Modified antimicrobial peptides

Abstract

This invention provides modified antimicrobial peptides, belonging to the field of antimicrobial peptide technology. The modified antimicrobial peptides include at least one of a first, second, third, fourth, fifth, and sixth antimicrobial peptide; the amino acid sequences of the first, second, third, fourth, fifth, and sixth antimicrobial peptides are shown sequentially as SEQ ID NO.1 to SEQ ID NO.6. 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 amino acid sequences, and optimizing the peptide structure. In vitro minimum inhibitory concentration (MIC) determination showed that, compared with the initial peptide, the modified antimicrobial peptides exhibited significantly enhanced antimicrobial activity against bacteria and fungi, and also showed significant bactericidal activity against clinically resistant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, and particularly relates to modified antimicrobial peptides. Background Technology

[0002] Prevotellin-2 is a candidate antibiotic. However, prevotellin-2 has a high MIC against pathogenic bacteria, indicating a weaker antibacterial effect. Summary of the Invention

[0003] The purpose of this invention is to provide a modified antimicrobial peptide, the modified antimicrobial peptide of this invention having significantly higher antimicrobial activity than prevotellin-2.

[0004] The present invention provides modified antimicrobial peptides, including 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 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.

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

[0007] The present invention also provides the application of the modified antimicrobial peptide described above in the preparation of drugs against pathogenic bacterial infections.

[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 clinically drug-resistant bacteria.

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

[0013] The present invention also provides a drug for combating pathogenic bacterial infections, comprising the modified antimicrobial peptide described in the above-described scheme and pharmaceutically acceptable excipients.

[0014] This invention provides modified antimicrobial peptides, including 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 sequentially as SEQ ID NO.1 to SEQ ID NO.6. 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 amino acid sequences, and optimizing the peptide structure. In vitro minimum inhibitory concentration (MIC) determinations showed that, compared to prevotellin-2, the modified antimicrobial peptides exhibited significantly enhanced antibacterial activity against bacteria and fungi, and also demonstrated significant bactericidal activity against clinically resistant bacteria. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The results of the paper disc method for inhibiting E. coli ATCC 8739 by different peptides and ampicillin;

[0017] Figure 2 The results of the paper disc assay for the antibacterial activity of different peptides and ampicillin against B. subtilis ATCC6633;

[0018] Figure 3 The results of the paper disc assay for the antibacterial activity of different peptides and ampicillin against P. aeruginosa ATCC 27853;

[0019] Figure 4 The results of the paper disc assay for the antibacterial activity of different peptides and ampicillin against S. aureus ATCC 6538;

[0020] Figure 5 The results of the antimicrobial peptide's inhibition against the standard strain Cryptococcus neoformans ATCC 32045 using the paper disc method;

[0021] Figure 6 The results of the antimicrobial peptide against the standard strain Cryptococcus neoformans BNCC 225501 using the paper disc method are shown.

[0022] Figure 7 The results of the antimicrobial peptide against the standard strain Cryptococcus neoformans BNCC 25033101 using the paper disc method are shown.

[0023] Figure 8 The results of the antimicrobial peptide's inhibition of the standard strain Candida albicans BNCC 186382 using the paper disc method. Detailed Implementation

[0024] The present invention provides modified antimicrobial peptides, including 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 peptide of this invention is obtained by optimizing the peptide structure based on prevotellin-2 by reducing negatively charged amino acids, increasing positively charged amino acids, introducing hydrophobic amino acids, and adjusting or deleting amino acid sequences. Compared with prevotellin-2, the antimicrobial peptide of this invention exhibits significantly enhanced antibacterial activity against bacteria and fungi, and also shows significant bactericidal activity against clinically resistant bacteria. The amino acid sequence of prevotellin-2 is shown in SEQ ID NO.7, specifically: MLNYLYDRDINRYRAIIKALGLRK-NH2.

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

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

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

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

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

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

[0032] In one 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 one implementation method, the modified antimicrobial peptide was synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0035] The present invention also provides the application of the modified antimicrobial peptide described above in the preparation of drugs against pathogenic bacterial infections.

[0036] In one implementation, the pathogenic bacteria include bacteria and / or fungi.

[0037] In one embodiment, the bacteria include Gram-positive 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.

[0038] In one embodiment, the *Escherichia coli* includes the standard strain *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] In one embodiment, the *Cryptococcus* species includes at least one of *Cryptococcus* ATCC 32045, *Cryptococcus* BNCC 225501, and *Cryptococcus* BNCC 25033101; the *Cryptococcus auris* species includes at least one of *Cryptococcus auris* BNCC356255, *Cryptococcus auris* BNCC 357785, and *Cryptococcus auris* BNCC 357784; the *Aspergillus fumigatus* species includes *Aspergillus fumigatus* BNCC 187486 and / or *Aspergillus fumigatus* BNCC 340016; the *Cryptococcus albus* species includes *Cryptococcus albus* BNCC 299343 and / or *Cryptococcus albus* BNCC 186382, further comprising *Cryptococcus albus* BNCC 186382.

[0041] In one implementation, the pathogenic bacteria include clinically drug-resistant bacteria.

[0042] As one implementation, the clinically resistant bacteria include methicillin-resistant Staphylococcus aureus.

[0043] In one embodiment, the methicillin-resistant Staphylococcus aureus (MRSA) 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; and 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 combating pathogenic bacterial infections, comprising the modified antimicrobial peptide described in the above-described scheme and pharmaceutically acceptable excipients.

[0045] To further illustrate the present invention, the modified antimicrobial peptides provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these 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 as follows:

[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. Clinical strains of methicillin-resistant Staphylococcus aureus (MRSA639SA1 and MRSA 639SA2) were obtained from Kunming Medical University. Different strains were cultured in Luria-Bertani (LB) medium with shaking at 37°C.

[0049] Cryptococcus neoformans ATCC 32045, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans BNCC25033101, Candida auris BNCC 356255, Candida auris BNCC357785, 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, while other different strains were cultured in Sabouraud Dextrose Broth (SDB) medium at 30°C with shaking for 24–48 h.

[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, served as a positive control.

[0059] The above-mentioned 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 E. Physiological saline (NC, sterile 0.9% NaCl) was used as a negative control.

[0062] 2. Analysis of bacterial inhibition zones

[0063] The inhibition zones of standard bacterial strains (E. coli ATCC 8739, B. subtilis ATCC 6633, P. aeruginosa ATCC 27853 and S. aureus ATCC 6538) were determined using the following steps:

[0064] To activate laboratory-preserved bacteria, inoculate them onto Luria-Bertani (LB) solid medium and incubate them upside down in a 37°C incubator. Once colonies have grown, use an inoculation loop to pick a single colony and inoculate it onto fresh liquid LB medium. Incubate this medium at 37°C and 180 rpm with shaking. After 3–5 hours, the bacteria reach the logarithmic growth phase. [The text then abruptly shifts to a different topic:] ...according to OD... 600 =1≈1×10 9 CFU / mL, dilute the above bacterial culture with physiological saline to 2×10⁻⁶. 8 CFU / mL. Take 50 μl of the above bacterial suspension and spread it evenly on LB solid medium using a spreader or glass beads. Attach a sterile, 0.5 cm diameter circular filter paper disc to the surface of the medium. Then, add 5 μl of the 2 mM test sample onto the filter paper disc. Incubate upside down in a 37°C incubator for 16–24 h and observe for the formation of inhibition zones. If inhibition zones form, it indicates that the test sample has antibacterial activity; the size of the inhibition zone can preliminarily indicate the strength of the antibacterial activity.

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

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

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

[0068] See results Figures 1-4 The antibacterial activity of prevotellin-2 and its derived peptides against three Gram-negative bacteria and two Gram-positive bacteria was detected by inhibition zone assay. The results showed that prevotellin-2 did not have a significant inhibition zone, while the modified antimicrobial peptides prevotellin-2d, prevotellin-2e, and prevotellin-2f all had significant inhibition zones against the above bacteria, consistent with the results of the minimum inhibitory concentration.

[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-mentioned antimicrobial peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0076] 2. Analysis of fungal inhibition zones

[0077] The inhibition zones of standard fungal strains (Cryptococcus neoformans ATCC 32045, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans BNCC 25033101, and Candida albicans BNCC186382) were determined using the following steps:

[0078] To activate laboratory-preserved fungi, inoculate them onto Sabouraud Dextrose Broth (SDB) solid medium and incubate them upside down in a 30°C incubator. Once colonies have grown, pick single colonies with an inoculation loop and inoculate them into fresh liquid SDB medium. Incubate at 30°C and 180 rpm with shaking for 3–5 hours until the fungi reach the logarithmic growth phase. (According to OD...) 600 =1≈1×10 7 CFU / mL, dilute the above bacterial culture with physiological saline to 2×10⁻⁶. 6 CFU / mL. Take 50 μl of the above bacterial suspension and spread it evenly on SDB solid medium using a spreader or glass beads. Attach a sterile, 0.5 cm diameter circular filter paper disc to the surface of the medium. Then, add 5 μl of the 2 mM test sample onto the filter paper disc. Incubate upside down in a 30℃ incubator for 16–24 h, observing for the formation of inhibition zones. The formation of inhibition zones indicates that the test sample has antibacterial activity; the size of the inhibition zone can preliminarily indicate the strength of the antibacterial activity. See results below. Figures 5-8The 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 detected by inhibition zone assay. Preoteollin-2d, preoteollin-2e, and preoteollin-2f showed good antibacterial effects against Cryptococcus neoformans (BNCC 225501), Cryptococcus neoformans (BNCC 25033101), and Candida albicans (BNCC 186382).

[0079] Example 3: Determination of the minimum inhibitory concentration (MIC) for bacteria

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

[0081] Standard bacterial strains: E. coli ATCC 8739, B. subtilis ATCC 6633, P. aeruginosa ATCC 27853, S. aureus ATCC 6538, and A. baumannii ATCC 1968.

[0082] The procedure is 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 strains were grown in Luria-Bertani (LB) medium at 37°C until they entered the exponential growth phase. [The text then abruptly shifts to a different topic:] ...according to OD... 600 =1≈1×10 9 CFU / mL, dilute the above bacterial culture with physiological saline to 2×10⁻⁶. 5CFU / mL. 100 μL of physiological saline was added to a sterile 96-well plate beforehand, and 200 μL of the test sample was added to the first row of wells. The test sample was then serially diluted twofold in the 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. 100 μL of a 2×10⁻⁶ CFU / mL solution was added to each well. 5 CFU / mL bacterial suspensions were cultured at 37°C for different times. The absorbance of the bacterial suspensions at 600 nm was measured using a microplate reader. The MIC value was defined as the concentration of the sample in wells where no microbial growth was detected and the average concentration of the sample in adjacent wells. The inhibitory activity of the above antimicrobial peptides against different standard strains was determined, and the results are shown in Table 1.

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

[0084]

[0085] As shown in Table 1, the prevotellin-2 antimicrobial peptides exhibited high MICs against various bacterial strains, indicating relatively weak antimicrobial activity. The antimicrobial activity of the prevotellin-2 antimicrobial peptides obtained through structural modification was significantly improved. The structurally optimized Prevotellin-2e and Prevotellin-2f showed better antimicrobial effects. Specifically, Prevotellin-2f had a MIC of 0.7813 μM against *Escherichia coli*, 0.3906 μM against *Staphylococcus aureus* and *Acinetobacter baumannii*, 1.5625 μM against *Pseudomonas aeruginosa*, and 0.7813 μM against *Bacillus subtilis*. Furthermore, compared to traditional antibiotics (such as ampicillin, polymyxin E, and vancomycin), especially against *Staphylococcus aureus* and some Gram-negative bacteria, the optimized Prevotellin-2f exhibited lower MIC values ​​and a broader spectrum of antimicrobial activity.

[0086] Example 4: Determination of the minimum inhibitory concentration (MIC) against clinically resistant bacteria.

[0087] The 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-mentioned antimicrobial peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

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

[0095] Clinically resistant bacteria: S. Aureus ATCC 6538, MRSA ATCC 43300, MRSA-Z, MRSA639SA1 and MRSA639SA2.

[0096] The MICs of antimicrobial peptides against clinically 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 strains.

[0098]

[0099] The antimicrobial activity of Prevotellin-2 and its modified antimicrobial peptides against methicillin-resistant Staphylococcus aureus (MRSA) and clinically 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 decreased 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 a MIC as low as 0.1953 μM against MRSA639SA1 and 639SA2, demonstrating activity stronger than traditional antibiotics Ampicillin and vancomycin. This indicates that the modified antimicrobial peptides of this invention have the potential to surpass traditional antibiotics and can be used to treat infections caused by clinically resistant bacteria.

[0100] Example 5: Determination of the minimum inhibitory concentration (MIC) against fungi

[0101] The antimicrobial peptide used was the same as in Example 1, diluted to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 0.7815, 0.3906, 0.1953, and 0.0977 μM in 96-well plates. Amphotericin B and fluconazole were used as positive controls. Amphotericin B was diluted to concentrations of 6.25, 3.125, 0.7815, 0.3906, 0.1953, 0.0977, 0.0488, 0.0244, and 0.0122 μM in 96-well plates, 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 BNCC25033101, Candida auris BNCC 356255, Candida auris BNCC357785, 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. Strains of Cryptococcus neoformans, Candida auris, and Candida albicans were grown in their respective media at 30°C until they entered the exponential growth phase. (Based on OD...) 600 =1≈1×10 7 CFU / mL, dilute the above bacterial culture with physiological saline to 2×10⁻⁶. 4CFU / mL. 100 μL of physiological saline was added to a sterile 96-well plate beforehand, and 200 μL of the test sample was added to the first row of wells. The test sample was then serially diluted twofold in the 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. 100 μL of a concentration of 2 × 10⁻⁶ CFU / mL was added to each well. 4 CFU / mL bacterial suspension was cultured at 37°C for different times. The absorbance of the bacterial suspension at 600 nm was measured using a microplate reader. The MIC value was taken as the average concentration of the sample in the well where no microbial growth was detected and the concentration of the sample in the adjacent well.

[0104] Different strains of *Aspergillus fumigatus* require the preparation of spore suspensions for MIC determination: In a sterile operating room, add 5–10 mL of sterile PBS + 0.05% Tween-80 to the colony surface. Gently scrape the spores using a sterile glass rod or L-shaped spreader, avoiding the introduction of hyphae. Transfer the spore-containing suspension to a sterile centrifuge tube, centrifuge at 3000 rpm for 5 min, and collect the supernatant to obtain the spore suspension. The remaining steps are the same as above.

[0105] Finally, the inhibitory activity of the above-mentioned antimicrobial peptides against standard strains of different fungi was determined, and the results are shown in Tables 3-6.

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

[0107] Petides CandidaalbicansBNCC299343 CandidaalbicansBNCC186382 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 AmphotericinB 0.1953 0.1953 Fluconazole 1.95 3.9

[0108] The results showed that prevotellin-2 had MIC values ​​exceeding 100 μM against both strains, indicating weak antifungal activity. In contrast, prevotellin-2a and prevotellin-2b had MIC values ​​of 100 μM and 25 μM, respectively, demonstrating moderate antifungal effects. Notably, prevotellin-2c, prevotellin-2e, and prevotellin-2f all showed strong antifungal activity against both strains, with MIC values ​​of 25 μM, 12.5 μM, and 6.25 μM, and 50 μM, 25 μM, 6.25 μM, and 3.125 μM, respectively. These results suggest that some prevotellin derivatives exhibit antifungal activity close to or even superior to traditional antifungal drugs, such as amphotericin B (MIC value 0.1953 μM) and fluconazole (MIC values ​​1.95 μM and 3.9 μM).

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

[0110]

[0111] The results showed that prevotellin-2, prevotellin-2a, and prevotellin-2d all had MIC values ​​exceeding 50 μM against both strains, indicating that these peptides had limited antibacterial effects against Aspergillus fumigatus. In contrast, prevotellin-2b had a MIC value of 50 μM against BNCC340016, showing some antibacterial activity. Prevotellin-2c had a MIC value of 50 μM against both strains, while prevotellin-2e and prevotellin-2f showed 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 conventional antifungal drug amphotericin B showed extremely high antifungal activity with a MIC of 0.09 μM against both strains. However, fluconazole showed MICs exceeding 50 μM against both strains, indicating poor antifungal efficacy against Aspergillus fumigatus. These results suggest that although some prevotellin derivatives have limited antifungal activity against Aspergillus fumigatus, prevotellin-2e and prevotellin-2f exhibit strong antifungal potential.

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

[0114]

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

[0116] In contrast, the traditional antifungal drug amphotericin B showed extremely high antifungal activity with a MIC of 0.09 μM against all tested strains. However, fluconazole showed MICs greater than 100 μM against all tested strains, indicating poor antifungal efficacy against *C. auris*. These results are consistent with previous findings that *C. auris* exhibits high resistance to azole and polyene antifungal drugs, thus highlighting the strong antifungal potential of the antimicrobial peptides prevotellin-2e and prevotellin-2f.

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

[0118]

[0119] The results showed that prevotellin-2 and prevotellin-2a had MIC values ​​greater than 50 μM against all tested strains, 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, showing extremely strong antibacterial effects.

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

[0121] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A modified antimicrobial peptide, characterized in that, a first antimicrobial peptide or a second antimicrobial peptide; the amino acid sequence of the first antimicrobial peptide is shown as SEQ ID NO. 1; the amino acid sequence of the second antimicrobial peptide is shown as SEQ ID NO.

2.

2. Use of the modified antimicrobial peptide of claim 1 in the preparation of a drug for resisting pathogenic bacteria infection. the pathogenic bacteria are bacteria and / or fungi; the bacteria are gram-positive bacteria and / or gram-negative bacteria; the gram-positive bacteria are Staphylococcus aureus and / or Bacillus subtilis; the gram-negative bacteria are at least one of Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa; the fungi are Cryptococcus neoformans and / or Staphylococcus albus.

3. Use according to claim 2, characterized in that, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

4. An agent for preventing or treating pathogenic bacterial infection, characterized by comprising the compound or salt according to claim 1. a pharmaceutical composition comprising the modified antimicrobial peptide of claim 1 and a pharmaceutically acceptable excipient.

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