Modified antimicrobial peptides from mealworm defensin source and their applications

Abstract

This invention discloses modified antimicrobial peptides derived from mealworm defensins and their applications. A series of modified antimicrobial peptides were designed by adding 2-3 consecutive pairs of tryptophan or leucine, arginine or lysine combinations to both ends of the 5th-11th amino acid fragment of the mealworm-derived insect defensin Z-d14CFR, and then synthesized via solid-phase synthesis. The modified antimicrobial peptides of this invention exhibit good bactericidal effects against a variety of Gram-negative and Gram-positive bacteria, especially against methicillin-resistant Staphylococcus aureus (MRSA), and are characterized by low cytotoxicity and non-hemolytic activity. This series of modified antimicrobial peptides can self-assemble into nanofiber networks to adhere to bacteria and kill bacteria in a short time by disrupting the bacterial membrane structure. They demonstrate excellent killing ability against various drug-resistant bacteria and have high application potential in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the modification of antimicrobial peptides from mealworm defensins and their applications. Background Technology

[0002] Antibiotics were once considered the most effective means of combating bacterial infections. However, with the use of antibiotics, the problem of bacterial resistance has become increasingly prominent. The resistance of bacteria such as Escherichia coli, Staphylococcus aureus, and Klebsiella is particularly noteworthy. In particular, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals poses a challenge to the prevention of secondary bacterial infections caused by surgery. Furthermore, with the use of vancomycin, the sensitivity of MRSA has gradually decreased, and due to the obstruction of systems such as biofilms, antibiotics have become more difficult to penetrate and kill the bacteria.

[0003] Antimicrobial peptides (AMPs) are a class of positively charged short-chain polypeptides that serve as important defense mechanisms in organisms. Widely distributed in animals and plants, their antibacterial activity primarily stems from direct disruption of bacterial membranes. This antibacterial action makes it difficult for bacteria to develop resistance, making it a crucial means of combating bacterial resistance. While insect antimicrobial peptides, primarily based on mealworm defensins, possess advantages such as good biocompatibility, their antibacterial efficiency is low in their natural sequences, and their effectiveness against Gram-positive bacteria, especially MRSA, is limited. Therefore, developing modified antimicrobial peptides with more efficient and broad-spectrum bactericidal functions based on mealworm defensins is an effective way to address this issue. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide modified from a mealworm defensin source and its application.

[0005] This invention increases structural symmetry and stability by adding 2-3 pairs of repeating sequences of tryptophan (W) or leucine (L), arginine (R) or lysine (K) combinations to both ends of the 5-11 amino acid fragment (CNSKSFC) of the insect defensin Z-d14CFR derived from barley worms. This resulted in the design of a series of modified antimicrobial peptides, whose amino acid sequences are shown in SEQ ID NO: 1-10. The peptides were then synthesized using a solid-phase synthesis method. After identification by mass spectrometry and purification by high-performance liquid chromatography, and further testing for antibacterial activity, cytotoxicity, and self-assembly ability, the peptides were finally named the modified antimicrobial peptides Z(WR)2, Z(WR)3, Z(WK)(WR), Z(WK)2, Z(WK)3, Z(LR)2, Z(LR)3, Z(LK)(LR), Z(LK)2, and Z(LK)3.

[0006] To achieve the objective of this invention, in a first aspect, this invention provides a modified antimicrobial peptide derived from mealworm defensin, wherein the antimicrobial peptide is Z(WR)2, Z(WR)3, Z(WK)(WR), Z(WK)2, Z(WK)3, Z(LR)2, Z(LR)3, Z(LK)(LR), Z(LK)2, or Z(LK)3 (SEQ ID NO:1-10):

[0007] Z(WR)2: WRWRCNSKSFCRWRW-NH2;

[0008] Z(WR)3: WRWRWRCNSKSFCRWRWRW-NH2;

[0009] Z(WK)(WR):WRWKCNSKSFCKWRW-NH2;

[0010] Z(WK)2:WKWKCNSKSFCKWKW-NH2;

[0011] Z(WK)3:WKWKWKCNSKSFCKWKWKW-NH2;

[0012] Z(LR)2:LKLKCNSKSFCKLKL-NH2;

[0013] Z(LR)3:LKLKLKCNSKSFCKLKLKL-NH2;

[0014] Z(LK)(LR):LRLKCNSKSFCKLRL-NH2;

[0015] Z(LK)2:LRLRCNSKSFCRLRL-NH2;

[0016] Z(LK)3: LLRLRCNSKSFCRLRLRL-NH2.

[0017] Furthermore, the molecular formulas of the modified antimicrobial peptides are shown in formulas (I) to (X):

[0018]

[0019]

[0020] In a second aspect, the present invention provides a nucleic acid molecule encoding the antimicrobial peptide.

[0021] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0022] Fourthly, the present invention provides a recombinant microorganism that expresses the antimicrobial peptide.

[0023] Fifthly, the present invention provides any of the following applications of the antimicrobial peptide:

[0024] 1) Used to inhibit bacteria;

[0025] 2) Used in the preparation of antibacterial agents;

[0026] 3) Used in the preparation of preservatives;

[0027] 4) Used in the preparation of antibacterial drugs.

[0028] The bacteria include Gram-negative and Gram-positive bacteria.

[0029] The Gram-negative bacteria are selected from Escherichia coli, Salmonella, Klebsiella, etc., such as Salmonella typhimurium and Klebsiella pneumoniae.

[0030] The Gram-positive bacteria are selected from Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus, etc., such as Streptococcus suis.

[0031] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] The modified antimicrobial peptides provided by this invention exhibit strong antimicrobial activity against common drug-resistant bacteria such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Furthermore, some of the modified antimicrobial peptides show excellent antimicrobial properties against MRSA and its clinical isolates. They exhibit almost no toxicity to macrophages, mammary epithelial cells, or renal epithelial cells, and do not cause hemolysis in animal blood cells. The antimicrobial peptides can self-assemble into nanofiber networks to adhere to bacteria. In addition, the modified antimicrobial peptides primarily exert their antimicrobial effect by disrupting bacterial membranes, demonstrating potential application against infections caused by drug-resistant bacteria. Attached Figure Description

[0033] Figure 1 This is a time-killing curve of the modified antimicrobial peptide in a preferred embodiment of the present invention.

[0034] Figure 2 The cytotoxicity of the modified antimicrobial peptide is shown in a preferred embodiment of the present invention.

[0035] Figure 3 This is a graph showing the hemolytic activity test results of the modified antimicrobial peptide in a preferred embodiment of the present invention.

[0036] Figure 4 This is a scanning electron microscope diagram illustrating the bactericidal mechanism of the modified antimicrobial peptide against E. coli CAU201919 in a preferred embodiment of the present invention.

[0037] Figure 5 This is a scanning electron microscope diagram illustrating the bactericidal mechanism of the modified antimicrobial peptide against S. aureus ATCC33591 in a preferred embodiment of the present invention.

[0038] Figure 6 The image shows a nanofiber network formed by modifying antimicrobial peptides under a transmission electron microscope in a preferred embodiment of the present invention. Detailed Implementation

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0040] Example 1: Design and Synthesis of Modified Antimicrobial Peptides

[0041] By adding 2-3 consecutive pairs of repeating sequences of tryptophan (W) or leucine (L), arginine (R), or lysine (K) combinations to both ends of the 5-11 amino acid fragment of the barley insect defensin Z-d14CFR, structural symmetry and stability were increased. This resulted in the design of modified antimicrobial peptides Z(WR)2, Z(WR)3, Z(WK)(WR), Z(WK)2, Z(WK)3, Z(LR)2, Z(LR)3, Z(LK)(LR), Z(LK)2, and Z(LK)3. The peptides were then synthesized using a solid-phase synthesis method. The peptides were identified by mass spectrometry and purified by high-performance liquid chromatography, followed by assays for antibacterial activity, cytotoxicity, cell penetration, and self-assembly ability.

[0042] The amino acid sequences of the modified antimicrobial peptides are as follows (Table 1):

[0043] Trp-Arg-Trp-Arg-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Arg-Trp-Arg-Trp-NH2 (SEQ ID NO:1), abbreviated as WRWRCNSKSFCRWRW-NH2, has a molecular weight of 2156.5; Trp-Arg-Trp-Arg-Trp-Arg-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Arg-Trp-Arg-Trp-Arg-Trp-NH2 (SEQ ID NO:2), abbreviated as WRWRWRCNSKSFCRWRWRW-NH2, has a molecular weight of 2841.9; Trp-Arg-Trp-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Trp-Arg-Trp-NH2 (SEQ ID NO:2), abbreviated as WRWRWRCNSKSFCRWRWRW-NH2, has a molecular weight of 2841.9; Trp-Arg-Trp-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Trp-Arg-Trp-NH2 (SEQ ID NO:2) NO:3), abbreviated as WRWKCNSKSFCKWRW-NH2, has a molecular weight of 2101.25;

[0044] Trp-Lys-Trp-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Trp-Lys-Trp-NH2 (SEQ ID NO:4), abbreviated as WKWKCNSKSFCKWKW-NH2, has a molecular weight of 2044.8; Trp-Lys-Trp-Lys-Trp-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Trp-Lys-Trp-Lys-Trp-NH2 (SEQ ID NO:4), with the single-letter abbreviation WKWKCNSKSFCKWKW-NH2, has a molecular weight of 2044.8; NO:5), with the single-letter abbreviation WKWKWKCNSKSFCKWKWKW-NH2 and a molecular weight of 2674; Leu-Lys-Leu-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Leu-Lys-Leu-NH2 (SEQ ID NO:6), with the single-letter abbreviation LKLKCNSKSFCKLKL-NH2 and a molecular weight of 1752.45; Leu-Lys-Leu-Lys-Leu-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Leu-Lys-Leu-Lys-Leu-NH2 (SEQ ID NO:6), with the single-letter abbreviation LKLKCNSKSFCKLKL-NH2 and a molecular weight of 1752.45; Leu-Lys-Leu-Lys-Leu-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Leu-Lys-Leu-Lys-Leu-NH2 (SEQ ID NO:6). NO:7), with the single-letter abbreviation LKLKLKCNSKSFCKLKLKL-NH2 and a molecular weight of 2235.4; Leu-Arg-Leu-Lys-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Lys-Leu-Arg-Leu-NH2 (SEQ ID NO:8), with the single-letter abbreviation LRLKCNSKSFCKLRL-NH2 and a molecular weight of 1808.4; Leu-Arg-Leu-Arg-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Arg-Leu-Arg-Leu-NH2 (SEQ ID NO:8), with the single-letter abbreviation LRLKCNSKSFCKLRL-NH2 and a molecular weight of 1808.4; Leu-Arg-Leu-Arg-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Arg-Leu-Arg-Leu-NH2 (SEQ ID NO:8). NO:9), with the single-letter abbreviation LRLRCNSKSFCRLRL-NH2 and a molecular weight of 1864.6; Leu-Arg-Leu-Arg-Leu-Arg-Cys-Asn-Ser-Lys-Ser-Phe-Cys-Arg-Leu-Arg-Leu-Arg-Leu-NH2 (SEQ ID NO:10), with the single-letter abbreviation LRLRLRCNSKSFCRLRLRL-NH2 and a molecular weight of 2403.3.

[0045] Table 1. Modified antimicrobial peptide sequences

[0046]

[0047]

[0048] Example 2: Determination of the antibacterial activity of the modified antimicrobial peptide

[0049] The minimum inhibitory concentration (MIC) of the modified peptide was determined using the standard microbroth dilution method. The specific method is as follows:

[0050] The bacteria were cultured in liquid medium to the logarithmic phase, and then the bacterial culture was serially diluted to 2 × 10⁻⁶. 5 CFU / mL. Mix 100 μL of modified antimicrobial peptides and original antimicrobial peptides (concentrations ranging from 1 to 512 μg / mL) with 100 μL of diluted bacterial culture in a 96-well plate and incubate at 37°C for 16–20 hours. Measure OD at 600 nm using a microplate reader. 600 The absorbance value at () was determined as the MIC. The experiment was independently repeated 3 times, with 3 replicates for each repetition. The results are shown in Table 2.

[0051] Table 2. Minimum inhibitory concentrations (μg / mL) of the modified antimicrobial peptides.

[0052]

[0053] Note: >512 μg / mL is considered ineffective. Among them, S. aureus ATCC33591, S. aureus CAU202012P, S. aureus CAU202011H, and S. aureus CAU20200JL are MRSA strains. The amino acid sequence of the original antimicrobial peptide Z-d14CFR is: RGCRCNSKSFCVCR-NH2.

[0054] As shown in Table 2, this type of modified peptide exhibits strong antibacterial activity against Escherichia coli, Salmonella or Klebsiella, Staphylococcus aureus, and Streptococcus, while the original antimicrobial peptide Z-d14CFR has no inhibitory effect on MRSA strains and Salmonella.

[0055] Example 3: Determination of the time-induced bactericidal curve of the modified antimicrobial peptide

[0056] The time-killing curve of the modified antimicrobial peptide was determined using a colony counting method, as detailed below:

[0057] Bacteria were cultured in liquid medium to the logarithmic growth phase. The bacterial suspension was then diluted with sterile phosphate-buffered saline solution. 100 μL of modified antimicrobial peptides of varying concentrations (from 1×MIC to 4×MIC) were then mixed with 100 μL of the diluted bacterial suspension, ensuring a bacterial concentration of 1×10⁻⁶ in the mixture. 6 CFU / mL was incubated at 37℃. Bacterial culture was taken at 0, 15, 30, 60, and 120 minutes, diluted to appropriate concentration, plated, and colony counts were recorded. The results were then logarithmically plotted. Figure 1It can be seen that modified antimicrobial peptides can kill bacteria in a short time.

[0058] Example 4: Cytotoxicity Study of Modified Antimicrobial Peptides

[0059] Mouse macrophages RAW264.7, bovine mammary epithelial cells MAC-T, and African green monkey kidney cells VERO were diluted to 2×10⁻⁶. 5 Cells / mL: 50 μL of diluted cells were added to 96-well plates and cultured in a 37°C incubator containing 5% CO2 until fully adherent. The cell culture medium was then removed, and DMEM medium containing gradient concentrations of modified antimicrobial peptides was added, and the plates were cultured for 24 hours. The medium was then removed again, replaced with fresh DMEM medium, and 10% CCK-8 solution was added. The plates were then placed in a 37°C incubator for 2-4 hours. OD values ​​at 450 nm were measured using a microplate reader. 450 The absorbance at the specified point is shown in the figure. In this experiment, a control group was set up without the use of antimicrobial peptides, and a blank control group was set up with cell-free DMEM and 10% CCK-8 solution. The above experiments were independently repeated three times.

[0060] Cell viability = (OD) 450肽处理组 -OD 450空白组 ) / (OD 450空白对照组 -OD 450空白组 )×100%

[0061] The results are as follows Figure 2 As shown, the modified antimicrobial peptide did not exhibit cytotoxicity at concentrations below 64 μg / mL.

[0062] Example 5: Hemolytic activity study of the modified antimicrobial peptide

[0063] Take an appropriate amount of defibrinated sheep blood, centrifuge at 3000g for 10 minutes at 4℃, discard the supernatant, and retain the lower layer of blood cells. Resuspend the lower layer of blood cells in sterile phosphate buffer solution, centrifuge at 3000g for 10 minutes at 4℃, repeat twice to obtain purified blood cells. Then, transfer 100μL of the blood cell suspension to a 96-well plate. Add 100μL of serially diluted modified antimicrobial peptide, and use phosphate buffer containing 0.2% Triton X-100 as a positive control. Incubate at 37℃ for 1 hour, remove the 96-well plate, centrifuge at 4℃ for 5 minutes at 3000g, transfer the supernatant to a new 96-well plate, and measure the OD at 567nm using a microplate reader. 567 The absorbance at () is measured. The above experiment was repeated three times independently.

[0064] Hemolysis rate = (OD 567肽处理组 -OD 567空白孔 ) / (OD 567 TritonX-100 -OD 567空白孔 )×100%

[0065] The results are as follows Figure 3 As shown, the modified antimicrobial peptide did not exhibit hemolytic activity against blood cells.

[0066] Example 6: Modification of the bactericidal mechanism of antimicrobial peptides

[0067] Inoculate E. coli CAU201919 or S. aureus 33591 into liquid medium and incubate overnight at 37°C. Then transfer to fresh MHB medium and incubate until the logarithmic growth phase is reached. Centrifuge the bacterial suspension, discard the MHB medium, collect the remaining bacteria, wash three times with sterile phosphate-buffered saline solution, and resuspend the bacteria to OD200. 600 =0.2. The antimicrobial peptide was incubated with bacteria at 37°C for 1 h (control group was untreated with antimicrobial peptide), then centrifuged to collect the bacteria, and the cells were resuspended in 600 μL of glutaraldehyde (2.5%) and fixed overnight at 4°C. The samples were dehydrated with different concentrations of ethanol (50%, 70%, 90%, and 100%). The samples were replaced with a 1:1 (v / v) mixture of ethanol and tert-butanol, and tert-butanol, respectively, for 15 min each. After lyophilizing the samples, they were adhered to a sample plate with conductive tape, and a metal coating was deposited on the sample surface using a coating apparatus. Finally, images were acquired using a scanning electron microscope.

[0068] The results are as follows Figure 4 and Figure 5 As shown, modified antimicrobial peptides can disrupt bacterial membrane structures, thereby killing bacteria.

[0069] Example 7: Modification of the self-assembly properties of antimicrobial peptides

[0070] The modified antimicrobial peptide was diluted to 32 μg / mL with sterile phosphate buffer and allowed to stand at room temperature for 1 hour. 10 μL of the peptide solution was dropped onto a 300-mesh carbon copper grid and dried with an infrared lamp. The copper grid was then negatively stained with 1% phosphotungstic acid for 30 seconds, rinsed with distilled water, dried, and observed using a transmission electron microscope.

[0071] The results are as follows Figure 6 As shown, the modified antimicrobial peptides can self-assemble into a dense nanofiber network structure.

[0072] In summary, the modified antimicrobial peptides provided by this invention exhibit strong antimicrobial activity against common drug-resistant bacteria such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Furthermore, some of these modified antimicrobial peptides demonstrate excellent antimicrobial properties against MRSA and its clinical isolates. They show almost no toxicity to macrophages, mammary epithelial cells, or renal epithelial cells, and exhibit no hemolytic activity against animal blood cells. The antimicrobial peptides can self-assemble into nanofiber networks to adhere to bacteria. In addition, the modified antimicrobial peptides primarily exert their antimicrobial effect by disrupting bacterial membranes, demonstrating potential application against infections caused by drug-resistant bacteria.

[0073] Example 8: Stability of modified antimicrobial peptides

[0074] The modified antimicrobial peptide was incubated at different temperatures (37℃, 60℃, 80℃, 90℃ and 100℃) for 1 hour. After the drug solution returned to room temperature, the MIC values ​​against E. coli ATCC25922 and S. aureus ATCC29213 were determined.

[0075] The modified antimicrobial peptide was incubated in buffer solutions of different pH values ​​(pH 2–12) for 1 hour, and then the pH of the solution was neutralized to 7.0. The MICs against E. coli ATCC25922 and S. aureus ATCC29213 were measured.

[0076] The modified antimicrobial peptide was co-incubated with proteases (pepsin, trypsin, papain, and α-chymotrypsin) for 1 hour, during which time the pH of the solution was adjusted to the optimal pH for protease activity. Subsequently, the solution was subjected to high-temperature treatment to inactivate the proteases, and the pH was adjusted to 7.0 to determine the MICs against E. coli ATCC25922 and S. aureus ATCC29213.

[0077] The results are shown in Tables 3, 4 and 5. The modified antimicrobial peptide has good stability under a wide range of temperature and pH conditions, and compared with the original antimicrobial peptide, the modified antimicrobial peptide has significantly improved resistance to proteases.

[0078] Table 3 Effect of different temperatures on the stability of antimicrobial peptides

[0079]

[0080]

[0081] Table 4. Effects of different pH values ​​on the stability of antimicrobial peptides

[0082]

[0083] Table 5. Effects of proteases on the stability of antimicrobial peptides

[0084]

[0085]

[0086] Note: A MIC value > 512 μg / mL can be considered invalid.

[0087] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Antimicrobial peptides modified from mealworm defensin source, characterized in that, The antimicrobial peptide is Z(WR)2 or Z(WR)3: Z(WR)2: WRWRCNSKSFCRWRW-NH2; Z(WR)3: WRWRWRCNSKSFCRWRWRW-NH2.

2. A nucleic acid molecule encoding the antimicrobial peptide of claim 1.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

4. A recombinant microorganism, characterized in that, The recombinant microorganism expresses the antimicrobial peptide of claim 1.

5. Any of the following applications of the antimicrobial peptide of claim 1: 1) Bacteriostatic agents used for purposes other than disease diagnosis and treatment; 2) Used in the preparation of antibacterial agents; 3) Used in the preparation of preservatives; 4) Used in the preparation of antibacterial drugs; The bacteria or bacterium mentioned are selected from Escherichia coli (Escherichia coli) Escherichia coli ),salmonella( Salmonella Klebsiella pneumoniae ( Klebsiella Staphylococcus aureus ( Staphylococcus aureus Streptococcus ( Streptococcus ).

6. The application according to claim 5, characterized in that, The bacteria or fungi mentioned are methicillin-resistant Staphylococcus aureus.

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