Antibacterial peptide p-1-SB and application thereof

Abstract

The invention belongs to the technical field of antibacterial products, and relates to an antibacterial peptide p-1-SB and application thereof. The amino acid sequence of the antibacterial peptide p-1-SB is as shown in SEQ ID NO.1, the antibacterial peptide p-1-SB is screened from a probiotic fungus Saccharomyces boulardii, and the antibacterial peptide p-1-SB has the advantages of low hemolysis, low cytotoxicity, high stability and efficient in-vivo antibacterial activity, and has application safety. The antibacterial peptide p-1-SB has strong antibacterial activity on Gram-negative bacteria enterohemorrhagic escherichia coli, urinary tract infection escherichia coli, shigella, salmonella and acinetobacter baumannii which are commonly infected by a human body, and has potential application value in development of prevention and treatment of bacterial infections such as intestinal diarrhea or urinary tract infection.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial product technology, specifically relating to an antibacterial peptide p-1-SB and its applications. Background Technology

[0002] Saccharomyces boulardii is a probiotic yeast that was originally isolated from the peels of lychee and mangosteen in Southeast Asia and has been widely used as a probiotic fungus.

[0003] Antimicrobial peptides (AMPs) are a class of naturally occurring immune defense molecules widely distributed in organisms, possessing broad-spectrum antibacterial, immunomodulatory, and anti-inflammatory functions. Due to their unique mechanisms of action and low risk of drug resistance, antimicrobial peptides are considered potential alternatives to traditional antibiotics and have significant applications in medicine, agriculture, and food preservation. Antimicrobial peptides can be classified according to their origin into natural antimicrobial peptides derived from animals, plants, and microorganisms, as well as artificially designed, synthesized, or modified peptides.

[0004] Among related technologies, the effectiveness and variety of antimicrobial peptides (AMPs) are limited, thus requiring urgent improvement. Utility Model Content

[0005] The purpose of this invention is to provide an antimicrobial peptide p-1-SB and its application. The antimicrobial peptide p-1-SB exhibits strong antimicrobial activity against Gram-negative bacteria that commonly cause human infections, and has potential application value in the development of prevention and treatment of skin bacterial infections.

[0006] In a first aspect, the present invention provides an antimicrobial peptide p-1-SB, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] In some embodiments, the amino acid sequence of the antimicrobial peptide p-1-SB has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 1, which is a functional homology.

[0008] In some embodiments, an amino acid sequence having the same protein activity is formed by adding, deleting, or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

[0009] Secondly, the present invention provides the use of the above-mentioned antimicrobial peptide p-1-SB in the preparation of products with antimicrobial and / or bactericidal activities.

[0010] In some embodiments, the bacteria include Gram-negative bacteria that can cause diarrhea or urinary tract infections.

[0011] In some embodiments, Gram-negative bacteria that can cause diarrhea or urinary tract infection include one or more of enterohemorrhagic Escherichia coli, urinary tract infection Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa, and Acinetobacter baumannii.

[0012] Thirdly, the present invention provides a product having antibacterial and / or bactericidal activity, the product comprising the above-mentioned antimicrobial peptide p-1-SB.

[0013] In some embodiments, the product includes excipients, including one or more of phosphate buffer, dimethyl sulfoxide, and physiological saline.

[0014] In some embodiments, the antimicrobial peptide p-1-SB has a mass concentration of 4 mg / mL to 6 mg / mL in the product.

[0015] In some embodiments, the product is an injection or a tablet.

[0016] The antimicrobial peptide p-1-SB provided by this invention has specific and highly effective antibacterial activity against Gram-negative bacteria causing diarrhea or urinary tract infections, while also exhibiting advantages such as low hemolytic activity, low cytotoxicity, and high stability. The antimicrobial peptide p-1-SB also shows strong antibacterial activity against common human Gram-negative bacteria such as Escherichia coli, Salmonella, Shigella, and Acinetobacter baumannii, and has potential application value in the development of treatments for bacterial infections causing intestinal diarrhea or urinary tract infections in humans. Attached Figure Description

[0017] 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.

[0018] Figure 1 The secondary structure diagram of the fungal antimicrobial peptide p-1-SB provided by the present invention is shown; Figure 2 The diagram shows the inhibition of Salmonella strain growth by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 3 The diagram shows the inhibition of Acinetobacter baumannii strain growth by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 4 The diagram shows the inhibition of Shigella strain growth by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 5 The diagram shows the inhibition of enterohemorrhagic Escherichia coli strains by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 6 The diagram shows the leakage of bacterial cell contents caused by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 7 The diagram shows the ATP leakage in bacterial cells caused by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 8 The figure shows the test results of the hemolytic activity of the fungal antimicrobial peptide p-1-SB provided by the present invention against sheep erythrocytes; Figure 9 The diagram shows the cytotoxicity results of the fungal antimicrobial peptide p-1-SB provided by the present invention against human lung cancer cells A549. Figure 10 The diagram showing the effect of the fungal antimicrobial peptide p-1-SB provided by the present invention on the expression of bacterial virulence genes is shown. Figure 11 The figure shows the effect of the fungal antimicrobial peptide p-1-SB provided by the present invention on the survival of the animal model wax moth after bacterial infection. Detailed Implementation

[0019] To make the inventive objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0020] The above description of the invention is not intended to describe every disclosed embodiment or implementation of the invention. Instead, the following description provides more specific examples of exemplary embodiments. Throughout this application, guidance is provided through a series of embodiments that can be used in various combinations. In each example, the examples are listed only as representative groups and should not be construed as exhaustive.

[0021] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number; "multiple" and "more than" in "one or more" mean two or more; and "one or more items" means two or more items.

[0022] The term "comprising" and its variations are not restrictive when they appear in the specification and claims.

[0023] The terms "preferred" and "ideal" refer to embodiments of the invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this disclosure.

[0024] The grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Each member of a group may be adopted and claimed individually, or in any combination with other members of that group or other elements found herein. It is foreseeable that one or more members of a group may be included in or removed from the group for convenience and / or patentability reasons.

[0025] The antimicrobial peptide p-1-SB of this invention is screened from probiotics, specifically Saccharomyces boulardii. For example, Saccharomyces boulardii can be any commercially available species of Saccharomyces boulardii.

[0026] Saccharomyces boulardii has the advantages of low hemolysis, low cytotoxicity, high stability and high efficiency of in vivo antibacterial activity, and is safe for application.

[0027] Studies have found that *Saccharomyces boulardii* can be used for the prevention and treatment of diarrhea and for regulating intestinal flora. Recent research has shown that *Saccharomyces boulardii* can be attacked by non-pathogenic *Escherichia coli* in the human body, thus affecting its colonization ability or activity in the host. Fungi extensively synthesize and secrete antimicrobial peptides and other antimicrobial substances, making them excellent natural hosts for antimicrobial components. Therefore, the strong resistance of *Saccharomyces boulardii* to gastric acid and bile salts provides potential for obtaining natural antimicrobial peptide sequences from its genomic information.

[0028] Whole-genome sequence analysis was performed on the probiotic Saccharomyces boulardii (Saccharomyces boulardii(nom.inval.)strainunique28scf7180000000067). Antimicrobial peptides with antimicrobial activity higher than 99.5% and fewer than 15 amino acids were screened using the optimized antimicrobial peptide sequence prediction program Deep-AmPEP30 (https: / / cbbio.online / AxPEP / ). Antimicrobial peptides with positive antimicrobial activity were screened using the DBAASP database (https: / / www.dbaasp.org / tools?page=linear-amp-prediction) based on the Gram-negative bacteria antimicrobial activity prediction index. Several antimicrobial peptides were screened out, and after further screening, the antimicrobial peptide p-1-SB of this application was obtained.

[0029] Saccharomyces boulardii exerts its probiotic effect on pathogenic Escherichia coli by capturing and inactivating lipopolysaccharides on the surface of these bacteria. However, recent studies have shown that non-pathogenic Escherichia coli can adsorb and disrupt the cell wall of Saccharomyces boulardii, thus affecting its function. Based on this, this invention utilizes the genome sequence of Saccharomyces boulardii and employs comprehensive computational and experimental methods for rapid characterization, screening a novel, highly efficient, and specific antimicrobial peptide, p-1-SB, from the fungal genome.

[0030] This application provides an antimicrobial peptide p-1-SB, the amino acid sequence of which is shown in SEQ ID NO. 1. It can be: LKIRRKIWKLLW.

[0031] In some embodiments, the amino acid sequence of the antimicrobial peptide p-1-SB has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 1, which is a functional homology.

[0032] In some embodiments, an amino acid sequence having the same protein activity is formed by adding, deleting, or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

[0033] The antimicrobial peptide p-1-SB of this invention is a natural antimicrobial peptide derived from probiotics. Specifically, the antimicrobial peptide p-1-SB of this invention is screened from the probiotic Saccharomyces boulardii.

[0034] The antimicrobial peptide p-1-SB of this invention contains 12 amino acids, has a molecular weight of 1653.11 g / mol, an isoelectric point of 12.43, and an instability index of 261.80. The arithmetic mean of hydrophobicity is -0.18, the transmembrane capacity is -6.0 kcal / mol, and the secondary structure is an α-helix. The antimicrobial peptide p-1-SB of this invention exhibits significant antimicrobial activity against Gram-negative bacteria that can cause intestinal diarrhea. It also possesses advantages such as low hemolysis, low cytotoxicity, high stability, and highly efficient in vivo antimicrobial activity, demonstrating application safety. It is of great significance for applications such as the defense against pathogenic Escherichia coli infections.

[0035] The antimicrobial peptide p-1-SB of this invention is effective against Gram-negative enterohemorrhagic Escherichia coli and Shigella, which can cause intestinal diarrhea, as well as CFT073 and UTI89, which cause urinary tract infections, including Escherichia coli O157:H7. O26:H11, O103:H2, O111:H-, O145:H28, Shigella M90T, Salmonella Ty2 and 14028, and Acinetobacter baumannii ATCC19606 all exhibited strong antibacterial activity, showing great promise for their development into treatments for Gram-negative bacterial infections causing intestinal diarrhea and urinary tract infections in humans.

[0036] The present invention also provides the application of the above-mentioned antimicrobial peptide p-1-SB in the preparation of products with antibacterial and / or bactericidal activities.

[0037] In this invention, the bacteria include Gram-negative bacteria that can cause intestinal diarrhea and urinary tract infections. Preferably, the Gram-negative bacteria include enterohemorrhagic Escherichia coli, urinary tract pathogenic Escherichia coli, Shigella, and Salmonella.

[0038] In this invention, enterohemorrhagic Escherichia coli EHECO157:H7, EHECO26:H11, and EHECO103: H2, EHECO111:H-, EHECO145:H28, Shigella strains preferably include M90T, urinary pathogenic Escherichia coli strains include UPECCFT073 and UPECUTI89, Salmonella strains preferably include Ty2 and 14028, and Acinetobacter baumannii strains preferably include ATCC19606.

[0039] The present invention also provides a product having antibacterial and / or bactericidal activity, the product comprising the antimicrobial peptide p-1-SB of the above-described technical solution.

[0040] In some embodiments, the product includes excipients, including one or more of phosphate buffer, dimethyl sulfoxide, and physiological saline.

[0041] In some embodiments, the mass concentration of the antimicrobial peptide p-1-SB in the product is 4 mg / mL to 6 mg / mL. Optionally, the mass concentration of the antimicrobial peptide p-1-SB in the product can be any value or a range thereof from 4.0 mg / mL, 4.1 mg / mL, 4.2 mg / mL, 4.3 mg / mL, 4.4 mg / mL, 4.5 mg / mL, 4.6 mg / mL, 4.7 mg / mL, 4.8 mg / mL, 4.9 mg / mL, 5.0 mg / mL, 5.1 mg / mL, 5.2 mg / mL, 5.3 mg / mL, 5.4 mg / mL, 5.5 mg / mL, 5.6 mg / mL, 5.7 mg / mL, 5.8 mg / mL, 5.9 mg / mL, and 6.0 mg / mL.

[0042] In some embodiments, the product is an injection or a tablet.

[0043] Products may include pharmaceuticals, animal feed, toiletries, hygiene products, or other sterilization products.

[0044] The present invention also provides a pharmaceutical composition comprising the antimicrobial peptide p-1-SB of the above-described technical solution and pharmaceutically acceptable excipients. In the present invention, the pharmaceutical composition preferably comprises a pharmaceutical composition having preventive and / or therapeutic effects against diseases caused by common Gram-negative bacterial infections in humans, and more preferably comprises a pharmaceutical composition having preventive and / or therapeutic effects against diseases caused by Gram-negative bacterial infections of the human intestinal or urinary tract.

[0045] To further illustrate the present invention, the following detailed description of an antimicrobial peptide p-1-SB and its applications, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention. Example

[0046] The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight, and all reagents used in the examples are commercially available or synthesized by conventional methods and can be used directly without further processing. The instruments used in the examples are also commercially available. Example

[0047] Fungal selection and screening: Fungal genome sequences were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore). To ensure the safety of natural antimicrobial peptides, the genomes of pathogenic fungi or conditionally pathogenic fungi were removed. Probiotics or potential probiotics were then specifically selected. Therefore, based on literature review, *Saccharomyces boulardii*, which is widely used commercially, was chosen.

[0048] Screening of fungal antimicrobial peptide p-1-SB: Whole-genome sequence analysis of the probiotic *Saccharomyces boulardii* (nom.inval.) strainunique28scf7180000000067 was performed. Using the optimized antimicrobial peptide sequence prediction program Deep-AmPEP30 (https: / / cbbio.online / AxPEP / ), 14 antimicrobial peptides with antimicrobial activity higher than 99.5% and fewer than 15 amino acids were screened. These were peptide A, peptide B, peptide C, and peptide p-1-SB, with the following amino acid sequences: Peptide A: WFRKRLKWK; Peptide B: LKYLFLHIYTWK. C-peptide: WLKFKKYHKR; D-peptide: LFFLIHWSKWVH; E-peptide: RRCFCCSWRIFIWL; F-peptide: KILTRTFWKKLI; G-peptide: LHLKLLLWHKKTK; H-peptide: HLLKLWHHFLGKYR; I-peptide: KKKKKKKKKIWL; J-peptide: KRLTNWRKTAKFWC; K-peptide: HRWRRWW; L-peptide: RFWWRRKKILWLLK; M-peptide: LIHKKWKKIF; and p-1-SB peptide: LKIRRKIWKLLW. Antimicrobial peptides with positive antimicrobial activity were screened using the DBAASP database (https: / / www.dbaasp.org / tools?page=linear-amp-prediction) to predict the antimicrobial activity of Gram-negative bacteria. Furthermore, CellPM (https: / / cellpm.org / cellpm_server) was used to predict the transmembrane activity of the antimicrobial peptides, screening for short peptide sequences with strong transmembrane activity (transfer energy ≤ -6.0). The identified peptides were: F peptide: KILTRTFWKKLI, M peptide: LIHKKWKKIF, and antimicrobial peptide p-1-SB: LKIRRKIWKLLW. These short peptide sequences obtained using this method have transmembrane activity between -6.0 and -8.0 kcal / mol, and their chemical synthesis purity is higher than 95%. For example, this method can be used to obtain the short peptide sequence antimicrobial peptide p-1-SB, with the amino acid sequence Leu-Lys-Ile-Arg-Arg-Lys-Ile-Trp-Lys-Leu-Leu-Trp, a transmembrane capacity of -6.0 kcal / mol, and a chemical synthesis purity of over 95%.

[0049] The fungal antimicrobial peptide p-1-SB of this invention has 12 amino acid residues, and its specific amino acid sequence is: Leu-Lys-Ile-Arg-Arg-Lys-Ile-Trp-Lys-Leu-Leu-Trp. The single-letter abbreviation of the amino acid sequence is LKIRRKIWKLLW. The molecular weight of the fungal antimicrobial peptide p-1-SB is 1653.11 g / mol, the isoelectric point is 12.43, and the arithmetic mean of hydrophobicity is -0.18. Figure 1 As shown, PEP-FOLD3 predicts that the secondary structure of this antimicrobial peptide is an α-helix.

[0050] Example 2: Determination of the antimicrobial activity of antimicrobial peptides The strains involved in this embodiment include Gram-negative bacteria: Escherichia coli, Salmonella typhi Ty2, 14028, Shigella M90T, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii ATCC19606, and Gram-positive bacteria Staphylococcus aureus Newman.

[0051] These strains (such as Gram-negative bacteria) were obtained from the -80°C incubator of the Biomedical Center Laboratory of Shenzhen Second People's Hospital | Shenzhen Institute of Translational Medicine. For example, Escherichia coli EHECO157:H7, EHECO26:H11, EHECO103:H2, EHECO111:H- and EHECO145:H28 were obtained from this laboratory.

[0052] The experiment used a two-fold serial dilution method to determine the minimum inhibitory concentration, which is the lowest drug concentration that can inhibit bacterial growth and reproduction.

[0053] MIC value determination: S1. Prepare freshly streaked bacterial colonies. Take a small amount of colony and place it in 1.5 mL of sterile water. Mix well by pipetting. Adjust the bacterial concentration to OD600 = 0.4 using a UV spectrophotometer. Dilute the bacterial solution 200 times with an appropriate volume of MH medium and adjust to OD600 = 0.002. Dispense 75 μL of bacterial solution / well into a 96-well plate. S2. Dissolve the fungal antimicrobial peptide p-1-SB in DMSO at a concentration of 4 mg / mL; prepare 300 μL of the antimicrobial peptide in MH medium with a two-fold serial dilution, from 0 μg / mL to 240 μg / mL; mix well by pipetting and add 75 μL / well to each well of a 96-well plate containing the bacterial culture to be tested, with three replicates for each concentration; S3. Place the 96-well plate in a constant temperature shaker at 37℃ and 180rpm for 24h. S4. Use an ELISA reader to detect the light absorbance of the bacterial solution at 600nm. The minimum inhibitory concentration (MIC) is the average of the sample concentrations in wells where no bacterial growth can be detected and in adjacent wells.

[0054] S5. The antimicrobial activity of the fungal antimicrobial peptide p-1-SB against *Escherichia coli*, *Salmonella*, *Shigella*, and *Acinetobacter baumannii* was verified through three repeated experiments, as shown in Table 1, with MIC values ​​ranging from 6.25 to 12.5 μg / mL. The antimicrobial activity against *Pseudomonas aeruginosa* and *Staphylococcus aureus* (a Gram-positive bacterium) is shown in Table 2, with MIC values ​​of 60 μg / mL and ≥200 μg / mL, respectively.

[0055] MIC (Minimum Inhibitory Concentration) refers to the lowest concentration of an antimicrobial drug that can inhibit the visible growth of a certain microorganism in in vitro experiments. The smaller the MIC value, the stronger the inhibitory effect of the antimicrobial peptide p-1-SB on bacteria. Conversely, the larger the MIC value, the lower the sensitivity of bacteria to the antimicrobial peptide p-1-SB, and the higher the concentration required to be effective.

[0056] Table 1. Antimicrobial activity of antimicrobial peptide p-1-SB against Escherichia coli, Salmonella, and Shigella. Table 2. Antimicrobial activity of antimicrobial peptide p-1-SB against Pseudomonas aeruginosa and Staphylococcus aureus.

[0057] In summary, this demonstrates the antimicrobial activity of the antimicrobial peptide p-1-SB against bacteria such as Escherichia coli, Salmonella, Shigella, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0058] Example 3: Determination of the growth inhibition curve induced by the fungal antimicrobial peptide p-1-SB. The strains involved in this embodiment are Escherichia coli EHECO157:H7, Salmonella 14028, Shigella M90T and Acinetobacter baumannii ATCC19606.

[0059] S1. Place the bacteria to be tested in LB medium and incubate at 37℃ and 220rpm for 12h; transfer 1:100 to 2mL MH medium and continue incubation at 37℃ and 220rpm for 2.5h, and measure OD600 using an ELISA reader to find it to be 0.6; dilute the bacterial culture to OD600=0.1. S2. Take 150 μL of LMH culture base into a 96-well plate, add 10 μL of the bacterial culture to be tested to each well, and add the antimicrobial peptide p-1-SB to each well in sequence at final concentrations of 0 μg / mL, 3.75 μg / mL, 7.5 μg / mL, 15 μg / mL and 30 μg / mL. Mix well, place in a microplate reader, and incubate at 37℃. Measure the OD600 reading every 1 h for 20 h; plot the growth curve.

[0060] The results are as follows Figure 2-5 As shown, where, Figure 2 The diagram shows the inhibition of Salmonella strain growth by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 3 The diagram shows the inhibition of Acinetobacter baumannii strain growth by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 4 The diagram shows the inhibition of Shigella strain growth by the fungal antimicrobial peptide p-1-SB provided by the present invention. Figure 5 The diagram shows the inhibition of enterohemorrhagic Escherichia coli strains by the fungal antimicrobial peptide p-1-SB provided by the present invention.

[0061] In summary, three repeated experiments showed that bacterial growth was inhibited with increasing concentration of the antimicrobial peptide p-1-SB. When the concentration of the antimicrobial peptide increased to 12.5 μg / mL, no bacterial growth was detected, indicating that bacterial growth was inhibited at this concentration.

[0062] Example 4: Determination of leakage of bacterial intracellular contents induced by the fungal antimicrobial peptide p-1-SB The strain involved in this embodiment is Escherichia coli EHECO157:H7.

[0063] S1. Place the bacteria to be tested in BHI medium and incubate at 37°C and 220 rpm for 12 h; then transfer them 1:100 to 2 mL of BHI medium and continue incubating at 37°C and 220 rpm for 6 h. S2. Adjust the bacterial concentration to OD600=0.1 with PBS; dispense 2.5 mL of bacterial solution into 5 mL EP tubes, add antimicrobial peptides to the EP tubes at final concentrations of 10 μg / mL and 20 μg / mL respectively, and incubate at room temperature on a mixing rack; use bacterial solution containing a final concentration of 0.5% Triton-X100 as a positive control; S3. At 0 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively, 200 μL of bacterial culture was taken into a 96-well plate (white plate), 2 μL of 1 mg / mL propidium iodide (PI) was added, and the mixture was mixed. The plate was then incubated at room temperature for 15 min. S4. Place the 96-well plate in a microplate reader and use an excitation wavelength of 540 nm and an emission wavelength of 621 nm to detect the staining of propidium iodide. like Figure 6 As shown in the three repeated experiments, the staining intensity of propidium iodide increased with the extension of the incubation time of the antimicrobial peptide p-1-SB, indicating that the antimicrobial peptide p-1-SB can cause leakage of intracellular contents of bacteria, thereby contributing to the antimicrobial effect.

[0064] Propidium iodide (PI) is a membrane-insoluble fluorescent dye that cannot normally enter the intracellular space of intact cells. When the cell membrane is damaged, PI enters the cell and binds to DNA / RNA, emitting red fluorescence (the fluorescence intensity is positively correlated with the degree of membrane damage). Deeper staining can be understood as an increase in PI signal over time → continuous loss of bacterial membrane integrity → leakage of intracellular substances (such as nucleic acids, ions, and metabolites) → bacterial death.

[0065] It is speculated that p-1-SB may insert into the bacterial membrane (negatively charged phospholipids) through cation-hydrophobic interactions, forming channels or dissolving the lipid bilayer, leading to membrane rupture, prolonged incubation time, and further binding of p-1-SB molecules to the membrane, resulting in cumulative membrane damage until complete bacterial lysis. This membrane-targeting mechanism is less likely to induce drug resistance and is effective against stationary bacteria and biofilms, suggesting that p-1-SB may possess broad-spectrum antibacterial potential.

[0066] Example 5: Assay of ATP Release from Bacterial Cells Induced by the Fungal Antimicrobial Peptide p-1-SB The strain involved in this embodiment is Escherichia coli EHECO157:H7.

[0067] S1. Place the bacteria to be tested in BHI medium and incubate at 37°C and 220 rpm for 12 h; then transfer them 1:100 to 2 mL of BHI medium and continue incubating at 37°C and 220 rpm for 6 h. S2. Adjust the bacterial culture concentration to OD600=0.1 with PBS, and dispense 0.5 mL of the bacterial culture into 1.5 mL EP tubes. Add the antimicrobial peptide p-1-SB to the EP tubes at a final concentration of 20 μg / mL, and take 100 μL as the zero point. Incubate the EP tubes at 37℃ and 220 rpm on a shaker. Take 100 μL of the sample at 5 min, 10 min, and 15 min and place it on ice (protected from light). S3. Centrifuge 9000g of the above sample at 4℃ for 5min, collect the supernatant and place it on ice (protected from light). S4 Take 100 μL of ATP detection solution into a 96-well plate (white plate), mix well, let stand at room temperature for 3-5 min, add 20 μL of the above sample and place it in an ELISA reader to detect the cold light value. like Figure 7 As shown in the figure, three repeated experiments showed that the amount of ATP in the supernatant increased with the extension of the incubation time of the antimicrobial peptide p-1-SB, indicating that the antimicrobial peptide p-1-SB can cause leakage of bacterial cells, thereby contributing to the antimicrobial effect.

[0068] ATP (adenosine triphosphate) is a core molecule in bacterial energy metabolism, normally present intracellularly (at a much higher concentration than extracellularly). An increase in ATP in the supernatant indicates cell membrane / wall disruption (e.g., pore formation, lipid bilayer dissolution), leading to passive ATP leakage. Bacteria lose vitality or die due to energy depletion and ion imbalance. Antimicrobial peptides bind to the bacterial membrane (negatively charged phospholipids) through cation-hydrophobic interactions. p-1-SB disrupts the integrity of the bacterial cell membrane or cell wall, causing intracellular ATP to leak into the external environment, directly demonstrating the membrane-targeted bactericidal mechanism of this antimicrobial peptide. This mechanism is consistent with most antimicrobial peptides (such as polymyxins and melittin) and is less likely to induce resistance. Prolonged incubation time allows more p-1-SB molecules to insert into the membrane, causing cumulative damage and exacerbating ATP leakage, ultimately leading to bacterial death. The membrane-targeting mechanism can kill bacteria in a short time.

[0069] Example 6: Determination of the thermal and pH stability of the fungal antimicrobial peptide p-1-SB The strain involved in this embodiment is Acinetobacter baumannii ATCC19606.

[0070] S1. Prepare a 0.1M sodium acetate solution and sterilize it using a 0.22μm filter membrane; prepare solutions with pH values ​​of 2, 4, 6, 8, 11, and 13 respectively using HCl or NaOH. S2. Prepare 500 μL of antimicrobial peptide p-1-SB containing 400 μg / mL using 1×PBS, mix well, and let stand at room temperature (25°C) for 1 h; prepare 500 μL of antimicrobial peptide p-1-SB containing 400 μg / mL using 1×PBS, mix well, and let stand at 4°C for 1 h; let stand at 25°C for 1 h; and heat in a water bath at 50°C, 70°C, and 90°C for 1 h respectively. S3. Using 0.1M sodium acetate solution to prepare solutions with pH values ​​of 2, 4, 6, 8, 11, and 13, prepare 500 μL of antimicrobial peptide p-1-SB containing 400 μg / mL, mix well, and let stand at room temperature (25°C) for 1 h. S4. The MIC value of the antimicrobial peptide p-1-SB treated above was determined using the method described in Example 2.

[0071] Table 3. Results of thermal stability of fungal antimicrobial peptide p-1-SB

[0072] Table 4. Results of pH stability of the fungal antimicrobial peptide p-1-SB in the alkaline environment.

[0073] As shown in Tables 3 and 4, three repeated experiments showed that the antimicrobial peptide p-1-SB was thermally stable. The stability of the antimicrobial peptide remained unchanged under neutral, acidic and weakly alkaline pH treatments (MIC value remained at 25 μg / mL), while the stability of the antimicrobial peptide p-1-SB was somewhat affected under strongly alkaline pH treatment (pH=13) (MIC value was 40 μg / mL).

[0074] In summary, the antimicrobial peptide p-1-SB has a lower MIC value under acidic conditions or in environments with a lower pH, which is more conducive to antibacterial activity.

[0075] Example 7: Determination of hemolysis rate of fungal antimicrobial peptide p-1-SB 1. Take an appropriate amount of sheep blood, dilute it 3 times with PBS buffer (pH 7.4), and aliquot 190 μL / well into a 96-well plate; 2. Prepare antimicrobial peptides at different concentration gradients using PBS buffer (pH 7.4): 0 μg / mL (negative control), 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, with 0.2% Triton X-100 as a positive control; 3. Add 10 μL of antimicrobial peptide to the 96-well plate containing sheep blood; incubate at 37°C for 60 min; 4. Remove the 96-well plate, centrifuge (3000×g, 5min, 4℃), transfer 100μL of supernatant to a new 96-well plate, and measure the absorbance at 567nm using a microplate reader; 5. The hemolysis rate of antimicrobial peptides is calculated as (OD567 of test wells - OD567 of negative wells) / (OD567 of positive wells - OD567 of negative wells) × 100%.

[0076] like Figure 8 As shown in the three repeated experiments, the OD567 results of incubation of the antimicrobial peptide solution and sheep red blood cell suspension showed that, compared with the control group, the antimicrobial peptide p-1-SB did not cause significant hemolysis of red blood cells at a concentration of 400 μg / mL, and the hemolysis rate was less than 10%.

[0077] Based on the experimental results (the hemolysis rate of the antimicrobial peptide at a concentration of 400 μg / mL was <10%), it is indicated that the antimicrobial peptide has a weak membrane-disrupting effect on eukaryotic cells (such as sheep erythrocytes) and exhibits selective toxicity, meaning it tends to target bacteria rather than host cells.

[0078] Furthermore, the low hemolysis rate (<10%) and the effective antibacterial concentration (MIC 6.25~12.5 μg / mL) are far lower than the hemolytic concentration (>400 μg / mL), indicating low potential toxicity and a wide therapeutic window. The low hemolytic properties of antimicrobial peptides are highly correlated with their selective membrane-targeting or non-membrane-dissolving antibacterial mechanisms. This characteristic enables them to effectively kill bacteria while avoiding damage to host cells, providing an important basis for the development of novel low-toxicity antibacterial drugs.

[0079] The combination of p-1-SB's in vivo efficacy and low hemolysis rate (<10%) suggests that p-1-SB may be suitable for intravenous treatment of serious infections such as sepsis. If its mechanism differs from traditional antibiotics (such as β-lactams), it may be effective against multidrug-resistant bacteria (such as MRSA and carbapenem-resistant Acinetobacter baumannii).

[0080] Example 8: Cytotoxicity assay of the fungal antimicrobial peptide p-1-SB The CellCounting Kit-8 (CCK-8) method was used to determine cytotoxicity in the experiment. Human lung cancer cells A549 were cultured in DMEM medium, counted, and the cell concentration was adjusted to 7×10^4 cells / mL. S1. Add 100 μL PBS to the outermost periphery of the 96-well plate to prevent cell sap evaporation and experimental errors. Seed 100 μL / well of cell suspension into the remaining 96-well plates. Add DMEM complete medium to the remaining column as a blank group. Incubate the 96-well plates overnight at 37°C and 5% CO2 (usually 12 h).

[0081] S2. Remove the 96-well plate, remove the cell supernatant, and add 100 μL of DMEM complete medium containing DMSO control group and gradient concentrations of antimicrobial peptides (10 μg / mL, 30 μg / mL, 50 μg / mL, and 100 μg / mL, respectively). Incubate at 37°C and 5% CO2 for 24 h.

[0082] S3. Remove the supernatant cell solution, add 100 μL of fresh DMEM complete culture medium to each well, and then add 10% CCK-8 solution sequentially, taking care not to generate air bubbles, otherwise it will affect the accuracy of the spectrophotometer readings. Incubate the 96-well plate at 37°C for 2 hours.

[0083] S4. Measure the absorbance at 450 nm using an ELISA reader. Repeat the experiment three times.

[0084] Cell viability (%) = (Antimicrobial peptide treatment group - Blank) / (DMSO control group - Blank) × 100% like Figure 9 As shown, after incubation of antimicrobial peptide p-1-SB solution with A549 cell suspension, OD450 assay results showed that antimicrobial peptide p-1-SB concentration less than or equal to 100 μg / mL did not exhibit cytotoxicity, and at a concentration of 200 μg / mL, the hemolysis rate was less than 10%, indicating that antimicrobial peptide p-1-SB has low cytotoxicity.

[0085] Example 9: Determination of the effect of fungal antimicrobial peptide p-1-SB on bacterial virulence gene expression The strain involved in this embodiment is Escherichia coli EHECO157:H7.

[0086] The experiment used real-time quantitative PCR to determine the expression of representative virulence genes of pathogenic bacteria by the fungal antimicrobial peptide p-1-SB; S1. Escherichia coli EHECO157:H7 was cultured overnight at 37℃ and 220rpm for 12h, then transferred 1:100 to 10mL LB medium. Antimicrobial peptide p-1-SB was added or not added to a final concentration of 10MIC (125μg / mL) and cultured until OD600=0.6 (approximately 2.5h). The bacterial culture was collected and RNA was extracted using Trizol. S2. cDNA was synthesized using a reverse transcription kit and detected by q-PCR using SBYRGreen. The target virulence gene was detected using the DDCt method, with 16S RNA used as an internal reference gene. The experiment was repeated three times.

[0087] like Figure 10 As shown, the antimicrobial peptide p-1-SB has no effect on the expression of virulence genes stxA1, nleF, espY2.

[0088] According to the experimental results, the antimicrobial peptide p-1-SB had no significant effect on the expression of virulence genes stxA1 (Shiga toxin), nleF (type III secretion system effector protein), and espY2 (virulence factor of enteropathogenic Escherichia coli), indicating that it mainly relies on direct bactericidal action rather than virulence inhibition. Its mechanism of action is independent of virulence gene regulation. Unlike traditional antibacterial mechanisms, the antibacterial activity of p-1-SB (MIC 6.25~12.5 μg / mL) may primarily depend on the aforementioned direct bactericidal mechanism, rather than weakening bacterial pathogenicity by regulating virulence gene expression. For example, some antibiotics (such as subinhibitory concentrations) may reduce bacterial pathogenicity by inhibiting virulence genes (such as quorum sensing systems), but p-1-SB acts differently, favoring rapid bactericidal action. Therefore, it may reduce the selective pressure of drug resistance, but may not alleviate toxin-mediated pathological damage.

[0089] Example 10: In vivo bactericidal activity assay of the fungal antimicrobial peptide p-1-SB The strain involved in this embodiment is Escherichia coli EHECO157:H7.

[0090] The experiment used an animal infection model, wax moth larvae, to determine the bactericidal activity of the fungal antimicrobial peptide p-1-SB in vivo; S1. Weigh 0.3~0.4g of wax moth larvae and place them in a 37℃ incubator for starvation for 12h; randomly assign 10 wax moth larvae to a petri dish; S2. Collect fresh bacterial culture that has been cultured at 37℃ and 220rpm for 12 hours, and adjust the bacterial culture to OD600=0.2 (approximately 1x10^8 colonies / mL) with PBS; take out 100μL of bacterial culture, add antimicrobial peptide to a final concentration of 200μg / mL, and mix well; inject 5μL of bacterial culture, bacterial culture + antimicrobial peptide into the left hind leg of each wax moth larva, 12 larvae per group; place the wax moths in a 37℃ incubator for culture; S3. Using PBS as a blank control group, 5 μL was injected into the left hind leg of the wax moth larvae, with 10 larvae in each group; the wax moths were placed in an incubator at 37°C for incubation. S4. Record the mortality of wax moth larvae every 12 hours, up to 120 hours; repeat the experiment three times.

[0091] like Figure 11 As shown, the antimicrobial peptide p-1-SB can significantly increase the survival rate of wax moth larvae after bacterial infection, indicating that it has in vivo bactericidal activity.

[0092] The wax moth larvae are a classic model for evaluating the in vivo activity of antibacterial drugs. The increased survival rate of p-1-SB indicates that p-1-SB can effectively inhibit bacterial proliferation in the larvae; it exerts its effects through direct bactericidal action or by enhancing host immunity; and it has no significant toxicity to the host (if the toxicity is high, the survival rate will decrease). In addition, p-1-SB has been confirmed to have significant antibacterial activity in vivo.

[0093] 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.

[0094] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An antimicrobial peptide p-1-SB, characterized in that, The amino acid sequence of the antimicrobial peptide p-1-SB is shown in SEQ ID NO.

1.

2. The antimicrobial peptide p-1-SB according to claim 1, characterized in that, The antimicrobial peptide p-1-SB has a functional homologous sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID No. 1; or, it is an amino acid sequence with one or more amino acids added, deleted, or substituted in the amino acid sequence shown in SEQ ID No. 1 and having the same protein activity.

3. The use of the antimicrobial peptide p-1-SB as described in claim 1 or 2 in the preparation of products for treating bacterial infections and / or eliminating bacteria.

4. The application according to claim 3, characterized in that, The bacteria include Gram-negative bacteria that cause diarrheal infections or urinary tract infections.

5. The application according to claim 4, characterized in that, The Gram-negative bacteria that can cause diarrheal infections or urinary tract infections include one or more of the following: enterohemorrhagic Escherichia coli, urinary tract infection Escherichia coli, Shigella, Salmonella, Pseudomonas aeruginosa, and Acinetobacter baumannii.

6. A product having antibacterial and / or bactericidal activity, characterized in that, The product includes the antimicrobial peptide p-1-SB as described in claim 1 or 2.

7. The product according to claim 6, characterized in that, The product includes excipients, which include one or more of phosphate buffer, dimethyl sulfoxide, and physiological saline.

8. The product according to claim 7, characterized in that, The antimicrobial peptide p-1-SB has a mass concentration of 4 mg / mL to 6 mg / mL in the product.

9. The product according to claim 6, characterized in that, The product is an injection or a tablet.

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