KR102296949B1 - Antimicrobial polypeptide from Monodelphis domestica with potent antibacterial activity against gram-positive bacteria and antiviral activity, and antimicrobial composition comprising the same - Google Patents

Abstract

The present invention provides a polypeptide of a specific sequence that exhibits strong antibacterial and antiviral activity against E. coli and gram-positive bacteria, and antimicrobial uses thereof.
The newly discovered antimicrobial peptide of the present invention is an antimicrobial material that exhibits strong antibacterial and antiviral activity against E. coli and Gram-positive bacteria, and can be usefully used as a next-generation natural antibiotic or antiviral material that can be applied to the pharmaceutical field. ..

Description

An antimicrobial polypeptide having strong antibacterial and antiviral activity against gram-positive bacteria derived from gray short-tailed opossum, and an antimicrobial composition comprising the same antimicrobial composition comprising the same}

The present invention relates to a gray short-tailed opossum-derived polypeptide and an antimicrobial composition comprising the same, and more particularly, to a gray short-tailed opossum-derived antimicrobial polypeptide having strong antibacterial and antiviral activity against Gram-positive bacteria and It relates to an antimicrobial composition comprising the same.

Antimicrobial peptides (AMPs), also known as host defense peptides, are considered as an alternative to antibiotics due to their functions such as antimicrobial activity and immunomodulatory responses, along with their low potential to induce bacterial drug resistance. is being accepted Cathelicidin is an AMP family with a characteristic structure comprising a well-conserved cathelin-like domain (CLD) with four characteristic cysteines and a mature peptide with biological activity in the C-terminal region. and has been identified as an innate immune defense in most vertebrates. Cathelicidin prepropeptide undergoes post-translational modification through proteolytic cleavage by specific enzymes, releasing an active antimicrobial domain. The number of cathelicidin genes varies from species to species, from 1 in humans to at least 12 in opossums.

Endogenous AMPs have been limitedly identified and identified in a small number of species. However, the availability of genomic sequences of various species combined with AMP databases and bioinformatics tools provides a great opportunity to identify novel AMPs.

Marsupials differ from eutherian mammals, particularly in their reproductive and developmental biology. Newborn marsupials are exposed to harsh environments caused by pathogenic bacteria in burrows or sacs infested with microbes. In this environment, cathelicidins expressed in milk and pup sac endothelium play an essential role in protecting the immunologically immature pups from harmful bacteria. Thus, during the evolution of marsupials, AMP was expanded and diversified. Cathelicidins from the gray short-tailed opossum ( Monodelphis domestica ) have been studied previously, but their genes have not been fully characterized and characterized.

LL-37 is human immunodeficiency virus (HIV), herpes simplex virus type 1 (HSV-1), influenza A virus and Zika virus (ZIKV). Although there are several studies showing that it has antiviral activity against

West Nile virus (WNV) is an arthropod-borne virus belonging to the genus Flavivirus, like Dengue, Zika or Yellow Fever viruses. WNV has recently emerged in many parts of the world and has become an increasing threat to public health. This mosquito-borne virus is associated with human infection and is considered the number one cause of viral encephalitis worldwide.

As the initial site of viral inoculation, the skin provides the first line of defense of the host against flaviviruses and leads to the initiation of the initial innate immune response. The most abundant epidermal cells, keratinocytes, are unable to defend against WNV and express inflammatory and antiviral proteins after infection. Therefore, there is still no antiviral agent that can fight WNV infection or a vaccine that can prevent this pathogen. Therefore, antimicrobial peptides may be a promising research area for novel antiviral and antibacterial therapies.

Peel et al., Microbiology, 2017, 163:1457-1465 (September 1, 2017) Belov et al., Genome Research, 2007, 17:982-991 (February 05, 2007)

The inventors of the present invention have discovered a novel antibacterial peptide that does not trigger antibiotic-resistant bacteria and has broad and strong antibacterial activity, low cytotoxicity and hemolysis, and high stability in plasma, discovery of a peptide exhibiting antiviral activity, and its mechanism In the course of the study, a novel cathelicidin-like sequence was selected through in silico analysis of the entire genome of gray short-tailed opossum, and the predicted antimicrobial activity domain and 2 Antimicrobial polypeptides of a specific sequence were discovered based on the primary structure, and it was found that the newly discovered antimicrobial polypeptides exhibit strong antibacterial and antiviral activity against E. coli and Gram-positive bacteria.

Accordingly, an object of the present invention is to provide a polypeptide having a specific sequence exhibiting strong antibacterial and antiviral activity against E. coli and Gram-positive bacteria, and antimicrobial use thereof.

According to one aspect of the present invention, an antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 5 is provided.

In one embodiment, the amino acid sequence may be derived from the genome of a gray short-tailed opossum.

In one embodiment, the antimicrobial polypeptide may have an amino acid sequence in which hydrophobic residues are 35%, and may have an amino acid sequence in which a net charge ratio is +5.

According to another aspect of the present invention, there is provided an antimicrobial pharmaceutical composition comprising the antimicrobial polypeptide.

In one embodiment, the antimicrobial polypeptide may have antibacterial or antiviral activity against E. coli and Gram-positive bacteria, Escherichia coli , Staphylococcus aureus , Bacillus cereus ) And Enterococcus faecalis ( Enterococcus faecalis ) It may have antibacterial activity against one or more bacteria selected from the group consisting of.

In one embodiment, the antimicrobial polypeptide may have a MIC of 6 μg/ml or less for E. coli and Gram-positive bacteria.

In one embodiment, the antimicrobial polypeptide may have antiviral activity against West Nile virus (WNV).

According to another aspect of the present invention, there is provided a natural preservative comprising the antimicrobial polypeptide.

According to another aspect of the present invention, there is provided a feed additive comprising the antimicrobial polypeptide.

According to another aspect of the present invention, there is provided a cosmetic composition comprising the antimicrobial polypeptide.

According to another aspect of the present invention, there is provided an antibacterial product for hygiene comprising the antimicrobial polypeptide.

According to another aspect of the present invention, there is provided a medical antibacterial product comprising the antimicrobial polypeptide.

According to the present invention, a polypeptide of a specific sequence derived from the genome of a gray short-tailed opossum exhibits strong antibacterial and antiviral activity against E. coli and Gram-positive bacteria, while exhibiting low cytotoxicity and hemolysis, and high stability in plasma. It turned out to have

Therefore, the newly discovered antimicrobial peptide of the present invention is an antimicrobial substance that exhibits strong antibacterial and antiviral activity against E. coli and Gram-positive bacteria. can

1 relates to bacterial membrane permeation disruption by ΔModoCath peptide. B. cereus (ATCC 10876), E. coli (ATCC 25922) and S. aureus (ATCC 6538) were treated with ΔModoCath1, 5, 6 at concentrations of 0.1x, 0.2x, and 1x MIC to TO-PRO-3 iodide and DiOC ₂ (3) fluorescence measured. Nisin and vancomycin were used as controls. (A) The fluorescence of TO-PRO-3 iodide was measured at _{λ ex} 640 nm and λ _{em 700 nm for membrane permeability.} (B) _{Fluorescence of DiOC 2} (3) was measured at _{λ ex} 480 nm and λ _em 530 nm for cell polarity. The log2 ratio of the detected signal between treated and untreated wells is plotted on the Y-axis. Error bars represent the standard deviation of three replicates. Modo 1, 5 and 6 represent ΔModoCath1, 5 and 6, respectively.
2 is a result of WNV replication in human primary keratinocytes treated with ΔModoCath1, 5 and 6. Primary keratinocytes were treated with a final concentration of 16 μg/ml of peptide before infection with WNV at a multiplicity of infection (MOI) of 0.1 for 24 h. Viral load was measured in cell supernatants (A, log10 viral RNA copies/ml) and cell lysates (B, log10 viral RNA copies/ng total RNA) from infected keratinocytes. Data are expressed as mean+SEM of 3 independent experiments. * p <0.05 compared to the infected control group without peptide. Modo 1, 5 and 6 represent ΔModoCath1, 5 and 6, respectively.
3 is a result of confirming the absorption spectrum of TO-PRO-3 iodide and DiOC _{2 (3).} In order to determine the optimal UV excitation wavelength for the two fluorescent dyes, absorbance spectra were measured from 400-700 nm to 10 nm intervals. Absorbance maxima for (A) TO-PRO-3 iodide and (B) DiOC ₂ (3) were detected at 640 nm and 480 nm, respectively.
4 is an in silico genome analysis strategy for identifying and characterizing AMPs. AMP-related sequences were downloaded from the UniProtKB/Swiss-Prot database, and BLAST was performed on the genome of gray short-tailed opossum using 420 representative sequences of 8 families with antimicrobial activity. A total of 56 AMP-like sequences, including 21 cathelicidin-like proteins, were obtained by performing BLAST with searches against NCBI and IDMM databases. Putative cathelicidin-like sequences were further analyzed using SignalP, HMMER, AMPA, DBAASP and PSIPRED. Finally, eight cathelicidin peptides with predicted antimicrobial domains and functional structures were selected as promising candidates with antimicrobial activity.
5 is a comparison of cathelicidin-like sequences in the genome of gray short-tailed opossum. Twenty cathelicidin-like sequences obtained from BLAST, NCBI and IDMM analyzes were compared by multiplex sequence alignment using MUSCLE. A gap and four conserved cysteines in the cathelin-like domain (CLD) are marked with dashes (-) and asterisks (*), respectively. The signal peptide cleavage site is underlined and the signal peptide region is indicated by a dotted line above the aligned sequence. Predicted sites for proteolytic cleavage by neutrophil elastase to form mature cathelicidins are highlighted in gray. The CLD sequences of the top four cathelicidin sequences are different from the rest of the sequences.
6 is a result of AMPA and PSIPRED analysis for prediction of antimicrobial activity key regions and secondary structures. Antimicrobial domain prediction sequences obtained from AMPA analysis are indicated in red. Cylinders, arrows and lines of different colors represent alpha-helix, beta-sheet and coil, respectively.
7 is a comparison of amino acid properties of predicted key sequences with antimicrobial activity among eight cathelicidins. A high degree of similarity in amino acid properties was observed between ΔModoCath1 and 2 and between ΔModoCath6 and 7.
8 is an evaluation of the antimicrobial activity of ΔModoCath1 and 5 after incubation with serum. ΔModoCath1 and 5 were incubated with 50% (v/v) fetal bovine serum (FBS) for 0, 60 and 120 minutes, and their antimicrobial activity against E. coli was evaluated by colorimetry. was determined by measuring the absorbance (OD) at 450 nm. The X and Y axes represent the peptide concentration and absorbance at 450 nm, respectively. Samples containing FBS in the absence of peptides were used as negative controls for intact bacterial growth. (A) ΔModoCath1 showed strong stability in serum even after incubation for 120 min. (B) The antimicrobial activity of ΔModoCath5 at 5 μg/ml was affected by incubation with serum, but was found to be quite stable at concentrations higher than this.
9 is a result of modulation of the WNV-induced antiviral response of human primary keratinocytes by ΔModoCath5. CXCL10, IFIT2, IL28A, ISG20 and viperin mRNA expression was quantified in human primary keratinocytes infected with WNV for 24 h at an MOI of 0.1 in the presence or absence of ΔModoCath 5 at a final concentration of 16 μg/ml. mRNA expression levels were expressed in folds increased over uninfected cultures. Data are expressed as mean+SEM of at least 3 independent experiments. Modo 5 stands for ΔModoCath5.
10 is an evaluation result of the virus killing properties of ΔModoCath1, 5 and 6 for WNV. WNV suspensions were titrated by endpoint dilution assay on Vero cells by pre-incubation at 37° C. for 1 h at a final concentration of 16 μg/ml in the presence or absence of ΔModoCath 1, 5 and 6. Virus titers were expressed as TCID50/ml. Data are expressed as mean+SEM of 3 independent experiments. Modo 1, 5 and 6 represent ΔModoCath1, 5 and 6, respectively.
11 is an in silico structural analysis result of a cathelicidin-like sequence of a gray short-tailed opossum genome using a database.

The present invention provides an antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 5.

In one embodiment, the antimicrobial polypeptide may be a polypeptide isolated from a biological species, expressed by gene cloning, or prepared by chemical synthesis, but is not limited thereto. Preferably, the amino acid sequence may be derived from the genome of a gray short-tailed opossum. Gray short-tailed opossum ( Monodelphis domestica ) belongs to marsupial, and newborn marsupials are expected to be suitable for cathelicidin screening because they grow in an environment exposed to pathogenic bacteria.

In one embodiment, the antimicrobial polypeptide may be from 28 to 200 amino acids, preferably from 28 to 100 amino acids, more preferably from 28 to 50 amino acids.

In one embodiment, the antimicrobial polypeptide may have an amino acid sequence in which hydrophobic residues are 33 to 41%, preferably 35%, and an amino acid having a net charge ratio of +3 to +12, preferably +5. It may have a sequence.

In the present invention, a total of 56 putative AMP-like sequences were identified in the genome of gray short-tailed opossum through in silico analysis, and among these, 19 cathelicidin-like sequences satisfying the characteristics of functional cathelicidin were identified. Nine antimicrobial activity-containing regions were discovered by predicting protein secondary structure and key regions for antimicrobial activity by in silico analysis. Among the 8 ModoCath peptides predicted to have antimicrobial activity, three peptides were selected based on the amino acid characteristics of the core sequence and the sequence homology to the rest of the opossum cathelicidins. Among them, the antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 5 was named ModoCath5, and then this name was used in the antimicrobial test.

Accordingly, the present invention provides an antimicrobial pharmaceutical composition comprising the antimicrobial polypeptide.

In one embodiment, the antimicrobial polypeptide comprises three Gram-negative bacteria [ Escherichia coli , Pseudomonas aeruginosa and Salmonella typhimurium ] and three Gram-positive bacteria [ Staphylococcus aureus ) , Bacillus cereus ( Bacillus cereus ), and Enterococcus faecalis [ , and, in particular, showed excellent antibacterial activity with a MIC of 2 μg/ml or less against Staphylococcus aureus and Enterococcus faecalis.

Gram-negative bacteria are bacteria that are stained red when stained by Gram staining. Most of them have a thin cell wall (about 10 nm) and a large amount of lipopolysaccharide (LPS) on the outside of the cell membrane, and LPS causes inflammation in the body. It causes sepsis and other diseases.

Examples of Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Staphylococcus aureus, Streptococcus pneumoniae, Bacterial yeast (Shigella spp.), Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella typhimurium, Clostridium difficile, Shigella, Yersinia pestis, Pseudomonas aeruginosa ( Psedomonas aeruginosa), Enterobacteriaceae, Haemophilus, Brucella, Legionella, Burkholderia cepacia, Vibrio, Stenopropomonas maltophilia Stenotrophomonas maltophilia), Neisseria.

Gram-positive bacteria are stained purple when stained by Gram staining and have a thick cell wall with a peptidoglycan layer. Gram-positive bacteria include Staphylococci, Streptococci, Enterococci, Bacillus, Corynabacterium, Listeria, Lactobacillus , Actinomyces, and the like, but is not limited thereto.

In general, gram-negative bacteria have a lower infection rate than gram-positive bacteria, but the mortality rate of infected patients is higher, which is a problem. In particular, in the case of nosocomial infections, Gram-positive bacteria such as Staphylococcus aureus and Streptococcus have a higher infection rate, but Gram-negative bacteria include Escherichia coli, Pneumococci, Pseudomonas aeruginosa, Acinetobacter baumanii, etc. It is mainly infected with terminal cancer patients with weakened immune systems, blood cancer patients, and the elderly, so the mortality rate after infection is higher.

In one embodiment, the antimicrobial polypeptide may have antiviral activity, and preferably may have antiviral activity against West Nile virus (WNV). The composition comprising the polypeptide having antiviral activity of the present invention can be used as a pharmaceutical composition having antiviral activity, including a vaccine composition against West Nile virus or a composition for treating West Nile virus infection.

In one embodiment, the pharmaceutical composition may include the antimicrobial polypeptide at a concentration of 1 to 1,000 μg/ml, preferably from 1 to 700 μg/ml, but is not limited thereto, but the subject's body weight, age, sex , health status, diet, administration time, administration method, excretion rate and severity of disease, etc. may vary. In addition, the antimicrobial pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method.

In one embodiment, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers selected from the group consisting of fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents and excipients. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and if necessary, antioxidants, buffers , and other conventional additives such as a bacteriostatic agent may be added.

In addition, the pharmaceutical composition of the present invention may be prepared in the form of an aqueous solution, suspension, emulsion, etc., in various dosage forms such as injection formulations, pills, capsules, granules, ointments, oral administration agents or tablets.

The present invention also provides a natural preservative comprising the antimicrobial polypeptide. As used in the present invention, the term "natural preservative" refers to a component derived from a natural substance for preventing the decay of substances and/or maintaining the freshness of food and beverages by inhibiting the growth of microorganisms such as fungi/bacteria, and generally food, cosmetic or It may be added to prevent deterioration of pharmaceuticals and the like and to maintain its purity during use or storage. The natural preservative composition of the present invention can be used as a pharmaceutical preservative (eg, acne treatment ointment, etc.), cosmetic preservative, food preservative, and the like.

The food preservative may be a food disinfectant or a food preservative, but is not limited thereto. The food preservatives include dihydroacetic acid, potassium sorbate, calcium sorbate, sodium benzoate, potassium benzoate, calcium benzoate, methyl paraoxybenzoate, propyl paraoxybenzoate, sodium propionate, calcium propionate, and the like, ingredients commonly acceptable in food additives. may be included, but is not limited thereto.

The present invention also provides a feed additive comprising the antimicrobial polypeptide. The feed additive may include ingredients commonly used in feed production, such as glucose, peptone, yeast extract, diphosphate, magnesium sulfate, cysteine, but is not limited thereto.

The present invention also provides a cosmetic composition comprising the antimicrobial polypeptide. The cosmetic composition may be in any one formulation selected from the group consisting of lotions, gels, lotions, ointments, creams, packs, emulsions, essences, patches, or sprays.

The present invention also provides an antibacterial product for hygiene comprising the antimicrobial polypeptide. The antibacterial product for hygiene may be a hygiene product generally sold in the market, such as oral hygiene products, feminine hygiene products, infant/child hygiene products, dog hygiene products, household hygiene products, and the like. Oral hygiene products include, for example, mouthwashes, toothbrushes, dental floss, toothpaste, etc., and feminine hygiene products include, for example, sanitary napkins, cotton pads, feminine hygiene products, bath products, etc., and infant/child hygiene products Examples include diapers, baby detergents, bibs, towels, bath products, etc., and dog hygiene products include diapers, toilet pads, mouthwashes, ear cleaners, tear stain erasers, eye cleaners, shampoos, conditioners, Includes toothpaste, etc., and household hygiene products include masks, cotton swabs, wet tissues, soap, shampoo, conditioner, detergent, bathing agent, sitz bath, deodorant, perfume, toilet paper, dishwashing detergent, laundry detergent, and the like.

The present invention also provides a medical antibacterial product comprising the antimicrobial polypeptide. The medical antibacterial product includes, for example, a disinfectant, a disinfectant swab, a bandage, a cotton gauze, a cotton swab, or a suture.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Description and method

1.1. In silico identification of AMP-like sequences in the genome of gray short-tailed opossum

1,173 non-overlapping AMP sequences of mammalian, avian and fish origins were retrieved from the database site [UniProtKB/Swiss-Prot ( http://www.uniprot.org/uniprot/ )] with the query "antimicrobial peptide AND reviewed: yes" downloaded using Consequently, alpha-defensin, apolipoprotein A2, beta-defensin, BPI/LBP superfamily, callycin, cathelicidin , 420 sequences corresponding to eight major AMP families were identified, including hepcidin and LEAP-2. Then, blast analysis [BLASTp and tBLASTn analysis (http://blast.ncbi.nlm.nih.gov)] was performed on the reference genome of opossum (GCF_000002295.2 MonDom5). In addition, a keyword search was performed with the query "cathelicidin AND monodelphis domestica " on the search site [NCBI and Immunome Database for Marsupials and Monotremes (IDMM)].

1.2. In silico functional characterization and naming of cathelicidin-like sequences in the gray short-tailed opossum genome

Exons/introns were predicted using the Splign Transcript to Genomic Alignment Tool ( https://www.ncbi.nlm.nih.gov/sutils/splign ). Signal peptides and CLDs were obtained using SignalP 4.1 server ( http://www.cbs.dtu.dk/services/SignalP/ ) and HMMER ( https://www.ebi.ac.uk/Tools/hmmer/), respectively. decided. DBAASP ( https://dbaasp.org/prediction ) and Antimicrobial Sequence Scanning System (AMPA, http://tcoffee.crg.cat/apps/ampa/do ) were used to predict possible antimicrobial activity. Protein secondary structure was analyzed using PSIPRED protein sequence analysis workbench ( http://bioinf.cs.ucl.ac.uk/psipred/). Proteolysis to determine mature peptide regions using ExPasy PeptideCutter ( http://web.expasy.org/peptide_cutter/ ) and PROtease Specificity Prediction servER (PROSPER; https://prosper.erc.monash.edu.au) The cleavage site was predicted. The mature peptide regions in the predicted cathelicidin sequence of the gray short-tailed opossum were named ModoCath 1 to 19, consistent with previous annotations in IDMM, where “Modo” and “Cath” refer to M. domestica and cathelicidin, respectively. do. ModoCath's hydrophobicity, net charge, molecular weight and sequence similarity can be measured using APD3 ( http://aps.unmc.edu/AP/prediction/prediction_main.php ) and Protparam tool ( https://web.expasy.org/protparam/ ). was used for analysis.

1.3. Bioinformation analysis of ModoCath expression using RNA-seq data

74 RNA-seq results of gray short-tailed opossum were downloaded from the NCBI SRA database ( https://www.ncbi.nlm.nih.gov/sra). The expression level of cathelicidin was determined based on EMC7 (accession, XM_001380762.4). The downloaded fastq sequences were mapped to the full-length coding sequences of 19 ModoCath and EMC7 using a BWA aligner (version 0.7.17). A sorted bam file with uniquely mapped parts was obtained using samtools (version 1.9). Mean depth and extent of expressed transcripts were calculated using bedtools (version 2.25.0) and R software (version 3.6.0).

1.4. Peptide synthesis and antibacterial activity evaluation

Predicted antimicrobial synthesis (solid phase peptide synthesis) of the biological domain via ModoCath1 (N-VKRTKRGARRGLTKVLKKIFGSIVKKAVSKGV-C), ModoCath5 (N-WYQLIRTFGNLIHQKYRKLLEAYRKLRD-C), and ModoCath6 (N-VRRSKRGIKVPSFVKKVLKDVVSESIS-C) biological domains via solid phase peptide synthesis and purified by high performance liquid chromatography (HPLC) using a commercial service (GenScript, Piscataway Township, NJ, USA). The MICs of the synthesized peptides were three gram-positive strains [ Staphylococcus aureus ATCC 6538 (ATCC, Manassas, VA, USA), Bacillus cereus ATCC 10876 and Enterococcus faecalis ATCC 29212] and three gram-negative strains [ Escherichia coli ATCC 25922, Pseudomonas aeruginosa]. ATCC 27853 and Salmonella typhimurium ATCC 14028]. Ampicillin (Ampicillin, Sigma Aldrich, St. Louis, MO, USA) and gentamicin sulfate (Sigma Aldrich) were used as controls for antimicrobial activity. MIC was measured using a colorimetric method specified by the Clinical and Laboratory Standards Institute (CLSI) guidelines (2018) and an assay kit (Microbial Viability Assay Kit-WST, Dojindo, Japan) according to the manufacturer's protocol. Briefly, 4 colonies of each bacteria were inoculated in 5 ml medium (Luria-Bertani (LB) broth medium) at 37°C for 4 to 6 hours. Cells were washed twice with sterile saline (0.9% NaCl) and seeded at a cell density ^{of 10 5 CFU/well into a single well of a 96-well plate.} Then 180 μl/well of fresh Mueller-Hinton broth (MHB) containing different concentrations of each peptide and reference antibiotic was added to the plate. Different concentrations of each peptide and reference antibiotic were serially diluted in 10 μl of MHB and added to each well. Plates were incubated at 37° C. for 6 hours. Cation-controlled MH broth (MH broth, CAMHB) was used for culturing Enterococcus faecalis ( E. faecalis ). Then, 10 μl of colorimetric reagent was added and cells were incubated at 37° C. for 2 hours. UV absorbance was measured for each well at 450 nm using a microplate spectrophotometer (xMark spectrophotometer; Bio-Rad, Hercules, CA, USA). MIC values were determined when the difference in optical density (OD) between treatment and blank (blank, medium and colorimetric reagents only) decreased to less than 0.05. The experiment was performed three times.

1.5. Serum stability of AMP in vitro ( in vitro ) analysis

AMP was dissolved in 50% (v/v) non-inactivated FBS (Hyclone ^™ ) and incubated at 37°C. Samples incubated as described above were used to evaluate antimicrobial activity against E. coli (ATCC 25922) by aliquoting three aliquots at 0, 60 and 120 minutes of incubation.

1.6. Determination of In Vitro Cytotoxicity to Mammalian Cells

Two mammalian cell lines, including HEK293T and human breast cancer cells (MCF7), were cultured in DMEM medium (Dulbecco's modified Eagle's medium, Hyclone™) supplemented with 10% FBS (Hyclone™) and 1% penicillin/streptomycin (Hyclone™). Incubated at 37 °C, 5% CO ₂ until 80% confluence. Cells attached to the substrate were isolated using an enzyme (Accutase, Innovative Cell Technologies, San Diego, CA, USA). ^{In total, 1 × 10 4} to 4 × 10 ⁴ cells in each well of a 96-well plate containing 8, 16, 32 and 64 μg/ml of ModoCath peptide were incubated at 37° C., 5% CO ₂ for 24 hours. . Triton X 100 (Sigma Aldrich) was used as a positive control for complete cytotoxicity and untreated cells as a negative control. After incubation, the medium was removed from the wells and 10 μl of a colorimetric solution (Cell Proliferation Reagent WST-1™; Sigma Aldrich) and 100 μl of DMEM (Hyclone ^™ ) were added to the wells according to the manufacturer's protocol. Absorbance was measured at 440 nm (peptide-treated and negative control) and 650 nm (background) for each well using a ^{microplate reader (xMark™ spectrophotometer; Bio-Rad) and recorded as OD.} Cell viability was calculated using Equation 1 below.

[Equation 1]

All experiments were performed in triplicate.

1.7. Double Dye Membrane Breakdown Assay

Four colonies of B. cereus (ATCC 10876), E. coli (ATCC 25922) and S. aureus (ATCC 6538) were each inoculated into 5 ml LB broth medium and incubated at 37° C. for 5 hours. The cell suspension was centrifuged at 25° C., 3000×g, washed, PBS+ (0.14 M NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO _{4 supplemented with 10 mM glucose and 0.5 mM MgCl 2 )} ; pH 7.4) was resuspended at _{0.1 OD 600.} Simultaneously, blank PBS+ samples without cells and nisin (Sigma Aldrich) and vancomycin (Sigma Aldrich) treated cells were used as controls. Then, TO-PRO-3 iodide (Sigma Aldrich) and DiOC ₂ (3) (Sigma Aldrich) dyes were added to the samples and controls to a final concentration of 625 nM and 10 μM, respectively. Plates were incubated at 25 °C in the dark for 5 min, then cells were treated with ModoCath peptides at final concentrations of 0.1x, 0.2x and 1x MIC, respectively. Then, absorbance spectra for TO-PRO-3 iodide and DiOC ₂ were measured ( FIG. 3 ).

_{The absorbance of each plate was measured at λ ex} 640 nm and λ _em 700 nm, DiOC ₂ (3) for TO-PRO-3 iodide using a fluorescence microplate reader (Gemini EM, Molecular Devices, Sunnyvale, CA, USA). For λ _ex 480 nm and λ _em 530 nm, λ _ex and λ _em mean wavelengths for excitation (λ _ex ) and emission (λ _em ), respectively.

1.8. Isolation and Culture of Normal Human Epidermal Keratinocytes from Skin Samples

The Ethics Committee of the Poitiers Hospital has approved the use of human skin samples for research. All entities provided written informed consent in accordance with the Helsinki Declaration Precautions. Normal abdominal or breast skin samples were obtained from plastic surgery patients and washed with calcium and magnesium-free PBS phosphate buffered saline solution (PBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) after fat removal. The skin sample was cut into pieces of ^{about 125 mm 2} using a scalpel blade. Skin samples were incubated overnight at 4° C. in dispase solution (25 U/ml; Life Technologies, Carlsbad, CA, USA). The epidermal sheet was removed from the dermis, and the keratinocytes were dissociated by trypsin-EDTA (Gibco) at 37° C. for 15 minutes. The cell suspension was then filtered through a 280 μm sterile filter. DMEM medium (Gibco) supplemented with 10% (vol/vol) FBS (Gibco) was added and the suspension was centrifuged at 25° C., 300×g for 10 min. Keratinocyte-serum-free medium supplemented with bovine pituitary extract (25 μg/ml) and recombinant epidermal growth factor (EGF) (0.25 ng/ml; all purchased from Invitrogen, Carlsbad, CA, USA) ( Keratinocyte-Serum Free Medium, K-SFM) was inoculated with keratinocytes at a density ^{of 10 7 cells in a 75-cm 2} tissue culture flask. Cultures were incubated at 37° C. in a humidified atmosphere of 5% CO ₂ until 80% confluence, and then stored frozen in liquid nitrogen until use. Finally, keratinocytes were inoculated in a sterile 24-well incubation plate at ^{a density of 10 5} cells/well in K-SFM supplemented with bovine pituitary extract and EGF and cultured until 80% confluence. The cells were then left in K-SFM alone overnight before the experiment.

1.9. Evaluation of viability of keratinocytes

^{Primary keratinocytes were cultured in a 96-well plate at 4 x 10 4} cells per well in 0.1 ml K-SFM until about 80% confluence, and treated with various concentrations of ModoCath peptide for 24 hours. Cell viability was assessed using Cell Proliferation Kit II (XTT; Roche, Basel, Switzerland) according to the manufacturer's protocol. The XTT label mixture was added after incubation for 24 hours in the presence or absence of the indicated concentrations of peptide.

1.10. WNV strain production

A strain 1 clinical strain of WNV isolated from the human brain during an epidemic in Tunisia in 1997 was isolated by Dr. Repak Gopart [Dr. I. Leparc Goffart (French National Reference Center on Arboviruses, Marseille, France)]. The virus stock was produced in clone cells ( Aedes . albopictus clone C6/36 cell, ATCC® CRL-1660 ^TM ). Cells were cultured in 75-cm ² tissue culture flasks to 50% confluence at 28 °C in Leibovitz's L-15 medium (Gibco) supplemented with 2% tryptose-phosphate (Gibco) and 5% FBS (Gibco). It was cultured until confluence and then infected for 72 hours at a multiplicity of infection (MOI) of 0.01. Cell supernatants of infected cells and uninfected C6/36 cells used as controls were isolated by centrifugation in 50 ml tubes at 1500×g for 15 minutes. The supernatant and virus suspension from the uninfected C6/36 suspension were then ultrafiltered using an Amicon Ultra-4 centrifugal filter unit 100 kDa (Dutscher) at 3000×g for 5 min. Supernatants and virus suspensions from uninfected C6/36 suspensions were frozen at -80°C in cryotubes containing 500 μl of Leibovitz L-15 medium supplemented with 0.5 M sucrose and 50 mM HEPES. ^{Final viral titers were 10 7.97} TCID50 per milliliter (ml) (50% tissue culture infective dose, 50% tissue) as measured using 10-fold serial dilutions of virus samples in Vero cell monolayers (described below) culture infective dose).

1.11. Virus quantification by endpoint dilution analysis

The day before titration, Vero cells were seeded in 96-well plates at a ratio ^{of 2 x 10 3} cells/well in DMEM (Gibco) supplemented with 2% SVF. The suspension was serially diluted from 10 ^{−1 to 10 −9} in DMEM medium supplemented with 2% SVF. Then, 100 μl of each dilution was placed in a row of 6 wells. Initial data were obtained after incubation at 5% CO ₂ , 37 °C for 120 hours. Wells containing cells exhibiting a cytopathic effect were considered positive for viral infection. The virus suspensions were then titered using Karber's method to assess TCID50.

1.12. virus infection

Human primary keratinocyte cultures (60-80% confluent) obtained from 3 different patients were infected at an MOI of 0.1 and incubated in K-SFM (Invitrogen) medium at 5% CO ₂ , 37 °C for 24 h. did. As described below, cell culture supernatants and cell monolayers were collected for virus quantification by RT-qPCR and transcript analysis of inflammatory markers.

1.13. Antiviral Assessment

The antiviral properties of ModoCath peptides were first evaluated by evaluating their effect on the growth kinetics of viruses inoculated into primary human keratinocytes. Keratinocyte cultures from three different patients were incubated with one of the three peptides at a final concentration of 16 μg/ml for 1 hour prior to addition of WNV at an MOI of 0.1 TCID50 per cell. Uninfected cultures with and without peptide were used as controls. _{After incubation for 24 hours at 37 °C in 5% CO 2} in K-SFM (Invitrogen) medium, cell culture supernatants and cell monolayers were collected for virus quantification and transcript analysis of inflammatory markers by RT-qPCR. The virus killing properties of ModoCath peptides directed against viruses were characterized by endpoint dilution using Vero cells by pre-incubating 0.1 ml of a virus stock (described above) with the peptides with the peptides for 1 h at 37 °C prior to titration, as described above. It was evaluated through analysis. The virus titers thus determined were compared with the values of untreated virus suspensions evaluated in the same way.

1.14. RNA extraction

To quantify viral RNA in cell supernatant, 200 μl of total DNA/RNA of keratinocyte supernatant was extracted using a NucliSENS easyMAG® automated system (bioMerieux, Marcy-l'Etoile, France) according to the manufacturer's instructions. To quantify intracellular viral RNA and evaluate the host inflammatory response, total RNA was extracted from the keratinocyte monolayer using a Nucleo-Spin RNA extraction kit according to the manufacturer's instructions (Macherey-Nagel, Duren, Germany). RNA concentration and purity were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific).

1.15. Virus quantification by RT-qPCR

Virus quantification in cell supernatant and keratinocytes was performed as described in Garcia M, et al., 2018. Frontiers in Cellular and Infection Microbiology 8 using a one-step real-time RT-PCR analysis. 12.5 µl of Master Mix (Invitrogen), 0.5 µl (0.2 µM) of forward (5'-GTGCGGTCTACGATCAGTTT-3') and reverse primer (5'-CACTAAGGTCCACACCATTCTC-3'), 0.25 µl (0.1 µM) of 5 'FAM and 3' Dark Quencher probe (5'-AATGTGGGAAGCAGTGAAGGACGA-3'), 0.5 μl SuperScript III reverse transcriptase (Invitrogen) and DNA polymerase Platinum Taq (Invitrogen), 0.5 μl RNase out (Invitrogen), and 5 μl of total RNA to the reaction mixture containing 5.25 μl water. Transcripts produced using a plasmid containing the WNV genome lacking the structural protein coding gene were used to determine the range of the calibration curve. Transcripts were diluted to obtain a calibration curve range for quantifying the viral load of ^{10 2} to 10 ^{7 RNA copies/ml.}

1.16. Transcript analysis of the innate antiviral immune response in keratinocytes

Total RNA (1 μg) was reverse transcribed using the SuperScript II kit (Invitrogen). Quantitative real-time PCR was performed in 96-well plates using a LightCycler 480 system (Roche). The reaction mixture contained 5 μl of AceQ SYBR Green qPCR Master Mix (Vazyme Biotech, Nanjing, China), 1 μM of forward and reverse primers designed using Primer 3 software, and 12.5 ng of cDNA template in a total volume. contained in 10 μl. The PCR conditions were as follows: 40 amplification cycles including 5 min at 95°C, 20 sec at 95°C, 15 sec at 64°C, and 20 sec at 72°C. Relative mRNA expression of target genes was normalized to two independent control housekeeping genes (GAPDH and 28S rRNA genes) and recorded as fold change in ^{RNA: 2ΔΔCT = 2 ΔCTsample-ΔCTreference using the ΔΔCT method.}

2. Results

2.1. gray short-tailed opossum ( Monodelphis domestica ) Identification of 56 AMP genes in genome in silico analysis

Figure 4 describes the strategy for in silico identification of AMP from the gray short-tailed opossum genome. 1 alpha-defensin, 1 apolipoprotein A2, 3 beta-defensin, 21 BPI/LBP superfamily from gray short-tailed opossum A total of 56 putative AMP genes were identified, including 7 callycin, 21 cathelicidin analogs, 1 hepcidin and 1 LEAP-2 (e-value <0.001). Seven of the 21 cathelicidins were not previously described, and the cathelicidin-related peptide Oh-Cath-like isoform X2 and secreted phosphoprotein 24, which did not meet the properties of functional cathelicidins, were After exclusion, they were assigned the putative names ModoCath 13 to 19 (FIG. 11). Conservation of the CLD domain and cysteine motif among 20 opossum cathelicidins is shown in FIG. 5 . The basic proline-rich protein-like isoform X1 was excluded because it had a longer sequence than the others.

2.2. In silico prediction of eight gray short-tailed opossum cathelicidins with antimicrobial activity

Antimicrobial Sequence Scanning System (AMPA, http://tcoffee.crg.cat/apps/ampa/do ), DBAASP (Antimicrobial Sequence Scanning System, http://tcoffee.crg.cat/apps/ampa/do) for 21 cathelicidin-like sequences of opossum to predict protein secondary structure and key regions of antimicrobial activity using the database of Antimicrobial Activity and Structure of Peptides , https://dbaasp.org/prediction) and PSIPRED (protein sequence analysis workbench, http://bioinf.cs.ucl.ac.uk/psipred/ ) database double Rico analyzed. In the above analysis, 8 sequences including ΔModoCath1, 2, 4 to 7, 12 and 19 were strongly predicted to have antimicrobial activity-related structures ( FIGS. 6 and 11 ). As a result of confirming the biochemical characteristics of the key sequence from 8 cathelicidins deduced using APD3 and Protparam, the length of the antimicrobial activity-containing region is 27 to 41 amino acids with a molecular weight of 3.04 to 4.85 kDa. was found to be (Table 1). The ratios of hydrophobic residues and net charge to the core region were 25 to 41% and +4 to +12, respectively. Their sequence similarity to known AMPs is less than 50%, indicating novelty. ΔModoCath12 and ΔModoCath19 shared the same core sequence (FIG. 6 and Table 1).

Table 1 below shows the properties of the antimicrobial active domains for the eight cathelicidins of gray short-tailed opossums selected according to their antimicrobial activity.

2.3. The difference in antimicrobial specificity of ΔModoCath1, 5 and 6

Of the 8 ModoCath peptides predicted to have antimicrobial activity, in the present invention, three types of peptides, ΔModoCath 1, 5 and 6, were chemically synthesized based on the amino acid characteristics of the core sequence and the sequence homology to the rest of the opossum cathelicidin ( 6 and 7). The antimicrobial activity of the peptide was determined by three Gram-negative strains [ Escherichia coli , Pseudomonas aeruginosa and Salmonella typhimurium ] and three Gram-positive strains [ Staphylococcus aureus , Bacillus cere Bacillus cereus , and Enterococcus faecalis ] were evaluated against a panel of bacteria. All three peptides exhibited strong antibacterial activity and showed a difference in bacterial strain specificity (Table 2). ΔModoCath1 showed the strongest activity against gram-positive and gram-negative bacteria with a MIC of 0.75 ~ 3 μg/ml except for B. cereus (30 μg/ml). ΔModoCath5 exhibited antibacterial activity against Gram-positive strains with a MIC of 1.5 to 6 μg/ml. ΔModoCath6 showed bactericidal activity only against E. coli among the tested bacterial panel.

Table 2 below shows the antimicrobial activity of three types of opossum cathelicidin compared to conventional antibiotics against standard bacterial strains.

2.4. Stability evaluation of bactericidal activity of ΔModoCath1 and 5 in serum

In order to apply AMP to the pharmaceutical field, stability in serum is important. In the present invention, the effect of serum on the antimicrobial activity of ΔModoCath1 and 5 at different concentrations was evaluated. Peptides were incubated in 50% fetal bovine serum (FBS; Hyclone ^™ , Logan, UT, USA) for 0 to 120 minutes, and the antimicrobial activity of the peptides against E. coli was evaluated over time ( FIG. 8 ). The antimicrobial activity of ΔModoCath1 was not affected even after 120 min of serum incubation. The activity of ΔModoCath5 was only affected at low concentrations (5-7.5 μg/ml). Therefore, from the above results, it was shown that two cathelicidins, ΔModoCath1 and 5, with broad antimicrobial activity, were not significantly sensitive to inhibitory substances in serum.

2.5. Low to moderate cytotoxicity of ΔModoCath1, 5 and 6 to mammalian cells

Cathelicidins with potent antimicrobial activity can negatively affect mammalian cells. Therefore, cells induced in human embryonic kidney cells (HEK293T), MCF7 cells and human primary keratinocytes by ModoCath by evaluating the viability of cells exposed to various concentrations (8 to 64 μg/ml) of ΔModoCath1, 5 and 6 The degree of damage was assessed (Table 3). For all cells tested for ΔModoCath1 and 6, cell viability was similar to or greater than 90% at concentrations up to 16 μg/ml, indicating that cells were minimally affected at these concentrations. However, ΔModoCath5 showed a change in cell viability from 67% to 96% at the above concentrations, indicating slightly higher cytotoxicity than other ModoCaths. In addition, the viability of the human breast cancer cell line, MCF7, was not significantly affected by the three types of ModoCath, indicating that there was no direct antitumor activity (Table 3).

Table 3 below shows the cytotoxicity of the three opossum cathelicidins to mammalian cells.

2.6. Disruption of bacterial membrane permeability by ModoCath

A fluorescent dye was used to determine the integrity of the membrane. The increased fluorescence intensity of TO-PRO-3 iodide and DiOC ₂ (3) in cells indicates that the dye penetrated into the cytoplasm due to damaged membrane and disruption of membrane potential. Treatment of B. cereus , E. coli and S. aureus with ΔModoCath1, 5, and 6 resulted in increased fluorescence in cells incubated with the dye regardless of cell type ( FIG. 1 ). These results are identical to those observed with the action of Nisin, a lantibiotic known to create pores that disrupt membrane permeability. The lower level of effect on membrane permeability of E. coli by nisin treatment than that of S. aureus and B. cereus is consistent with the activity preference of nisin for gram-positive bacteria rather than gram-negative bacteria.

The disruption of membrane potential indicated by _{DiOC 2} (3) fluorescence after peptide treatment was different for each bacterial strain. However, the degree of change was consistent with the intensity of the bactericidal activity of ModoCath (Table 2). In contrast, vancomycin, which does not destroy the cell membrane, showed no change in the fluorescence results of any of the dyes. Therefore, it was concluded that the antimicrobial activity of the three types of ModoCath identified in the present invention is mediated by the increase in membrane permeability following membrane disruption.

2.7. Strong inhibition of West Nile virus replication by ΔModoCath5

Before examining the activity of ModoCath on whether it inhibits West Nile virus replication, the cytotoxic effect on human primary keratinocytes was first evaluated (Table 3). The antiviral activity of ΔModoCath1, 5 and 6 was tested at a concentration of 16 μg/ml with a cell viability exceeding 90% for the three peptides. As a result of the test, ΔModoCath5 was found to have a strong inhibitory effect on WNV replication in primary keratinocytes, and ModoCath1 and 6 did not exhibit antiviral properties ( FIG. 2 ). The concentration of viral RNA in the supernatant of cells treated with ΔModoCath5 was significantly reduced, reducing virus production by about 500-fold. In addition, in the cell monolayer, ΔModoCath5 treatment showed about 1-log (93%) reduction in WNV viral load.

In the second step, in order to evaluate the potential immunomodulatory properties for the antiviral activity of ModoCath5, the cell innate immune response to WNV infection in the presence and absence of the peptide was identified. The expression profiles of the factors involved in the antiviral response of cells were studied by transcriptomic analysis. The mRNA expression levels of molecules known to have antiviral activity, such as type III interferon interleukin 28A (IL28A), the chemokine CXCL10 and three interferon-stimulating genes (ISG: IFIT2, ISG20 and viperin) were monitored (Fig. 9) ). ΔModoCath5 treatment in keratinocytes during WNV infection did not significantly modulate cellular antiviral responses compared to untreated infected cells. The presence of ΔModoCath5 decreased CXCL10, IFIT2 and IL28A mRNA levels, which may be related to decreased viral replication in keratinocytes treated with the peptide. Although viperin expression was not altered, ΔModoCath5 tended to increase ISG20 expression in WNV-infected keratinocytes.

In addition, the virus killing properties of ΔModoCath1, 5 and 6 were evaluated by measuring WNV infectivity titers after pre-incubation in the presence of these peptides for 1 hour at a final concentration of 16 μg/ml. The infectivity titers measured in the presence of the peptide were compared with those of the untreated virus suspension. A direct virus killing effect of the ModoCath peptide was not observed ( FIG. 10 ). Considering the above results, it can be seen that ΔModoCath5 can strongly inhibit WNV replication in primary human keratinocytes, and the related activation mechanism requires further study.

3. Discussion

Endogenous AMPs are natural substances encoded by various groups of genes in vertebrates to control host microbes or eliminate pathogens. Although a large number of AMPs have been discovered in various species, detailed analyzes of their biological activities critical for their development into clinically viable applications are still lacking. In the present invention, by characterizing the AMP list of gray short-tailed opossums using in silico analysis for cathelicidin, which is widely distributed within the species compared to that of eutherian species, this deficiencies were addressed. . The inventors of the present invention determined eight key sequences of opossum cathelicidin predicted to have potent antimicrobial activity. The selected three peptides, ΔModoCath1, 5 and 6, were shown to have potent antimicrobial activity with different antimicrobial spectra, including potent antiviral activity against WNV.

The antimicrobial activity of ModoCath4, a type of opossum cathelicidin, was confirmed in a previous study [Peel E, et al., 2017. Microbiology 163:1457-1465.]. The determination of the active key sequence for cathelicidin propeptide depends on the predicted enzymatic cleavage site, secondary structure, cationic charge and hydrophobicity. In the present invention, by adjusting the neutrophil elastase cleavage site, the core sequence of three opossum cathelicidins previously thought to have no antibacterial activity was slightly modified. Interestingly, antimicrobial activity against pathogenic bacteria could be detected for all three peptides tested in the present invention, suggesting that accurate determination of the active domain sequence is crucial in evaluating the activity of cathelicidin. These results suggest that 5 sequences not tested in Table 1 may also exhibit antimicrobial activity. Therefore, together with ModoCath4 in the previous study, antimicrobial activity was confirmed against four types of opossum cathelicidin.

Comparison of AMP gene numbers between eutherian and marsupial species revealed a widespread expansion of cathelicidins in marsupials during evolution. 19, 7, 11 and koala (Phascolarctos cinereus ) in gray short-tailed opossum ( Monodelphis domestica ), tammar wallaby ( Macropus eugenii ), Tasmanian devil ( Sarcophilus harrisii ) and koala ( Phascolarctos cinereus ), respectively. Eleven cathelicidin genes were identified. This suggests the importance of cathelicidins in marsupials compared to one gene in humans and mice, and is intended to compensate for the lack of an adaptive immune system in newborn marsupials.

Using publicly available RNAseq data, the analysis of ModoCath1, 2, 4, 5, 7, 8, 16 and 18 from 5 tissues: placenta, lung, spleen, kidney and Meckel's cartilage and anterior malleus. Expression of eight cathelicidins was detected. In particular, ModoCath4 and 8 were expressed at higher levels than others. The diversity of cathelicidin expression was highest in the spleen.

Although the antiviral properties of cathelicidin have been studied less intensively than their bactericidal effects, several compounds including LL-37, protegrin-1, SMAP-29, BMAP-27 and frog temporin Cathelicidin showed antiviral activity against several pathogenic viruses in humans. In the present invention, it was first confirmed that ΔModoCath5 has strong antiviral activity against WNV. In the present invention, only the antiviral activity against WNV was tested, but ΔModoCath5 can exhibit a wide range of antiviral activity against other flaviviruses and enveloped viruses. The antimicrobial spectrum of ModoCath5 appears to be similar to that of LL-37, which exhibits both antibacterial and antiviral activity. LL-37 acts through direct inactivation of viral particles or upregulation of the antiviral response of cells. However, in contrast to LL-37, ΔModoCath5 did not show a direct virus killing effect at the tested concentration ( FIG. 10 ). In addition, ΔModoCath5 did not regulate the production of inflammation mediators in WNV-infected human primary keratinocytes ( FIG. 9 ). Therefore, further experiments are needed to determine the mechanism of antiviral activity of ΔModoCath5 evaluated during keratinocyte infection.

Despite the great interest in the therapeutic use of endogenous AMPs and the great progress to date in the characterization of novel AMPs, including studies on the efficacy and specificity, mechanism of action and mammalian cytotoxicity against pathogens, research on this It is still limited. Animals such as opossums, which possess a large catalog of cathelicidins, could be an interesting model to study the systematic effects of cathelicidins. Given its broad spectrum of bactericidal and antiviral activity, ΔModoCath5 could be an interesting candidate for therapeutic applications.

3. Conclusion

In the present invention, 19 cathelicidin genes (ModoCath) including 7 novel cathelicidin genes were reported by in silico analysis of the opossum genome, and their antimicrobial activity was predicted. The three synthesized peptides corresponding to the mature regions of ModoCath1, 5 and 6 showed different spectra of antimicrobial activity against Gram-positive and negative bacteria and West Nile virus (WNV).

Cytotoxicity to mammalian cells was low to moderate and showed stability in serum. According to the analysis of the present invention, it was confirmed that the bactericidal and antiviral activity of ModoCath was exhibited by membrane disruption and inhibition of viral replication, respectively. ModoCath and its derivatives will be promising candidates for antibacterial and antiviral therapy.

4. Summary

It is an object of the present invention to characterize cathelicidins from gray short-tailed opossum by in silico analysis, and to experimentally verify their antimicrobial effects against various pathogenic bacteria and West Nile virus (WNV). will do A recent genome-wide in silico analysis of the genome assembly of gray short-tailed opossums identified 56 antimicrobial peptides (AMPs) comprising 8 different families. Among the AMPs, in silico analysis was performed to predict the presence of antimicrobial domains for 19 cathelicidins, ie, ModoCath1 to 19. Three of them (ModoCath1, 5 and 6) were selected and experimentally evaluated for their antimicrobial activity. All three peptides exhibited a diverse spectrum of antimicrobial activity against a panel of gram-positive and negative bacterial strains. Cytotoxicity to mammalian cells was low to moderate and showed stability in serum. It was confirmed that the bactericidal activity of ModoCath was induced by membrane disruption by dual-dye membrane disruption assay. In addition, ModoCath5 exhibited strong antiviral activity against West Nile virus (WNV) by inhibiting viral replication, which suggests that possum cathelicidin can develop antimicrobial endogenous substances considering the large number of mammals. This suggests that it can be a useful source. Expression of 8 ModoCaths from 5 tissues was confirmed by analysis of publicly available RNA-seq data. ModoCath or its derivatives could be promising candidates for controlling multidrug-resistant pathogens and inhibiting viral replication, including WNV.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Antimicrobial polypeptide from Monodelphis domestica with potent antibacterial activity against gram-positive bacteria and antiviral activity, and antimicrobial composition comprising the same <130> P190753 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 32 <212> PRT <213> Monodelphis domestica <400> 1 Val Lys Arg Thr Lys Arg Gly Ala Arg Arg Gly Leu Thr Lys Val Leu 1 5 10 15 Lys Lys Ile Phe Gly Ser Ile Val Lys Lys Ala Val Ser Lys Gly Val 20 25 30 <210> 2 <211> 30 <212> PRT <213> Monodelphis domestica <400> 2 Val Lys Arg Thr Lys Arg Gly Ile Lys Lys Gly Ile Ser Lys Val Leu 1 5 10 15 Lys Lys Phe Phe Ser Ser Ser Met Ile Lys Lys Ala Val Ser Lys 20 25 30 <210> 3 <211> 16 <212> PRT <213> Monodelphis domestica <400> 3 Ser Lys Lys Lys Gly Lys Lys Lys Leu Pro Tyr Thr Val Ile Phe Gly 1 5 10 15 <210> 4 <211> 12 <212> PRT <213> Monodelphis domestica <400> 4 Gly Ile Arg Gly Phe Trp Asn Gly Phe Arg Gly Arg 1 5 10 <210> 5 <211> 28 <212> PRT <213> Monodelphis domestica <400> 5 Trp Tyr Gln Leu Ile Arg Thr Phe Gly Asn Leu Ile His Gln Lys Tyr 1 5 10 15 Arg Lys Leu Leu Glu Ala Tyr Arg Lys Leu Arg Asp 20 25 <210> 6 <211> 27 <212> PRT <213> Monodelphis domestica <400> 6 Val Arg Arg Ser Lys Arg Gly Ile Lys Val Pro Ser Phe Val Lys Lys 1 5 10 15 Val Leu Lys Asp Val Val Ser Glu Ser Ile Ser 20 25 <210> 7 <211> 27 <212> PRT <213> Monodelphis domestica <400> 7 Ile Val Arg Arg Ser Lys Arg Gly Ile Lys Val Pro Gly Phe Val Lys 1 5 10 15 Lys Phe Leu Lys Asp Val Val Ser Glu Thr Ile 20 25

Claims (18)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete A pharmaceutical composition for treating West Nile virus (WNV) infection comprising a polypeptide represented by SEQ ID NO: 5 as an active ingredient. 16. The pharmaceutical composition of claim 15, wherein the composition inhibits the replication of West Nile virus. The pharmaceutical composition of claim 15, wherein the concentration of the polypeptide represented by SEQ ID NO: 5 in the composition is 16 μg/ml. A composition for anti-West Nile virus (WNV) comprising a polypeptide represented by SEQ ID NO: 5 as an active ingredient.

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