1. Patent Search
  2. Details

ANTIMICROBIAL PEPTIDE SPAMPCIN56-86 FROM SCYLLA PARAMAMOSAIN AND APPLICATIONS THEREOF

20230404076 ยท 2023-12-21

    Inventors

    Cpc classification

    International classification

    Abstract

    A Spampcin.sub.56-86 is provided. A molecular formula of Spampcin.sub.56-86 is C.sub.154H.sub.256N.sub.54O.sub.33S.sub.3, and an amino acid sequence of Spampcin.sub.56-86 is shown in SEQ ID NO: 01.

    Claims

    1. An antimicrobial peptide Spampcin.sub.56-86 from Scylla paramamosain (Spampcin.sub.56-86), wherein: a molecular formula of Spampcin.sub.56-86 is C.sub.154H.sub.256N.sub.54O.sub.33S.sub.3, and an amino acid sequence of Spampcin.sub.56-86 is shown in SEQ ID NO: 01.

    2. A nucleic acid molecule encoding the Spampcin.sub.56-86 according to claim 1.

    3. A method for preparing the Spampcin.sub.56-86 according to claim 1, comprising: synthesizing the Spampcin.sub.56-86 by a chemical solid-phase method.

    4. An application for using the Spampcin.sub.56-86 according to claim 1, comprising: preparing an antimicrobial agent using the Spampcin.sub.56-86, wherein: the antimicrobial agents have inhibitory or bactericidal effects on Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Pseudomona aeruginosa, Aeromonas hydrophila, Aeromonas sobria, Edwardsiella tarda, Pseudomonas fluorescens, Acinetobacter baumannii, drug-resistant Staphylococcus aureus, drug-resistant Acinetobacter baumannii, and drug-resistant Escherichia coli.

    5. An application for using the Spampcin.sub.56-86 according to claim 1, comprising: preparing an anti-fungal agent using the Spampcin.sub.56-86, wherein: the anti-fungal agent has inhibitory or bactericidal effects on Fusarium oxysporum, Fusarium graminearum, or Fusarium solani.

    6. An application for using the Spampcin.sub.56-86 according to claim 1, comprising: preparing a feed additive using the Spampcin.sub.56-86 in preparation.

    7. An application for using the Spampcin.sub.56-86 according to claim 1, comprising: inhibiting or killing Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Pseudomona aeruginosa, Aeromonas hydrophila, Aeromonas sobria, Edwardsiella tarda, Pseudomonas fluorescens, Acinetobacter baumannii, drug-resistant Staphylococcus aureus, drug-resistant Acinetobacter baumannii, and drug-resistant Escherichia coli using the Spampcin.sub.56-86.

    8. An application for using the Spampcin.sub.56-86 according to claim 1, comprising: inhibiting or killing Fusarium oxysporum, Fusarium graminearum, or Fusarium solani using the Spampcin.sub.56-86.

    Description

    BRIEF DESCRIPTION OF THE DRAWING

    [0027] FIGS. 1A, 1B, 1C, and 1D respectively show bactericidal kinetics of the antimicrobial peptide Spampcin.sub.56-86 of Scylla paramamosain (i.e., Spampcin.sub.56-86) against Staphylococcus aureus (i.e., S. aureus), Pseudomonas aeruginosa (i.e., P. aeruginosa), Aeromonas hydrophila (i.e., A. hydrophila), and Escherichia coli (i.e., E. coli) in Embodiment 3 of the present disclosure. The X-axes are time (minutes), and the Y-axes are bactericidal rates (%);

    [0028] FIGS. 2A and 2B respectively show the thermal stability of Spampcin.sub.56-86 against S. aureus and P. aeruginosa in Embodiment 4 of the present disclosure. The X-axes are time (hours), and the Y-axes are the OD.sub.600 values;

    [0029] FIGS. 3A, 3B, and 3C show the inhibition of Spampcin.sub.56-86 against Fusarium graminearum (i.e., F. graminearum) spore germination in Embodiment 5 of the present disclosure. The concentrations of Spampcin.sub.56-86 are 0 Mol (M), 6 M, and 12 M, respectively;

    [0030] FIGS. 4A, 4B, and 4C show the inhibition of Spampcin.sub.56-86 against Fusarium oxysporum (i.e., F. oxysporum) spore germination in Embodiment 5 of the present disclosure. The concentrations of Spampcin.sub.56-86 are 0 M, 6 M, and 12 M, respectively;

    [0031] FIG. 5A shows morphological changes of S. aureus without treatment, and FIG. shows morphological changes of S. aureus after treatment with 12 M Spampcin.sub.56-86 in Embodiment 6 of the present disclosure under scanning electron microscope (SEM) observation.

    [0032] FIG. 6A shows morphological changes of P. aeruginosa without treatment, and FIG. 6B illustrates morphological changes of P. aeruginosa after treatment with 12 M of Spampcin.sub.56-86 under SEM observation in Embodiment 6 of the present disclosure.

    [0033] FIGS. 7A and 7B illustrate graphs of a cytotoxicity test of Spampcin.sub.56-86 using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-phenazine methosulfate (MTS-PMS) assay. The X-axes are concentrations (M) of Spampcin.sub.56-86, and the Y-axes are cell proliferation rates (%).

    DETAILED DESCRIPTION OF THE EMBODIMENTS

    [0034] The technical solution of the present disclosure will be further described below in combination with the accompanying embodiments and drawings.

    Embodiment 1: Preparation of an Antimicrobial Peptide Spampcin.SUB.56-86 .from Scylla paramamosain (i.e., Spampcin.SUB.56-86.)

    [0035] An amino acid sequence of Spampcin.sub.56-86 is as follows:

    [0036] Arg-Arg-Ala-Ala-His-Gly-Leu-Leu-Pro-Arg-Leu-Arg-Ala-Pro-Pro-Pro-Phe-His-Lys-Arg-Cys-Val-Cys-Leu-Cys-Arg-Thr-Ala-Pro-Pro-Pro (SEQ ID NO: 01)

    [0037] The Spampcin.sub.56-86 was entrusted to Kingsley (Jiangsu) Co., Ltd. for chemical solid-phase synthesis, and the Spampcin.sub.56-86 was obtained with a purity of more than 95%. Physicochemical parameters of the Spampcin.sub.56-86 are shown in Table 1.

    TABLE-US-00001 TABLE 1 Physicochemical parameters of the Spampcin.sub.56-86 Physicochemical parameters Spampcin.sub.56-86 Amino acid residue 31 Molecular weight 3488.25 Molecular formula C.sub.154H.sub.256N.sub.54O.sub.33S.sub.3 Isoelectric point 11.71 Net charge +7 Hydrophobicity 41% Total average hydrophilicity 0.410 Molar extinction coefficient 125

    [0038] The Spampcin.sub.56-86 is a positively charged cationic peptide with a small molecular weight and good stability, as shown in Table 1.

    Embodiment 2: Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Spampcin.SUB.56-86

    [0039] Bacterial strains involved in Embodiment 2 are as follows: Staphylococcus aureus (i.e., S. aureus), Staphylococcus epidermidis (i.e., S. epidermidis), Enterococcus Faecium (i.e., E. Faecium), Enterococcus Faecalis (i.e., E. Faecalis), Listeria Monocytogenes (i.e., L. Monocytogenes), Escherichia coli (i.e., E. coli), Pseudomona aeruginosa (i.e., P. aeruginosa), Aeromonas hydrophila (i.e., A. hydrophila), Aeromonas sobria (i.e., A. sobria), Edwardsiella tarda (i.e., E. tarda), Pseudomonas fluorescens (i.e., P. fluorescens), Acinetobacter baumannii (i.e., A. baumannii), drug-resistant S. aureus, drug-resistant A. baumannii, drug-resistant E. coli, Fusarium oxysporum (i.e., F. oxysporum), Fusarium graminearum (i.e., F. graminearum), or Fusarium solani (i.e., F. solani). A. sobria and E. tarda are obtained from Freshwater Fisheries Research Institute of Fujian. The drug-resistant S. aureus, the drug-resistant A. baumannii, and the drug-resistant E. coli are clinical isolates and are provided by the Second Affiliated Hospital of Fujian Medical University, and the rest of the bacterial strains are purchased from the China General Microbiological Culture Collection Center (CGMCC).

    [0040] The specific method was as follows:

    [0041] (1) Bacteria, such as S. aureus, S. epidermidis, E. faecium, E. faecalis, L. monocytogenes, E. coli, P. aeruginosa, A. hydrophila, A. sobria, E. tarda, P. fluorescens, A. baumannii, drug-resistant S. aureus, drug-resistant A. baumannii, and drug-resistant E. coli were inoculated on nutrient broth plates and cultured at appropriate temperatures for 18-24 hours. Fungi, such as F. oxysporum, F. graminearum, and F. solani were inoculated on potato dextrose and cultured at 28 C. for 1-7 days.

    [0042] (2) The bacteria and the fungi were then inoculated on corresponding culture mediums: the bacteria and the fungi were further cultured for 18-24 hours. The bacteria were washed away from the corresponding culture medium with 10 mMol/L (mM) sodium phosphate buffer (pH=7.4) to obtain a suspension of the bacteria. The suspension of the bacteria was diluted with a mixture of Mueller Hinton (MH) medium and the sodium phosphate buffer to a final concentration of 3.3*10.sup.4 CFU/mL. Spores of the fungi were washed away from the corresponding culture medium with 10 mM of a sodium phosphate buffer (pH=7.4), and were then diluted with a mixture of the potato dextrose liquid and the sodium phosphate buffer. The concentration of the spores was determined under an optical microscope and adjusted to a final concentration of 5*10.sup.4 cells/mL.

    [0043] (3) The Spampcin.sub.56-86 was dissolved in sterilized MilliQ water, filtered by a 0.22 m pore size membrane, diluted to concentrations of 3 M, 6 M, 12 M, 12 M, 24 M, 48 M, and 96 M, and placed on ice for use.

    [0044] (4) A blank control group, a negative control group, and an experimental group of each of the bacteria and the fungi were set on 96-well cell culture plates, and each of the blank control group, the negative control group, and the experimental group has three parallel samples.

    [0045] The blank control group a: 50 L of the Spampcin.sub.56-86 with 50 L of the corresponding culture medium;

    [0046] The negative control group b: 50 L of sterilized MilliQ water with 50 L of the suspension of the bacteria or the suspension of the fungi; and

    [0047] The experimental group c: 50 L of the Spampcin.sub.56-86 with 50 L of the suspension of the bacteria or the suspension of the fungi.

    [0048] (5) The 96-well cell culture plates were placed in an incubator at 28 C. and cultured for 1-2 days.

    [0049] The results of MIC and MBC of the Spampcin.sub.56-86 are shown in Table 2.

    TABLE-US-00002 TABLE 2 Anti-bacterial activity and anti-fungal activity of the Spampcin.sub.56-86 Microorganism CGMC NO. MIC MBC Gram-positive bacterium S. aureus 1.2465 1.5-3.sup. 3-6 L. monocytogenes 1.10753 1.5-3.sup. 1.5-3.sup. E. faecalis 1.2135 1.5-3.sup. 1.5-3.sup. E. faecium 1.131 .sup.0-1.5 1.5-3.sup. S. epidermidis 1.4260 3-6 3-6 S. aureus QZ19133 6-12 6-12 S. aureus QZ19132 6-12 12-24 Gram-negative bacterium P. aeruginosa 1.2421 1.5-3.sup. 3-6 A. baumannii 1.6769 3-6 3-6 A. sobria 3-6 3-6 A. hydrophila 1.2017 6-12 6-12 E. coli 1.2389 3-6 3-6 E. tarda 3-6 24-48 P. fluorescens 1.3202 1.5-3.sup. 1.5-3.sup. E. coli QZ20147 6-12 6-12 E. coli QZ20148 6-12 6-12 A. baumannii QZ20142 3-6 3-6 A. baumannii QZ20143 3-6 6-12 Fungi F. oxysporum 3.6785 3-6 3-6 F. graminearum 3.4521 1.5-3.sup. 1.5-3.sup. F. solani 3.5840 1.5-3.sup. 3-6 Note: a-b represent minimum inhibitory concentration (MIC) (M) and minimum bactericidal concentration (MBC) (M). a: The highest concentration of the Spampcin.sub.56-86 that induce visible growth of microorganisms. b: The lowest concentration of the Spampcin.sub.56-86 that does not induce visible growth of microorganisms.

    Embodiment 3: A bactericidal Kinetic Curve of the Spampcin.SUB.56-86

    [0050] Staphylococcus Aureus (i.e., S. aureus), Pseudomona Aeruginosa (i.e., P. aeruginosa), Aeromonas hydrophila (i.e., A. hydrophila), and Escherichia coli (i.e., E. coli) were selected to test bactericidal kinetics of the Spampcin.sub.56-86.

    [0051] A specific method in Embodiment 3 is similar to the antimicrobial activity assay described in Embodiment 2. A final concentration of the Spampcin.sub.56-86 was adjusted to 1MBC (S. aureus: 6 Mol/L (M); P. aeruginosa: 6 M; A. hydrophila: 12 M; and E. coli: 6 M).

    [0052] At 2, 8, 10, 20, 25, and 30 minutes of incubation, 6 L of a suspension of S. aureus was diluted into 600 L of Dulbecco's phosphate-buffered saline (DPBS) to obtain a first solution, 20 L of the first solution was coated on a nutrient broth plate, and cultured at 37 C. for 1-2 days to record the number of S. aureus monoclonal, and the percentage of Colony-Forming Units (CFU) was calculated.

    [0053] At 2, 4, 5, 10, 15, 20, and 30 minutes of incubation, 6 L of the suspension of P. aeruginosa was diluted using 600 L of the DPBS to obtain a second solution, 40 L of the second solution was coated on the nutrient broth plate, and cultured at 37 C. for 1-2 days to record the number of P. aeruginosa monoclonal, and the percentage of CFU was calculated.

    [0054] At 10, 30, 60, 120, and 150 minutes of incubation, 6 L of the suspension of A. hydrophila was diluted using 720 L of the DPBS to obtain a third solution, and 20 L of the third solution was coated on the nutrient broth plate, and cultured at 28 C. for 1-2 days to record the number of A. Hydrophila monoclonal, and the percentage of CFU was calculated.

    [0055] At 10, 15, 20, 30, and 60 minutes of incubation, 6 L of the suspension of E. coli l was diluted using 720 L of the DPBS to obtain a fourth solution, and 20 L of the fourth solution was coated on the nutrient broth plate, and cultured at 37 C. for 1-2 days to record the number of E. coli monoclonal, and the percentage of CFU was calculated.

    [0056] Referring to FIGS. 1A, 1B, 1C, and 1D, the percentage of CFU is defined relative to the CFU obtained in the control group.

    Embodiment 4: The Thermal Stability of Anti-Bacterial Activity of the Spampcin.SUB.56-86

    [0057] Staphylococcus aureus (i.e., S. aureus) and Pseudomona aeruginosa (i.e., P. aeruginosa) were selected to test the thermal stability of the anti-bacterial activity of the Spampcin.sub.56-86.

    [0058] A specific method in Embodiment 4 is similar to the antimicrobial activity assay described in Embodiment 2. A final concentration of the Spampcin.sub.56-86 was adjusted to 1MBC (S. aureus: 6 Mol/L (M), P. aeruginosa: 6 M) to obtain a Spampcin.sub.56-86 solution. The Spampcin.sub.56-86 solution was heated in boiling water for 10, 20, and 30 minutes, and then placed on ice for use. The Spampcin.sub.56-86 was co-cultured with S. aureus or P. aeruginosa. OD.sub.600 values were measured with a microplate reader at 0, 12, 24, 36, and 48 hours, and the results are shown in FIGS. 2A and 2B.

    Embodiment 5: Microscopic Observations of Spore Germination of Fungi after Treatment with the Spampcin.SUB.56-86

    [0059] Fusarium oxysporum (i.e., F. oxysporum) and Fusarium graminearum (i.e., F. graminearum) were selected to evaluate the effects of the Spampcin.sub.56-86 on the spore germination of the fungi.

    [0060] A specific method in Embodiment 5 is similar to the antimicrobial activity assay described in Embodiment 2. The concentration of the Spampcin.sub.56-86 was adjusted to be 6 Mol/L (M) and 12 M. A final concentration of spores of F. oxysporum and F. graminearum was adjusted to be 5*10.sup.4 cells/mL. Each of the Spampcin.sub.56-86 with the concentration of 6 M and 12 M and a corresponding one of the spores of F. oxysporum and F. graminearum were mixed to even in 96-well cell culture plates, and cultured for 24 hours at 28 C. The spore germination of F. oxysporum and F. graminearum was observed under an optical microscope, as shown in FIGS. 3A, 3B, 3C, 4A, 4B, and 4C.

    Embodiment 6: Scanning Electron Microscope (SEM) Observation of Bacteria after Treatment with the Spampcin.SUB.56-86

    [0061] Staphylococcus aureus (i.e., S. aureus) and Pseudomona aeruginosa (i.e., P. aeruginosa) were selected as testing strains, and a method for preparing SEM samples comprises the following steps:

    [0062] (1) A suspension of S. aureus and P. aeruginosa (OD.sub.600=0.4) are prepared according to the method described in Embodiment 2.

    [0063] (2) The Spampcin.sub.56-86 was dissolved with sterilized pure water and placed on ice for use, and the concentration of the Spampcin.sub.56-86 was adjusted to be 12 Mol/L (M) Spampcin.sub.56-86.

    [0064] (3) The suspension of S. aureus was treated with the 12 M Spampcin.sub.56-86 with the same volume as the suspension of S. aureus at 37 C. for 10 minutes, and the suspension of P. aeruginosa was treated with the 12 M Spampcin.sub.56-86 with the same volume as the suspension of P. aeruginosa at 37 C. for 30 minutes.

    [0065] (4) A fixative solution of glutaraldehyde with the same volume as the 12 M Spampcin.sub.56-86 was added, fixed at 4 C. for 2 hours to obtain first samples.

    [0066] (5) The first samples were dehydrated in a series of concentration of 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100% (volume/volume (v/v)) of ethanol for 15 minutes to obtain second samples.

    [0067] (6) After a gold spray, the second samples are observed and photographed by a scanning electron microscope (SEM). The results are shown in FIGS. 5A, 5B, 6A, and 6B.

    Embodiment 7: Determination of Cytotoxicity of the Spampcin.SUB.56-86

    [0068] Human embryonic kidney 293T cells (HEK-293T) and hemocytes of Scylla paramamosain (i.e., S. paramamosain) were selected to test the cytotoxicity effects of the Spampcin.sub.56-86.

    [0069] (1) The hemocytes of S. paramamosain and HEK-293T were harvested, and the cell concentrations of the hemocytes of the S. paramamosain and HEK-293T are adjusted to 110.sup.5 cells/mL to obtain cell suspensions. 100 L of the cell suspensions were seeded in 96-well cell culture plates, and incubated at an appropriate temperature.

    [0070] (2) The hemocytes of the S. paramamosain and HEK-293T were treated with the Spampcin.sub.56-86 with different concentrations of (0, 3, 6, 12, 24, and 48 M) for 24 hours.

    [0071] (3) The hemocytes of the S. paramamosain and HEK-293T were treated with 20 L of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-phenazine methosulfate (MTS-PMS) reagent for another 2 hours, and then an absorbance value of each well of the 96-well cell culture plates was measured at 492 nm using a microplate reader to evaluate the cytotoxicity of the Spampcin.sub.56-86, and the results are shown in FIGS. 7A and 7B.

    [0072] The aforementioned embodiments are merely some embodiments of the present disclosure, and the scope of the disclosure is not limited thereto. Thus, it is intended that the present disclosure cover any modifications and variations provided they are made without departing from the appended claims and the specification of the present disclosure.