Pig intestinal microorganism source antibacterial peptide and application thereof

Abstract

The invention discloses a porcine intestinal microorganism source antibacterial peptide and application thereof, and belongs to the technical field of biology. The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1, and the antibacterial peptide is prepared through pig intestinal metagenome data mining, bioinformatics prediction screening and solid-phase chemical synthesis. The antibacterial peptide is effective to gram-negative and gram-positive bacteria and fungi; no obvious hemolytic activity exists below 64 mu M, the safety is high, and the stability in a physiological salt environment is high. Meanwhile, the antibacterial peptide shows low hemolytic activity and low cytotoxicity, has no in-vivo toxicity to mice within the dosage range of 10-40 mg / kg and can also regulate serum metabolism indexes in a targeted manner, and the action mechanism of the antibacterial peptide is that the antibacterial effect is achieved by reducing the membrane fluidity, increasing the outer membrane permeability, destroying the plasma membrane potential and increasing the intracellular active oxygen level. The antibacterial peptide serves as an antibiotic replacement therapy and provides an innovative solution for green animal husbandry development.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antimicrobial peptide derived from porcine intestinal microorganisms and its application. Background Technology

[0002] The deterioration of pig health caused by intensive farming is becoming increasingly prominent, and the spread of drug resistance due to the overuse of traditional antibiotics is exacerbating the industry crisis. Exploring new disease control strategies is now urgently needed. Antimicrobial peptides, as naturally occurring immunomodulatory molecules in organisms, have become one of the most promising candidates for antibiotic alternatives due to their unique mechanism of action (primarily acting on bacterial cell membranes, less likely to induce drug resistance), rapid metabolism with no residue, and low toxicity to host cells. The gut microbiota, as a key regulator of host health, not only participates in nutrient metabolism and immune regulation but also produces a large number of antimicrobial metabolites through interspecies competition or host interactions. Among these, gut microbial-derived antimicrobial peptides, due to their adaptation to the host's physiological environment and strong specificity, have become a core resource for the research and development of novel antimicrobial peptides.

[0003] Existing research has successfully identified hundreds of novel antimicrobial peptides from the gut microbiomes of model animals such as mice and rabbits using deep learning algorithms. However, systematic research on antimicrobial peptides derived from the gut microbiota of livestock (especially pigs) is still in its early stages. On the one hand, reported antimicrobial peptides from livestock mainly focus on host-secreted defensins and cathelicidin families, with insufficient exploration of antimicrobial peptides derived from gut microbiota. On the other hand, some discovered microbial-derived antimicrobial peptides suffer from narrow antimicrobial spectrum, high hemolytic activity, and poor stability under physiological saline conditions, making it difficult to meet practical application requirements. Therefore, it is necessary to screen and optimize novel antimicrobial peptides from the pig gut microbiota that possess broad-spectrum antimicrobial activity, low toxicity, and high physiological stability. This has significant theoretical and applied value for promoting the development of green animal husbandry and solving the problem of antibiotic resistance. Summary of the Invention

[0004] In view of the above shortcomings, the purpose of this invention is to provide an antimicrobial peptide derived from porcine intestinal microorganisms. This antimicrobial peptide is obtained by screening and verification from porcine intestinal metagenomics, and has high bactericidal activity and high physiological salt stability, while also having low hemolytic activity, low cytotoxicity and high in vivo safety.

[0005] The technical solution adopted in this invention is as follows: The amino acid sequence of a porcine intestinal microbial antimicrobial peptide P13 is shown in SEQ ID NO.1.

[0006] Furthermore, its molecular formula is shown in formula (Ⅰ):

[0007]

[0008] Formula (I).

[0009] Another object of the present invention is to provide the use of porcine intestinal microbial-derived antimicrobial peptide P13 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria, and / or Candida albicans.

[0010] Furthermore, the Gram-negative bacteria mentioned are Escherichia coli, Salmonella typhimurium, Salmonella pullorum, or Acinetobacter baumannii.

[0011] Furthermore, the Gram-positive bacteria mentioned are Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus suis, methicillin-resistant Staphylococcus aureus, or Listeria monocytogenes.

[0012] Another object of the present invention is to provide a medicament suitable for treating and / or preventing infections by Gram-negative bacteria, Gram-positive bacteria, and / or Candida albicans, said medicament containing a porcine intestinal microbial-derived antimicrobial peptide P13 as described above.

[0013] This invention has the following advantages and beneficial effects: This invention obtains highly efficient intestinal microbial-derived antimicrobial peptide P13 from the pig intestinal metagenomics through screening and verification. It has both broad-spectrum antimicrobial activity and strong physiological salt stability, and has low hemolytic activity, low cytotoxicity and high in vivo safety, providing an innovative solution for antibiotic alternative therapy and green animal husbandry. Attached Figure Description

[0014] Figure 1 This is the mass spectrum of the antimicrobial peptide P13.

[0015] Figure 2 This is a high-performance liquid chromatogram of the antimicrobial peptide P13.

[0016] Figure 3 This is a graph showing the hemolytic activity of the antimicrobial peptide P13.

[0017] Figure 4 This is a graph showing the cytotoxicity of the antimicrobial peptide P13.

[0018] Figure 5 The graph shows the effect of antimicrobial peptide P13 on mouse body weight and organ weight.

[0019] Figure 6 The figure shows the effect of antimicrobial peptide P13 on blood biochemical indicators in mice.

[0020] Figure 7 The figure shows the effect of antimicrobial peptide P13 on bacterial membrane fluidity.

[0021] Figure 8 This figure shows the effect of antimicrobial peptide P13 on the permeability of bacterial and fungal outer membranes.

[0022] Figure 9 The figure shows the effect of antimicrobial peptide P13 on bacterial and fungal plasma membrane depolarization.

[0023] Figure 10 This figure shows the effect of antimicrobial peptide P13 on the level of intracellular reactive oxygen species in bacteria and fungi. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0026] Example 1

[0027] This embodiment obtains highly efficient gut microbial antimicrobial peptide P13 from porcine gut metagenomics through screening and validation. A porcine gut microbial metagenomic dataset (ID: CNP0000824) was downloaded from the CNGBdb database. This dataset covers samples from feces, cecum, ileum, and jejunum. Macrel software was used to predict cationic antimicrobial peptides, obtaining candidate cationic peptide sequences. CD-Hit software was used to remove redundant sequences, and a sequence similarity threshold of 70% was set to obtain non-redundant candidate sequences. Amplify and amPEPpy software were then used on the non-redundant sequences to obtain high-confidence antimicrobial peptide sequences. High-confidence sequences were screened according to the following criteria to determine candidate peptides: ≤30 amino acids, and ≤3 violations of solubility and synthesis rules (wherein, solubility rule violations included: charged and / or hydrophobic amino acid percentage >45%, absolute total charge of the peptide >1 at pH=7, glycine or proline >1, the first or last amino acid of the peptide carrying a charge, and any amino acid accounting for >25% of the sequence; synthesis rule violations included: two consecutive proline residues in the sequence, a continuous dipeptide motif of aspartic acid followed by glycine and / or aspartic acid followed by proline, the sequence ending with asparagine or glutamine residues, charged residues appearing in every 5 amino acids, and the presence of easily oxidized amino acids including methionine, cysteine, and tryptophan). The minimum inhibitory concentration (MIC) of the candidate peptides against common pathogenic bacteria was determined using the microbroth dilution method, and the obtained antimicrobial peptide P13 showed the best overall antimicrobial activity.

[0028] Table 1. Sequence, actual molecular weight, average hydrophobicity, and charge of antimicrobial peptide P13

[0029]

[0030] The molecular formula is shown in formula (Ⅰ):

[0031]

[0032] Formula (I).

[0033] Example 2

[0034] Solid-phase chemical synthesis of antimicrobial peptides: The peptide backbone was prepared sequentially from the C-terminus to the N-terminus using a peptide synthesizer. 1 eq of the first protecting amino acid, 1.5 eq of DIEA, and DMF were reacted with the resin for 2 h. The resin was dried, washed three times with DMF, and then capped with methanol and DIEA for 1 h. The resin was washed again, and Fmoc was removed with a 20% piperidine DMF solution, reacting for 10 min, repeated twice. After washing the resin, 3 eq of the second amino acid, 3 eq of HOBT, and 3 eq of DIC were reacted with DMF for 1.5 h. These two steps were repeated until the last N-terminal amino acid residue was attached and the N-terminal Fmoc was removed. The resin was washed and dried. The resin and peptide side chain protecting groups were cleaved with 95% TFA + 2% Tis + 2% EDT + 1% H2O, reacting for 2 h. The resin was filtered, the filtrate was washed with ice-cold diethyl ether, centrifuged, and the precipitate was collected as the crude product. The crude product was purified by liquid chromatography and lyophilized. The obtained antimicrobial peptides were analyzed by electrospray ionization mass spectrometry (see appendix). Figure 1 The purity of the antimicrobial peptides is greater than 99% (see appendix). Figure 2 ).

[0035] Example 3

[0036] In vitro antimicrobial activity and physiological salt stability assay of antimicrobial peptides:

[0037] (1) In vitro antibacterial activity assay: Bacteria were streaked onto agar medium, and single colonies were picked and inoculated into broth medium and cultured overnight at 220 rpm and 37 °C. The overnight bacterial strain was inoculated into fresh broth medium and cultured at 220 rpm and 37 °C for 4 h to the logarithmic growth phase. The concentration was adjusted to an OD value of 0.1 at 600 nm and then further diluted 1000 times. Fungi were inoculated into yeast extract peptone agar plates and cultured in a 30 °C incubator for 48 h. The concentration of RPMI-1640 containing morpholine propanesulfonic acid as a buffer was adjusted to an OD value of 0.4 at 600 nm and then further diluted 1000 times. 50 μL of antimicrobial peptides of different concentrations (final concentration 1-64 μM) were added to each well of a 96-well plate, along with an equal volume of bacterial suspension. A negative control (culture medium only) and a positive control (bacteria and culture medium) were also set up and incubated in a 37 °C incubator for 18-20 h. The absorbance was measured at 492 nm using an ELISA reader to determine the minimum inhibitory concentration. The results are shown in Table 2.

[0038] Table 2. In vitro antibacterial activity of antimicrobial peptide P13

[0039]

[0040] Note: "-" indicates that no antibacterial activity was detected within the test range, and the same applies below.

[0041] As shown in Table 2, the antimicrobial peptide P13 exhibits high antimicrobial activity against common pathogens.

[0042] (2) Salt ion stability determination: Escherichia coli ATCC 25922 was selected as a Gram-negative bacterial model, and Staphylococcus aureus ATCC 43300 was selected as a Gram-positive bacterial model to detect the stability of antimicrobial peptides under physiological conditions. Salt powder was dissolved in 0.2% BSA solution, and the minimum inhibitory concentration of antimicrobial peptides was determined, following the steps described in Example 3 (1). Final salt concentrations: NaCl 150 mM, KCl 4.5 mM, MgCl2 1 mM, NH4Cl 6 mM, CaCl2 2 mM, ZnCl2 8 mM, FeCl3 4 mM.

[0043] Table 3. Minimum inhibitory concentration (µM) of antimicrobial peptide P13 after salt ion treatment against Escherichia coli and Staphylococcus aureus.

[0044]

[0045] Table 3 shows that, under physiological concentration conditions, the monovalent ion Na+... + K + and NH4 + divalent Zn 2+ and trivalent ions Fe 3+ It had no significant effect on the activity of antimicrobial peptide P13, only Ca 2+ This resulted in reduced antibacterial activity against E. coli ATCC 25922, while exhibiting higher salt ion stability.

[0046] Example 4

[0047] Safety assessment of antimicrobial peptides:

[0048] (1) Hemolytic activity assay: Fresh human erythrocyte suspension was collected and diluted 10-fold with PBS (pH=7.4). 50 μL of human erythrocyte suspension and an equal volume of antimicrobial peptides of different concentrations (final concentration 1-64 μM) were added to a 96-well plate. A negative control (untreated human erythrocyte suspension) and a positive control (human erythrocyte suspension treated with 0.1% Triton X-100) were also set up. The plates were incubated at 37 ℃ for 1 h. After centrifugation (1000 g, 5 min, 4 ℃), 50 μL of supernatant was collected from the mixture and transferred to a new 96-well plate. The absorbance was measured at OD value of 570 nm using a microplate reader, and the hemolysis rate was calculated. The minimum hemolytic concentration was defined as the concentration of antimicrobial peptide that caused a 10% hemolysis rate. Formula: Hemolysis rate (%) = [(sample OD value)] / [(sample OD value)] 570 nm- Negative control OD 570 nm ) / (Positive control OD 570 nm - Negative control OD 570 nm )]×100%. See attached results. Figure 3 The lower the hemolysis rate, the safer the peptide. Antimicrobial peptide P13 showed no significant hemolytic activity at concentrations below 64 μM.

[0049] (2) Cytotoxicity assay: Mouse macrophages RAW 264.7 and human embryonic kidney cells HEK 293T were cultured in high-glucose DMEM medium, and porcine small intestinal epithelial cells IPEC J2 were cultured in DMEM / F12 mixed medium at 37 ℃ in an incubator containing 5% CO2. The medium contained 10% fetal bovine serum and 1% penicillin-streptomycin solution. After 24 h, the cells were digested with trypsin and collected, and the concentration was adjusted to 1×10⁻⁶. 4 -5×10 4 Cells / mL. 100 μL of cell suspension was seeded into sterile 96-well plates and cultured until 70% confluence. The culture medium was then replaced with 100 μL of fresh medium containing different concentrations of antimicrobial peptides. A negative control (pure culture medium) and a positive control (cell suspension) were also included. After 24 h of culture, 50 μL of 5 mg / mL thiazolyl blue (MTT) solution was added to each well, and the plates were cultured for 3 h. Then, the MTT was removed, and 100 μL of dimethyl sulfoxide solution was added to each well. The plates were incubated at room temperature for 15 min. The absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated. Formula: Cell viability (%) = [(sample OD value)] / [sample OD value]. 570 nm - Negative control OD 570 nm ) / (Positive control OD 570 nm - Negative control OD 570 nm )]×100%. See attached results. Figure 4 At a concentration of 32 µM, the survival rate of all three cell types was above 70%, and even at a concentration of 64 µM, the cell survival rate remained above 50%, indicating that P13 has low cytotoxicity.

[0050] (3) In vivo safety assay: Twenty-four 6-8 week old female ICR mice, weighing 20.85±0.56 g, were given free access to water and a maintenance diet for 3 days, and then randomly divided into 4 groups (n=6): a control group and different concentrations of peptide treatment groups (10, 20, and 40 mg / kg antimicrobial peptide). The peptide treatment groups were intraperitoneally injected with 10, 20, and 40 mg / kg P13, respectively, while the control group was intraperitoneally injected with an equal volume of physiological saline. The weight of the mice was measured at 0 and 48 h. After 48 h, the mice were anesthetized and sacrificed. The liver, spleen, lung, and kidney of the mice were weighed, and the relative organ weight was calculated. Formula: Relative organ weight (%) = organ weight / body weight × 100%. Serum was collected, and biochemical indicators were measured. The results are shown in the appendix. Figure 5 ,6 Different letters represent significant differences (p<0.05), and ns indicates no significant difference. Compared with the control group, there were no significant changes in mouse body weight after treatment with different concentrations of antimicrobial peptide P13, and there were no significant differences in the relative organ weights of the spleen, lungs, and kidneys among the groups. Blood biochemical indicators showed no significant differences in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CR) levels among the groups. Compared with the control group, urea nitrogen (BUN) levels decreased after treatment with 10 mg / kg P13, and alkaline phosphatase (ALP) levels decreased after treatment with 20 and 40 mg / kg P13, demonstrating dose-regulating ability. These results indicate that antimicrobial peptide P13 has no significant in vivo toxicity within the dose range of 10-40 mg / kg and has the potential to target and regulate serum metabolic indicators, thus possessing potential clinical application value.

[0051] Example 5

[0052] Detection of the mechanism of action of antimicrobial peptides:

[0053] (1) Membrane fluidity detection: Escherichia coli ATCC 25922 was selected as a Gram-negative bacterial model, and Staphylococcus aureus ATCC 43300 was selected as a Gram-positive bacterial model. After bacterial culture to mid-log phase, the bacterial solution was centrifuged and resuspended until the absorbance at OD value of 600 nm was 0.4. The bacterial solution was incubated with 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan) probe (final concentration 20 μM) at 37 ℃ in the dark for 1 h. 50 μL of different concentrations of antimicrobial peptides were added to an equal volume of bacterial solution and Laurdan mixture in a 96-well plate and incubated at 37 ℃ in the dark for 1 h. The fluorescence intensity was measured using a microplate reader at emission wavelengths of 435 nm, 490 nm, and excitation wavelength of 350 nm, and the generalized polarization (GP) was calculated. Formula: GP = (I 435 nm -I 490 nm ) / (I 435 nm +I 490 nm (Results are attached.) Figure 7 Compared with the control, as the concentration of antimicrobial peptide P13 increased, the fluidity of bacterial cell membranes decreased and their rigidity increased, and a dose-dependent effect was observed.

[0054] (2) Outer membrane permeability detection: *Escherichia coli* ATCC 25922 was selected as the bacterial model, and *Candida albicans* CGMCC 2.2086 was selected as the fungal model. *Escherichia coli* ATCC 25922 and *Candida albicans* CGMCC 2.2086 were grown to mid-log phase. After centrifugation, the bacterial suspensions were washed three times with HEPES buffer and resuspended in HEPES buffer until the absorbance at OD value of 600 nm was 0.2 and 0.4, respectively. The bacterial suspensions were incubated with NPN dye (final concentration 10 μM) at 37 ℃ in the dark for 30 min. 50 μL of antimicrobial peptides of different concentrations and an equal volume of bacterial suspension mixed with NPN were added to a 96-well plate. The fluorescence intensity was measured using a microplate reader at an emission wavelength of 420 nm and an excitation wavelength of 350 nm. The results are shown in the appendix. Figure 8 The antimicrobial peptide P13 can increase the permeability of bacterial and fungal cell membranes in a dose-dependent manner, indicating that it can penetrate bacterial and fungal cell membranes.

[0055] (3) Detection of plasma membrane depolarization: *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 43300, and *Candida albicans* CGMCC 2.2086 were grown to mid-log phase. The bacterial culture was centrifuged, washed three times with HEPES buffer, and resuspended in HEPES buffer until the absorbance at 600 nm (OD value) was 0.05. The bacterial culture was incubated with DiSC3-5 dye (final concentration 0.4 μM) at 37 °C in the dark for 1 h, then saturated KCl solution (final concentration 100 mM, to balance potassium ion concentrations inside and outside the cell membrane) was added, and incubated at 37 °C in the dark for 30 min. 2 mL of the bacterial culture was added to a 24-well plate, and the fluorescence intensity was measured until the values ​​stabilized. Then, different concentrations of antimicrobial peptides were added to the wells, and the fluorescence intensity was measured using a microplate reader at an emission wavelength of 670 nm and an excitation wavelength of 620 nm. Results are shown in the appendix. Figure 9 The antimicrobial peptide P13 can rapidly increase fluorescence intensity and exhibits a dose- and time-dependent effect, indicating that it forms ion channels in the plasma membranes of bacteria and fungi, thereby disrupting the membrane potential.

[0056] (4) Detection of intracellular reactive oxygen species (ROS) levels: *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 43300, and *Candida albicans* CGMCC 2.2086 were grown to mid-log phase. The bacterial suspensions were centrifuged, washed three times with PBS, and resuspended until the absorbance at 600 nm (OD value) was 0.4. 2',7'-dichlorofluorescein diacetate (DCFH-DA) (final concentration 10 μM) was added to the bacterial suspensions and incubated at 37 °C in the dark for 15 min. 50 μL of different concentrations of antimicrobial peptides were mixed with an equal volume of bacterial suspension and DCFH-DA and added to a 96-well plate, incubated at 37 °C in the dark for 1 h. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Results are shown in the appendix. Figure 10 The antimicrobial peptide P13 can increase the level of reactive oxygen species in bacterial and fungal cells in a dose-dependent manner.

Claims

1. A porcine intestinal microbial-derived antimicrobial peptide P13, characterized in that: Its amino acid sequence is shown in SEQ NO.

1.

2. The porcine intestinal microbial-derived antimicrobial peptide P13 according to claim 1, characterized in that, Its molecular formula is shown in formula (Ⅰ): Formula (I).

3. The use of the porcine intestinal microbial-derived antimicrobial peptide P13 according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria, and / or Candida albicans.

4. The application according to claim 3, characterized in that: The Gram-negative bacteria mentioned are Escherichia coli, Salmonella typhimurium, Salmonella pullorum, or Acinetobacter baumannii.

5. The application according to claim 3, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus suis, methicillin-resistant Staphylococcus aureus, or Listeria monocytogenes.

6. A drug suitable for treating and / or preventing infections caused by Gram-negative bacteria and / or Gram-positive bacteria, characterized in that, The drug contains a porcine intestinal microbial-derived antimicrobial peptide P13 as described in claim 1.

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