptide, the fifth antibacterial peptide and the sixth antibacterial peptide are shown as SEQ ID NO.1-SEQ ID NO.6 in sequence. On the basis of prevotelin-2, the modified antibacterial peptide is obtained by reducing negative charge amino acid, increasing positive charge amino acid, introducing hydrophobic amino acid, adjusting or deleting an amino acid sequence and optimizing a polypeptide structure. In-vitro minimum inhibitory concentration determination results show that compared with an initial peptide, the modified antibacterial peptide has significantly enhanced antibacterial activity on bacteria and fungi, and also has an obvious bactericidal effect on clinical drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and in particular relates to modified antimicrobial peptides. Background Art

[0002] Prevotellin-2 is a candidate antibiotic, but its MIC against pathogenic bacteria is high and its antibacterial effect is weak. Summary of the Invention

[0003] The object of the present invention is to provide a modified antimicrobial peptide, wherein the antimicrobial activity of the modified antimicrobial peptide is significantly higher than that of prevotellin-2.

[0004] The present invention provides a modified antimicrobial peptide, comprising at least one of a first antimicrobial peptide, a second antimicrobial peptide, a third antimicrobial peptide, a fourth antimicrobial peptide, a fifth antimicrobial peptide, and a sixth antimicrobial peptide; the amino acid sequences of the first antimicrobial peptide, the second antimicrobial peptide, the third antimicrobial peptide, the fourth antimicrobial peptide, the fifth antimicrobial peptide, and the sixth antimicrobial peptide are shown in SEQ ID NO.1 to SEQ ID NO.6, respectively.

[0005] Preferably, it comprises at least one of the first antimicrobial peptide, the second antimicrobial peptide, the third antimicrobial peptide, the fourth antimicrobial peptide and the fifth antimicrobial peptide.

[0006] Preferably, it comprises a first antimicrobial peptide and / or a second antimicrobial peptide.

[0007] The present invention also provides the use of the modified antimicrobial peptide described in the above scheme in the preparation of drugs for resisting pathogenic bacteria infection.

[0008] Preferably, the pathogenic bacteria include bacteria and / or fungi.

[0009] Preferably, the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria include Staphylococcus aureus and / or Bacillus subtilis; the Gram-negative bacteria include at least one of Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa.

[0010] Preferably, the fungus includes at least one of Cryptococcus neoformans, Candida auris, Aspergillus fumigatus and Candida albicans.

[0011] Preferably, the pathogenic bacteria include clinical drug-resistant bacteria.

[0012] Preferably, the clinical drug-resistant bacteria include methicillin-resistant Staphylococcus aureus.

[0013] The present invention also provides a drug for resisting pathogenic bacteria infection, comprising the modified antimicrobial peptide described in the above scheme and pharmaceutically acceptable excipients.

[0014] The present invention provides modified antimicrobial peptides, comprising at least one of a first antimicrobial peptide, a second antimicrobial peptide, a third antimicrobial peptide, a fourth antimicrobial peptide, a fifth antimicrobial peptide, and a sixth antimicrobial peptide; the amino acid sequences of the first, second, third, fourth, fifth, and sixth antimicrobial peptides are shown in SEQ ID NOs. 1 to 6, respectively. Based on prevotellin-2, the modified antimicrobial peptides were obtained by reducing negatively charged amino acids, increasing positively charged amino acids, introducing hydrophobic amino acids, adjusting or deleting the amino acid sequence, and optimizing the polypeptide structure. In vitro minimum inhibitory concentration assays demonstrated that the modified antimicrobial peptides exhibited significantly enhanced antimicrobial activity against bacteria and fungi compared to prevotellin-2, and also exhibited significant bactericidal activity against clinically drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The antibacterial results of different peptides and ampicillin on E.coliATCC 8739 were obtained by disk method.

[0017] Figure 2 The antibacterial results of different peptides and ampicillin on B. subtilis ATCC6633 were obtained by disk method.

[0018] Figure 3 The antibacterial results of different peptides and ampicillin on P. aeruginosa ATCC 27853 were obtained by the disc method.

[0019] Figure 4 The antibacterial results of different peptides and ampicillin on S. aureus ATCC 6538 were obtained by the disc method.

[0020] Figure 5 The antibacterial results of the antimicrobial peptides on the standard strain of Cryptococcus neoformans ATCC 32045 were obtained by the disc method;

[0021] Figure 6 The antibacterial results of the antimicrobial peptides against the standard strain of Cryptococcus neoformans BNCC 225501 were obtained by the disc method.

[0022] Figure 7 The antibacterial results of the antimicrobial peptides against the standard strain of Cryptococcus neoformans BNCC 25033101 were obtained by the disc method;

[0023] Figure 8 The antibacterial results of the antimicrobial peptides against the standard strain Candida albicans BNCC 186382 were obtained by the disc method. DETAILED DESCRIPTION

[0024] The present invention provides a modified antimicrobial peptide, comprising at least one of a first antimicrobial peptide, a second antimicrobial peptide, a third antimicrobial peptide, a fourth antimicrobial peptide, a fifth antimicrobial peptide, and a sixth antimicrobial peptide; the amino acid sequences of the first antimicrobial peptide, the second antimicrobial peptide, the third antimicrobial peptide, the fourth antimicrobial peptide, the fifth antimicrobial peptide, and the sixth antimicrobial peptide are shown in SEQ ID NO.1 to SEQ ID NO.6, respectively.

[0025] The modified antimicrobial peptides of the present invention are derived from prevotellin-2 by reducing negatively charged amino acids, increasing positively charged amino acids, introducing hydrophobic amino acids, adjusting or deleting the amino acid sequence, and optimizing the polypeptide structure. Compared to prevotellin-2, the antimicrobial peptides of the present invention exhibit significantly enhanced antimicrobial activity against bacteria and fungi, and also demonstrate significant bactericidal activity against clinically drug-resistant bacteria. The amino acid sequence of prevotellin-2 is shown in SEQ ID NO. 7: MLNYLYDRDINRYRAIIKALGLRK-NH2.

[0026] In the present invention, the amino acid sequence of the first antimicrobial peptide is shown in SEQ ID NO. 1, specifically: RLKKLYRRWWRGYRGWWKGLG-NH2, and is named: Prevotellin-2f.

[0027] In the present invention, the amino acid sequence of the second antimicrobial peptide is shown in SEQ ID NO. 2, specifically: MLKYLYRRWWRRYRGWWKGLGLRK-NH2, and is named: Prevotellin-2e.

[0028] In the present invention, the amino acid sequence of the third antimicrobial peptide is shown in SEQ ID NO. 3, specifically: MLKYLYRRWIRRYRGIIKGLGLRK-NH2, and is named: Prevotellin-2d.

[0029] In the present invention, the amino acid sequence of the fourth antimicrobial peptide is shown in SEQ ID NO. 4, specifically: MLKYLYRRWIRRYRAIIKALGLRK-NH2, and is named: Prevotellin-2c.

[0030] In the present invention, the amino acid sequence of the fifth antimicrobial peptide is shown in SEQ ID NO. 5, specifically: MLNYLYRRWINRYRAIIKALGLRK-NH2, named: Prevotellin-2b.

[0031] In the present invention, the amino acid sequence of the sixth antimicrobial peptide is shown in SEQ ID NO. 6, specifically: MLNYLYRRDINRYRAIIKALGLRK-NH2, and is named: Prevotellin-2a.

[0032] As an embodiment, the modified antimicrobial peptide includes at least one of a first antimicrobial peptide, a second antimicrobial peptide, a third antimicrobial peptide, a fourth antimicrobial peptide and a fifth antimicrobial peptide.

[0033] In one embodiment, the modified antimicrobial peptide includes a first antimicrobial peptide and / or a second antimicrobial peptide.

[0034] As an embodiment, the modified antimicrobial peptide is synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0035] The present invention also provides the use of the modified antimicrobial peptide described in the above scheme in the preparation of drugs for resisting pathogenic bacteria infection.

[0036] As an embodiment, the pathogenic bacteria include bacteria and / or fungi.

[0037] As an embodiment, the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria include Staphylococcus aureus (S. aureus) and / or Bacillus subtilis (B. subtilis); the Gram-negative bacteria include at least one of Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa).

[0038] As an embodiment, the Escherichia coli includes the standard strain of Escherichia coli ATCC 8739; the Staphylococcus aureus includes Staphylococcus aureus ATCC 6538; the Acinetobacter baumannii includes Acinetobacter baumannii ATCC 1968; the Bacillus subtilis includes Bacillus subtilis ATCC 6633; and the Pseudomonas aeruginosa includes Pseudomonas aeruginosa ATCC 27853.

[0039] In one embodiment, the fungus includes at least one of Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans.

[0040] As an embodiment, the Cryptococcus neoformans includes at least one of Cryptococcus neoformans ATCC 32045, Cryptococcus neoformans BNCC 225501 and Cryptococcus neoformans BNCC 25033101; the Candida auris includes at least one of Candida auris BNCC356255, Candida auris BNCC 357785 and Candida auris BNCC 357784; the Aspergillus fumigatus includes Aspergillus fumigatus BNCC 187486 and / or Aspergillus fumigatus BNCC 340016; the Candida albicans includes Candida albicans BNCC 299343 and / or Candida albicans BNCC 186382, further being Candida albicans BNCC 186382.

[0041] As an embodiment, the pathogenic bacteria include clinical drug-resistant bacteria.

[0042] As an embodiment, the clinical drug-resistant bacteria include methicillin-resistant Staphylococcus aureus.

[0043] As an embodiment, the methicillin-resistant Staphylococcus aureus includes at least one of methicillin-resistant Staphylococcus aureus (MRSA, ATCC 443300), methicillin-resistant Staphylococcus aureus (MRSA-Z), methicillin-resistant Staphylococcus aureus (MRSA 639SA1) and methicillin-resistant Staphylococcus aureus (MRSA639SA2); the methicillin-resistant Staphylococcus aureus (MRSA, ATCC 443300) and methicillin-resistant Staphylococcus aureus (MRSA-Z) are from the Kunming Institute of Zoology, Chinese Academy of Sciences; the methicillin-resistant Staphylococcus aureus (MRSA639SA1) and methicillin-resistant Staphylococcus aureus (MRSA639SA2) are from Kunming Medical University.

[0044] The present invention also provides a drug for resisting pathogenic bacteria infection, comprising the modified antimicrobial peptide described in the above scheme and pharmaceutically acceptable excipients.

[0045] To further illustrate the present invention, the modified antimicrobial peptide provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] The strains and culture conditions used in the following examples and comparative examples are:

[0047] Standard strains of Escherichia coli (E. coli, ATCC 8739), Staphylococcus aureus (S. aureus, ATCC 6538), Acinetobacter baumannii (A. baumannii, ATCC 1968), Bacillus subtilis (B. subtilis, ATCC 6633), Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853), and different strains of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, and Pseudomonas aeruginosa were cultured in Luria-Bertani (LB) medium with shaking at 37°C.

[0048] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 443300) and methicillin-resistant Staphylococcus aureus (MRSA-Z) were obtained from the Kunming Institute of Zoology, Chinese Academy of Sciences. Methicillin-resistant Staphylococcus aureus clinical strains (MRSA639SA1 and MRSA 639SA2) were obtained from Kunming Medical University. The different strains were cultured in Luria-Bertani (LB) medium at 37°C with shaking.

[0049] Cryptococcus neoformans ATCC 32045, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans BNCC 25033101, Candida auris BNCC 356255, Candida auris BNCC 357785, Candida auris BNCC 357784, Aspergillus fumigatus BNCC 187486, Aspergillus fumigatus BNCC 340016, Candida albicans BNCC 299343, Candida albicans BNCC 186382. Aspergillus fumigatus was cultured in Sabouraud Dextrose Broth (SDB) solid medium at 30°C, and other different strains were cultured in Sabouraud Dextrose Broth (SDB) medium at 30°C with shaking for 24 to 48 hours.

[0050] Example 1

[0051] 1. Antimicrobial peptides used

[0052] Prevotellin-2f:RLKKLYRRWWRGYRGWWKGLG-NH2.

[0053] Prevotellin-2e:MLKYLYRRWWRRYRGWWKGLGLRK-NH2.

[0054] Prevotellin-2d:MLKYLYRRWIRRYRGIIKGLGLRK-NH2.

[0055] Prevotellin-2c:MLKYLYRRWIRRYRAIIKALGLRK-NH2.

[0056] Prevotellin-2b:MLNYLYRRWINRYRAIIKALGLRK-NH2.

[0057] Prevotellin-2a:MLNYLYRRDINRYRAIIKALGLRK-NH2.

[0058] The initial peptide Prevotellin-2: MLNYLYDRDINRYRAIIKALGLRK-NH2 was used as a positive control.

[0059] The above antimicrobial peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0060] The concentration of antimicrobial peptides such as prevotellin-2 was 2 mM.

[0061] Positive controls were set up as follows: 2 mM ampicillin, 2 mM vancomycin, and 2 mM colistin. Normal saline (NC, sterile 0.9% NaCl) was used as a negative control.

[0062] 2. Bacterial inhibition zone analysis

[0063] The inhibition zone of standard strains of bacteria (E. coli ATCC 8739, B. subtilis ATCC 6633, P. aeruginosa ATCC 27853, and S. aureus ATCC 6538) was determined as follows:

[0064] Activate the bacteria stored in the laboratory, inoculate the bacteria into Luria-Bertani (LB) solid medium, and invert the culture in a 37°C constant temperature incubator. After the colony grows, pick a single colony with an inoculation loop and inoculate it into fresh liquid LB medium. Place it in a constant temperature incubator at 37°C and 180 rpm / min and shake it. After 3 to 5 hours, the bacteria will reach the logarithmic growth phase. 600 =1≈1×10 9 CFU / mL, dilute the above bacterial solution with normal saline to 2×10 8 CFU / mL. Evenly spread 50 μl of the bacterial solution onto LB solid medium using a spreading rod or glass beads. Place a sterilized, 0.5 cm diameter circular filter paper on the surface of the medium. Then, drop 5 μl of the sample at a 2 mM concentration onto the filter paper. Incubate inverted in a 37°C incubator for 16-24 hours and observe for the formation of an inhibition zone. The formation of an inhibition zone indicates that the sample has antibacterial activity, and the size of the inhibition zone provides a preliminary indication of the strength of the antibacterial activity.

[0065] When the inhibition zones of E. coli ATCC 8739 and B. subtilis ATCC 6633 were measured, ampicillin was used as a positive control and normal saline (NC, sterile 0.9% NaCl) was used as a negative control.

[0066] When the inhibition zone of P. aeruginosa ATCC 27853 was measured, colistin was used as a positive control, and normal saline (NC, sterile 0.9% NaCl) was used as a negative control.

[0067] When the inhibition zone of S. aureus ATCC 6538 was measured, vancomycin was used as a positive control, and normal saline (NC, sterile 0.9% NaCl) was used as a negative control.

[0068] Results see Figures 1 to 4 The antibacterial activity of prevotellin-2 and its derivative peptides against three Gram-negative bacteria and two Gram-positive bacteria was tested using an inhibition zone assay. The results showed that prevotellin-2 had no significant inhibition zone, while the modified antimicrobial peptides prevotellin-2d, prevotellin-2e, and prevotellin-2f all exhibited significant inhibition zones against the aforementioned bacteria, consistent with the minimum inhibitory concentration (MIC) results.

[0069] Example 2

[0070] 1. Antimicrobial peptides used

[0071] Prevotellin-2f:RLKKLYRRWWRGYRGWWKGLG-NH2.

[0072] Prevotellin-2e:MLKYLYRRWWRRYRGWWKGLGLRK-NH2.

[0073] Prevotellin-2d:MLKYLYRRWIRRYRGIIKGLGLRK-NH2.

[0074] The initial peptide Prevotellin-2 was used as a positive control.

[0075] The above antimicrobial peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0076] 2. Fungal inhibition zone analysis

[0077] The inhibition zone of standard fungal strains (Cryptococcus neoformans ATCC 32045, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans BNCC 25033101, and Candida albicans BNCC186382) was determined as follows:

[0078] To activate the fungi stored in the laboratory, inoculate the fungi on Sabouraud Dextrose Broth (SDB) solid medium and invert the culture in a 30°C constant temperature incubator. After the colonies grow, pick a single colony with an inoculation loop and inoculate it into fresh liquid SDB medium. Place it in a constant temperature incubator at 30°C and 180 rpm / min and shake it until the fungi reach the logarithmic growth phase after 3 to 5 hours. 600 =1≈1×10 7 CFU / mL, dilute the above bacterial solution with normal saline to 2×10 6 CFU / mL. Take 50μl of the above bacterial solution and evenly spread it on the SDB solid culture medium with a coating rod or glass beads, and stick a sterilized circular filter paper with a diameter of 0.5cm on the surface of the culture medium. Then take 5μl of the sample with a concentration of 2mM and drop it on the filter paper. Incubate it upside down in a constant temperature incubator at 30℃ for 16-24h to observe whether there is an inhibition zone. If an inhibition zone is formed, it means that the test sample has antibacterial activity. The size of the inhibition zone can preliminarily indicate the strength of the antibacterial activity. For results, see Figures 5 to 8The antibacterial activity of prevotellin-2 and its derivative peptides (prevotellin-2d, prevotellin-2e, and prevotellin-2f) against two fungi (Cryptococcus neoformans and Candida albicans) was tested by inhibition zone assay. It was found that preoteollin-2d, preoteollin-2e, and preoteollin-2f had a good antibacterial effect against Cryptococcus neoformans (BNCC 225501), Cryptococcus neoformans (BNCC 25033101), and Candida albicans (BNCC 186382).

[0079] Example 3 Determination of Minimum Inhibitory Concentration (MIC) of Bacteria

[0080] The antimicrobial peptides used were the same as in Example 1 and were diluted in a 96-well plate to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, 0.1953, and 0.0977 μM. Additionally, ampicillin, colistin, and vancomycin were used as positive controls and diluted in a similar 96-well plate to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, 0.1953, and 0.0977 μM.

[0081] Bacterial standard strains: E. coli ATCC 8739, B. subtilis ATCC 6633, P. aeruginosa ATCC 27853, S. aureus ATCC 6538 and A. baumanni, ATCC 1968.

[0082] The steps are as follows: According to the Clinical and Laboratory Standards Institute (CLSI) protocol, the minimum inhibitory concentration (MIC) is the lowest drug concentration at which an antibiotic inhibits the growth and reproduction of a bacterial strain. Different bacterial strains were grown in Luria-Bertani (LB) medium at 37°C until they entered the exponential growth phase. The OD values were calculated. 600 =1≈1×10 9 CFU / mL, dilute the above bacterial solution with normal saline to 2×10 5CFU / mL. 100 μL of saline was added to a sterile 96-well plate in advance, and 200 μL of the sample to be tested was added to the first column of wells. The sample to be tested was diluted two-fold in a series to a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, 0.1953, and 0.0977 μM in the 96-well plate. 100 μL of a concentration of 2×10 5 Bacterial suspensions with a concentration of 100 CFU / mL were incubated at 37°C for various periods of time. The absorbance of the suspension at 600 nm was measured using a microplate reader. The average concentration of samples in wells where no microbial growth was detected and adjacent wells was used as the MIC. The inhibitory activity of the antimicrobial peptides against various standard strains was determined. The results are shown in Table 1.

[0083] Table 1 MIC (μM) of different antimicrobial peptides against different bacteria

[0084]

[0085] As can be seen from the data in Table 1, the MIC of Prevotellin-2 antimicrobial peptide against various strains is high and the antibacterial effect is weak. The antibacterial activity of the antimicrobial peptide obtained by structural modification of Prevotellin-2 is significantly improved. The structurally optimized Prevotellin-2e and Prevotellin-2f showed good antibacterial effects, among which the MIC of Prevotellin-2f against Escherichia coli was 0.7813μM, 0.3906μM against Staphylococcus aureus and Acinetobacter baumannii, respectively, 1.5625μM against Pseudomonas aeruginosa, and 0.7813μM against Bacillus subtilis. Moreover, compared with traditional antibiotics (such as ampicillin, polymyxin E and vancomycin), especially against Staphylococcus aureus and some Gram-negative bacteria, the optimized Prevotellin-2f showed lower MIC values and broader antibacterial activity.

[0086] Example 4 Determination of the Minimum Inhibitory Concentration (MIC) of Clinical Drug-Resistant Bacteria

[0087] Antimicrobial peptides used:

[0088] Prevotellin-2f:RLKKLYRRWWRGYRGWWKGLG-NH2.

[0089] Prevotellin-2e:MLKYLYRRWWRRYRGWWKGLGLRK-NH2.

[0090] Prevotellin-2d:MLKYLYRRWIRRYRGIIKGLGLRK-NH2.

[0091] Prevotellin-2c:MLKYLYRRWIRRYRAIIKALGLRK-NH2.

[0092] Prevotellin-2b:MLNYLYRRWINRYRAIIKALGLRK-NH2.

[0093] The above antimicrobial peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0094] In addition, ampicillin and vancomycin were used as positive controls.

[0095] Clinical drug-resistant bacteria: S.AureusATCC 6538, MRSAATCC 43300, MRSA-Z, MRSA639SA1 and MRSA639SA2.

[0096] The MICs of antimicrobial peptides against clinical drug-resistant bacteria were determined, and the results are shown in Table 2.

[0097] Table 2 MIC (μM) of different antimicrobial peptides against different Staphylococcus aureus

[0098]

[0099] The antimicrobial activity of Prevotellin-2 and its modified antimicrobial peptides against methicillin-resistant Staphylococcus aureus and clinically drug-resistant bacteria was determined. The results showed that the MIC values of Prevotellin-2b, 2c, and 2d were generally around 0.78 μM, while the MIC of Prevotellin-2e dropped to 0.39 μM against some MRSA strains (MRSA639SA1 and 639SA2). Notably, Prevotellin-2f exhibited extremely high bactericidal activity against various Staphylococcus aureus strains, with an MIC as low as 0.1953 μM against MRSA639SA1 and 639SA2. This activity was even stronger than that of the traditional antibiotics ampicillin and vancomycin, demonstrating that the modified antimicrobial peptides of this invention have the potential to surpass traditional antibiotics and be used to treat infections caused by clinically drug-resistant bacteria.

[0100] Example 5 Determination of Minimum Inhibitory Concentration (MIC) of Fungi

[0101] The antimicrobial peptides used were the same as those in Example 1 and were diluted in a 96-well plate to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, 0.1953, and 0.0977 μM. Amphotericin B and fluconazole were used as positive controls. Amphotericin B was diluted in a 96-well plate to concentrations of 6.25, 3.125, 0.7815, 0.3906, 0.1953, 0.0977, 0.0488, 0.0244, and 0.0122 μM, and fluconazole was diluted to concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, and 0.1953 μM.

[0102] Strains: Cryptococcus neoformans ATCC 32045, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans BNCC 25033101, Candida auris BNCC 356255, Candida auris BNCC 357785, Candida auris BNCC 357784, Aspergillus fumigatus BNCC 187486, Aspergillus fumigatus BNCC 340016, Candida albicans BNCC 299343, Candida albicans BNCC 186382).

[0103] According to the Clinical and Laboratory Standards Institute (CLSI) protocol, the minimum inhibitory concentration (MIC) is the lowest drug concentration at which an antibiotic inhibits the growth and reproduction of a bacterial strain. C. neoformans, C. auris, and C. albicans strains were grown in their respective culture media at 30°C until they entered the exponential growth phase. The OD values were calculated. 600 =1≈1×10 7 CFU / mL, dilute the above bacterial solution with normal saline to 2×10 4CFU / mL. 100 μL of saline solution was added to a sterile 96-well plate in advance, and 200 μL of the sample to be tested was added to the first column of wells. 100 μL of saline solution was added to a sterile 96-well plate in advance, and 200 μL of the sample to be tested was added to the first column of wells. The sample to be tested was diluted two-fold in a 96-well plate to a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, 0.1953, and 0.0977 μM. 100 μL of a 2×10 4 CFU / mL of bacterial solution was incubated at 37°C for different periods of time. The absorbance of the bacterial solution at 600 nm was measured using a microplate reader. The average concentration of the sample in the well where no microbial growth was detected and the adjacent wells was used as the MIC value.

[0104] Prepare a spore suspension of different strains of Aspergillus fumigatus for MIC determination: In a sterile laboratory, add 5-10 mL of sterile PBS + 0.05% Tween-80 to the surface of the colony. Gently scrape the spores using a sterile glass rod or L-shaped spreader, avoiding any contamination with mycelium. Transfer the spore suspension to a sterile centrifuge tube and centrifuge at 3000 rpm for 5 minutes. Remove the supernatant to obtain the spore suspension. Follow the same steps as above.

[0105] Finally, the inhibitory activities of the above antimicrobial peptides against standard strains of different fungi were determined. The results are shown in Tables 3 to 6.

[0106] Table 3 MIC (μM) of different antimicrobial peptides against different Candida albicans strains

[0107] Petides Candida albicans BNCC299343 Candida albicans BNCC186382 prevotellin-2 ＞100 ＞100 prevotellin-2a 100 50 prevotellin-2b 25 25 prevotellin-2c 25 12.5 Prevotellin-2d 50 25 prevotellin-2e 12.5 6.25 prevotellin-2f 6.25 3.125 Amphotericin B 0.1953 0.1953 Fluconazole 1.95 3.9

[0108] The results showed that the MIC values of prevotellin-2 against both strains exceeded 100 μM, indicating weak antibacterial activity. In contrast, the MIC values of prevotellin-2a and prevotellin-2b were 100 μM and 25 μM, respectively, indicating moderate antibacterial effects. In particular, prevotellin-2c, prevotellin-2e, and prevotellin-2f showed strong antibacterial activity against both strains, with MIC values of 25 μM, 12.5 μM, 6.25 μM, and 50 μM, 25 μM, 6.25 μM, and 3.125 μM, respectively. These results indicate that certain prevotellin derivatives have antibacterial activities close to or even superior to those of traditional antifungal drugs, such as amphotericin B (MIC value of 0.1953 μM) and fluconazole (MIC values of 1.95 μM and 3.9 μM).

[0109] Table 4 MIC (μM) of different antimicrobial peptides against different Aspergillus fumigatus

[0110]

[0111] The results showed that the MIC values of prevotellin-2, prevotellin-2a, and prevotellin-2d against both strains exceeded 50 μM, indicating that these peptides have limited antibacterial activity against Aspergillus fumigatus. In contrast, prevotellin-2b had an MIC of 50 μM against BNCC340016, showing some antibacterial activity. Prevotellin-2c had an MIC of 50 μM against both strains, while prevotellin-2e and prevotellin-2f exhibited stronger antibacterial activity, with MIC values of 12.5 μM / 6.25 μM and 50 μM / 25 μM against BNCC 187486 and BNCC 340016, respectively.

[0112] As a control, the traditional antifungal drug amphotericin B had an MIC value of 0.09 μM against both strains, showing extremely high antibacterial activity. However, fluconazole had an MIC value of over 50 μM against both strains, indicating poor antibacterial activity against A. fumigatus. These results indicate that although some prevotellin derivatives have limited antibacterial activity against A. fumigatus, prevotellin-2e and prevotellin-2f exhibit strong antibacterial potential.

[0113] Table 5 MIC (μM) of different antimicrobial peptides against different Candida auris

[0114]

[0115] The results showed that the MIC values of prevotellin-2, prevotellin-2a, prevotellin-2b, prevotellin-2c, and prevotellin-2d against BNCC 356255 and BNCC 357784 were 25 μM, respectively, while the MIC value against BNCC 357785 was greater than 100 μM, indicating that they had good inhibitory effects on the first two strains, but the inhibitory effect on the third strain was weak. In particular, prevotellin-2e and prevotellin-2f showed lower MIC values of 25 μM, 50 μM, 25 μM and 25 μM, 12.5 μM, and 12.5 μM, respectively, indicating that these two peptides have strong antibacterial activity against all tested C. auris strains.

[0116] In contrast, the traditional antifungal drug amphotericin B had an MIC value of 0.09 μM against all tested strains, demonstrating extremely high antibacterial activity. However, fluconazole had MIC values greater than 100 μM against all tested strains, indicating poor antibacterial activity against C. auris. These results are consistent with previous studies showing that C. auris is highly resistant to azole and polyene antifungal drugs, suggesting that the antimicrobial peptides prevotellin-2e and prevotellin-2f exhibit potent antibacterial potential.

[0117] Table 6 MIC (μM) of different antimicrobial peptides against different Cryptococcus neoformans

[0118]

[0119] The results showed that the MIC values of prevotellin-2 and prevotellin-2a against all tested strains were greater than 50 μM, indicating weak antibacterial activity. In contrast, prevotellin-2b, prevotellin-2c, prevotellin-2d, prevotellin-2e, and prevotellin-2f exhibited significant antibacterial activity, especially prevotellin-2f, with MIC values of 1.5625 μM, 0.78125 μM, and 0.78125 μM against the tested strains, respectively, demonstrating a very strong antibacterial effect.

[0120] The traditional antifungal drug amphotericin B exhibited a high antibacterial activity with an MIC of 0.02 μM against all tested strains. Fluconazole exhibited moderate antibacterial activity with an MIC of 25 μM against all tested strains. These results suggest that although some prevotellin derivatives have limited antibacterial activity against Cryptococcus neoformans, prevotellin-2f exhibits strong antibacterial potential and warrants further research and development.

[0121] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A modified antimicrobial peptide, characterized in that: The invention comprises at least one of a first antimicrobial peptide, a second antimicrobial peptide, a third antimicrobial peptide, a fourth antimicrobial peptide, a fifth antimicrobial peptide and a sixth antimicrobial peptide; the amino acid sequences of the first antimicrobial peptide, the second antimicrobial peptide, the third antimicrobial peptide, the fourth antimicrobial peptide, the fifth antimicrobial peptide and the sixth antimicrobial peptide are shown in SEQ ID NO.1 to SEQ ID NO.6, respectively.

2. The modified antimicrobial peptide according to claim 1, characterized in that The invention comprises at least one of a first antimicrobial peptide, a second antimicrobial peptide, a third antimicrobial peptide, a fourth antimicrobial peptide and a fifth antimicrobial peptide.

3. The modified antimicrobial peptide according to claim 1, characterized in that It includes a first antimicrobial peptide and / or a second antimicrobial peptide.

4. Use of the modified antimicrobial peptide according to any one of claims 1 to 3 in the preparation of drugs for resisting pathogenic bacterial infection.

5. The use according to claim 4, characterized in that The pathogenic bacteria include bacteria and / or fungi.

6. The use according to claim 5, characterized in that The bacteria include Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria include Staphylococcus aureus and / or Bacillus subtilis; the Gram-negative bacteria include at least one of Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa.

7. The use according to claim 5, characterized in that The fungus includes at least one of Cryptococcus neoformans, Candida auris, Aspergillus fumigatus and Candida albicans.

8. The use according to claim 4, characterized in that The pathogenic bacteria include clinical drug-resistant bacteria.

9. The use according to claim 8, characterized in that The clinical drug-resistant bacteria include methicillin-resistant Staphylococcus aureus.

10. A drug for resisting pathogenic bacteria infection, characterized in that: The invention comprises the modified antimicrobial peptide according to any one of claims 1 to 3 and pharmaceutically acceptable excipients.

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