KR20210038017A - Antibacterial polypeptide from Monodelphis domestica with potent antibacterial activity against gram-negative bacteria and antibacterial composition comprising the same - Google Patents

Abstract

The present invention provides a polypeptide of a specific sequence exhibiting strong antibacterial activity on Gram-negative bacteria and an antibacterial use thereof. The newly discovered antibacterial peptide of the present invention is an antibacterial substance exhibiting strong antibacterial activity on Gram-negative bacteria, thereby being able to be usefully used as a next-generation natural antibiotic that can be applied to a pharmaceutical field.

Description

{Antibacterial polypeptide from Monodelphis domestica with potent antibacterial activity against gram-negative bacteria and antibacterial composition comprising the same}

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

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

Endogenous AMP has been limitedly identified and identified in a few species. However, the availability of genome sequences of various species combined with AMP databases and bioinformatics tools provides a good opportunity to identify novel AMPs.

Marsupials are different from eutherian mammals, especially in reproductive and developmental biology. Newborn marsupials are exposed to the harsh environment of pathogenic bacteria in microbial pouch or burrow. In this environment, cathelicidin, expressed in the endothelium of milk and pups, plays an essential role in protecting immunologically underdeveloped pups from harmful bacteria. Therefore, in the process of marsupial evolution, AMP has been expanded and diversified. Cathelicidin from the gray short-tailed opossum (Mondelphis domestica) has been studied previously, but its gene has not been fully identified and characterized.

LL-37 is a human immunodeficiency virus (HIV), herpes simplex virus type 1 (HSV-1), influenza A virus and Zika virus (ZIKV) Although several studies have shown that it has antiviral activity against antiviral activity, the list and characterization of AMPs exhibiting antiviral activity is still limited.

West Nile virus (WNV) is an arthropod-borne virus belonging to the flavivirus genus, like Dengue, Zika, or Yellow Fever virus. WNV has recently emerged in many parts of the world, making it an increasingly threat to public health. This mosquito-borne virus is linked to human infections and is considered the first 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. Keratinocytes, the most abundant epidermal cells, cannot 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 can be a promising field of research for novel antiviral and antimicrobial therapies.

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

In the process of discovering a novel antimicrobial peptide having high stability in plasma and studying its mechanism, the inventors of the present invention have strong antibacterial activity against Gram-negative bacteria without triggering antibiotic-resistant bacteria. , A novel cathelicidin-like sequence was selected through in silico analysis of the entire genome of gray short-tailed opossum, and based on the antimicrobial active domain and secondary structure predicted from this sequence, specific An antimicrobial polypeptide of the sequence was discovered, and it was found that the newly discovered antimicrobial polypeptide exhibited strong antibacterial activity against Gram-negative bacteria.

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

According to one aspect of the present invention, an antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 6 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 having a hydrophobic residue of 37%, and may have an amino acid sequence having a net charge ratio of +6.

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 an antimicrobial activity against Gram-negative bacteria, and preferably may have an antimicrobial activity against Escherichia coli.

In one embodiment, the antimicrobial polypeptide may be MIC 5 μg/ml or less for Escherichia coli.

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

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

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 antimicrobial 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, it was found that a polypeptide having a specific sequence derived from the genome of a gray short-tailed opossum exhibits strong antimicrobial activity against Gram-negative bacteria, has low cytotoxicity, low hemolysis, and high stability in plasma.

Accordingly, the newly discovered antimicrobial peptide of the present invention is an antibacterial substance exhibiting strong antibacterial activity against Gram-negative bacteria and can be usefully used as a next-generation natural antibiotic that can be applied to the field of medicine.

1 relates to the destruction of bacterial membrane permeation by the ΔModoCath peptide. TO-PRO-3 iodide by treating B. cereus (ATCC 10876), E. coli (ATCC 25922) and S. aureus (ATCC 6538) with ΔModoCath1, 5, 6 at concentrations of 0.1x, 0.2x and 1x MIC And the fluorescence of _{DiOC 2 (3)} It was measured. Nisin and vancomycin were used as controls. (A) For membrane permeability, fluorescence of TO-PRO-3 iodide was measured at _{λ ex} 640 nm and λ _{em 700 nm.} (B) For cell polarity, _{fluorescence of DiOC 2} (3) was measured at _{λ ex} 480 nm and λ _em 530 nm. The log2 ratio of the detected signal between the treated and untreated wells is plotted on the Y-axis. Error bars represent standard deviations of 3 replicate experiments. Modo 1, 5 and 6 represent ΔModoCath1, 5 and 6, respectively.
Figure 2 shows the results 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 hours. Viral load was measured in cell supernatants (A, log 10 viral RNA copies/ml) and cell lysates (B, log 10 viral RNA copies/ng total RNA) from infected keratinocytes. Data are expressed as mean+SEM of 3 independent experiments. * p <0.05 is the result compared to the infected control without peptide. Modo 1, 5 and 6 represent ΔModoCath1, 5 and 6, respectively.
3 is a result of confirming the absorption spectra of TO-PRO-3 iodide and DiOC _{2 (3).} In order to determine the optimal UV excitation wavelengths for the two fluorescent dyes, absorbance spectra were measured at 10 nm intervals at 400-700 nm. The maximum absorbances 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 AMP. By downloading the AMP-related sequence from the UniProtKB/Swiss-Prot database, BLAST was performed on the genome of the gray short-tailed opossum using 420 representative sequences composed of 8 families having antimicrobial activity. By performing BLAST with a search on the NCBI and IDMM databases, 56 AMP-like sequences were obtained, including a total of 21 cathelicidin-like proteins. Putative cathelicidin-like sequences were further analyzed using SignalP, HMMER, AMPA, DBAASP and PSIPRED. Finally, 8 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 multiple sequence alignment using MUSCLE. The gaps and conserved four cysteines in the catelin-like domain (CLD) are marked with a dash (-) and an asterisk (*), respectively. The signal peptide cleavage site is underlined, and the signal peptide region is indicated by a dotted line above the aligned sequence. The predicted site of proteolytic cleavage by neutrophil elastase to form mature cathelicidin is indicated by gray highlights. The CLD sequence of the top four cathelicidin sequences is different from the rest of the sequences.
6 is a result of AMPA and PSIPRED analysis for prediction of key areas of antimicrobial activity and secondary structure. Antimicrobial domain predictive sequences obtained from AMPA analysis are indicated in red. Different colored cylinders, arrows and lines represent alpha-helix, beta-sheet and coil, respectively.
7 is a comparison of the amino acid properties of predicted core sequences with antimicrobial activity among 8 cathelicidins. A high 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 measured 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. In the absence of peptide, a sample containing FBS was used as a negative control for intact bacterial growth. (A) ΔModoCath1 showed strong stability in serum even after incubation for 120 minutes. (B) Antimicrobial activity at 5 μg/ml of ΔModoCath5 was affected by incubation with serum, but it was found to be quite stable at higher concentrations.
9 is a result of regulation of WNV-induced antiviral response of human primary keratinocytes by ΔModoCath5. CXCL10, IFIT2, IL28A, ISG20 and viperin mRNA expressions were quantified in human primary keratinocytes infected with WNV for 24 hours with an MOI of 0.1 in the presence or absence of ΔModoCath 5 at a final concentration of 16 μg/ml. The mRNA expression level was expressed in an increased fold compared to the non-infected 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 against WNV. WNV suspensions were titrated by endpoint dilution assay for Vero cells by pre-incubating at 37° C. for 1 hour at a final concentration of 16 μg/ml with or without 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 structure 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: 6.

In one embodiment, the antimicrobial polypeptide may be isolated from an organism, 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. The gray short-tailed opossum ( Mondelphis domestica ) belongs to marsupial, and newborn marsupials grow in an environment exposed to pathogenic bacteria, so it was expected to be suitable for cathelicidin detection.

In one embodiment, the antimicrobial polypeptide may be 27 to 200 amino acids, preferably 27 to 100 amino acids, and more preferably 27 to 50 amino acids.

In one embodiment, the antimicrobial polypeptide may have an amino acid sequence having a hydrophobic residue of 33 to 41%, preferably 37%, and an amino acid sequence having a net charge ratio of +3 to +12, preferably +6. It may be to have.

In the present invention, a total of 56 putative AMP-like sequences were identified in the genome of a gray short-tailed opossum through in silico analysis, and then 19 sequences satisfying the characteristics of functional cathelicidin in 21 cathelicidin-like sequences were identified. Nine antimicrobial activity-containing regions were identified by predicting the protein secondary structure and key areas of antimicrobial activity by in silico analysis. Of the eight ModoCath peptides predicted for antimicrobial activity, three peptides were selected based on the amino acid properties of the core sequence and sequence homology to the remaining possum cathelicidin. Among them, the antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 6 was named ModoCath6, and then this name was used in the antibacterial test.

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

In one embodiment, the antimicrobial polypeptide is 3 Gram-negative bacteria [ Escherichia coli ), Pseudomonas aeruginosa and Salmonella typhimurium ] and 3 Gram-positive bacteria [ Staphylococcus aureus ), Bacillus cereus (Bacillus cereus), and Enterococcus faecalis ] as a result of evaluating the antibacterial activity against a panel of bacteria, including E. coli , MIC 5 μg / ml or less excellent antimicrobial activity Indicated.

Gram-negative bacteria are bacteria that stain red when stained with the Gram staining method, and the cell wall is thin (about 10 nm) and the cell membrane contains a large amount of lipopolysaccharide (LPS) on the outside, and LPS is an inflammation in the body. By causing sepsis and other diseases.

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

Gram-positive bacteria are stained purple when stained with the Gram stain method, and have a thick cell wall made of a layer of peptidoglycan. Types of Gram-positive bacteria include Staphylococci, Streptococci, Enterococci, Bacillus, Corinabacterium, Listeria, and Lactobacillus. , Actinomyces, and the like, but are 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 infection, Gram-positive bacteria such as Staphylococcus aureus or Streptococcus have a higher infection rate, but Gram-negative bacteria include Escherichia coli, Pneumococcal, Pseudomonas aeruginosa, Acinetobacter Baumanii, and the infection rate is gradually increasing. The mortality rate after infection is higher because it is mainly infected with terminal cancer patients with weakened immune systems, blood cancer patients, and the elderly.

In one embodiment, the pharmaceutical composition may include the antimicrobial polypeptide at a concentration of 1 to 1,000 μg/mL, preferably 1 to 700 μg/mL, but is not limited thereto, and the weight, age, sex, and health of the individual It may vary depending on the condition, diet, administration time, administration method, excretion rate, and severity of disease. 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. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and at least one of these components, and antioxidants, buffer solutions, if necessary. , Other conventional additives such as bacteriostatic agents 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 administrations or tablets.

The present invention also provides a natural preservative comprising the antimicrobial polypeptide. The term "natural preservative" used in the present invention refers to a component derived from a natural substance for preventing spoilage of substances and/or maintaining the freshness of food and beverages by inhibiting the growth of microorganisms such as fungi/bacteria, and generally It can be added to prevent deterioration of medicines, etc. and to maintain its purity during use or preservation. The natural preservative composition of the present invention can be used as a pharmaceutical preservative (eg, acne treatment ointment, etc.), cosmetic preservatives, food preservatives, 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, etc. It 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 manufacturing, such as glucose, peptone, yeast extract, diphosphate, magnesium sulfate, and cysteine, but is not limited thereto.

The present invention also provides a cosmetic composition comprising the antimicrobial polypeptide. The cosmetic composition may be any one formulation selected from the group consisting of lotion, gel, lotion, ointment, cream, pack, emulsion, essence, patch, or spray.

The present invention also provides an antimicrobial product for hygiene comprising the antimicrobial polypeptide. The hygiene antibacterial product may be a hygiene product generally sold on the market, such as oral hygiene products, feminine hygiene products, infant/child hygiene products, dog hygiene products, and life hygiene products. Oral hygiene products include, for example, mouthwash, toothbrush, floss, toothpaste, and feminine hygiene products include, for example, sanitary napkins, cotton pads, feminine cleaners, bath products, and infant/child hygiene products Examples include diapers, baby detergents, bibs, towels, bath products, etc., and dog hygiene products include diapers, bowel pads, mouthwashes, ear cleaners, tear stain erasers, eye cleaners, shampoos, rinses, It includes toothpaste, and household hygiene products include masks, cotton swabs, wet tissues, soap, shampoo, conditioner, detergent, bath agent, sitz bath agent, deodorant, perfume, tissue paper, dish detergent, and laundry detergent.

The present invention also provides a medical antibacterial product comprising the antimicrobial polypeptide. The medical antibacterial products include, for example, disinfectants, disinfectants, bandages, cotton gauze, cotton swabs or sutures.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating 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-redundant AMP sequences of mammalian, avian and fish origin were obtained from the database site [UniProtKB/Swiss-Prot ( http://www.uniprot.org/uniprot/ )] and the query “antimicrobial peptide AND reviewed: yes”. Downloaded using. As a result, alpha-defensin, apolipoprotein A2, beta-defensin, BPI/LBP superfamily, calycin, cathelicidin , Hepcidin and LEAP-2, 420 sequences corresponding to eight major AMP families were identified. 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 " in 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 Splign Transcript to Genomic Alignment Tool ( https://www.ncbi.nlm.nih.gov/sutils/splign). Signal Peptides and CLDs, respectively, using SignalP 4.1 server ( http://www.cbs.dtu.dk/services/SignalP/ ) and HMMER ( https://www.ebi.ac.uk/Tools/hmmer/) I decided. Possible antimicrobial activity was predicted using DBAASP ( https://dbaasp.org/prediction ) and Antimicrobial Sequence Scanning System (AMPA, http://tcoffee.crg.cat/apps/ampa/do). Protein secondary structure was analyzed using the 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 of IDMM, where "Modo" and "Cath" mean M. domestica and cathelicidin, respectively. do. ModoCath's hydrophobicity, net charge, molecular weight, and sequence similarity were determined by APD3 ( http://aps.unmc.edu/AP/prediction/prediction_main.php ) and Protparam tool ( https://web.expasy.org/protparam/ ). Analyzed using.

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

74 RNA-seq search results of gray short-tailed opossums 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 sequence was mapped to the full-length coding sequence of 19 ModoCath and EMC7 using the BWA aligner (version 0.7.17). Aligned bam files with uniquely mapped parts were obtained using samtools (version 1.9). The average 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

ModoCath1 (N-VKRTKRGARRGLTKVLKKIFGSIVKKAVSKGV-C), ModoCath5 (N-WYQLIRTFGNLIHQKYRKLLEAYRKLRD-C), and ModoCath6 (N-VRRSKRGIKVPSFVKKVLKDVVSESIS-C). And purified by high performance liquid chromatography (HPLC) using a commercial service (GenScript, Piscataway Township, NJ, USA). MIC of the synthesized peptides were 3 Gram-positive strains [ Staphylococcus aureus ATCC 6538 (ATCC, Manassas, VA, USA), Bacillus cereus ATCC 10876 and Enterococcus faecalis ATCC 29212] and 3 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 the colorimetric method specified by the Clinical and Laboratory Standards Institute (CLSI) guideline (2018) and an assay kit (Microbial Viability Assay Kit-WST, Dojindo, Japan) according to the manufacturer's protocol. Briefly, 4 colonies of each bacterium were inoculated in 5 ml medium (Luria-Bertani (LB) broth medium, Luria-Bertani (LB) broth medium) for 4-6 hours at 37°C. Cells were washed twice with sterile saline (0.9% NaCl) and seeded in a single well of a 96-well plate at ^{a cell density of 10 5} CFU/well. Subsequently, 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. The plate was incubated at 37° C. for 6 hours. Cation-controlled MH broth (CAMHB) was used to cultivate Enterococcus faecalis. Then, 10 μl of colorimetric reagent was added and the 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). The MIC value was determined when the difference in optical density (OD) between the treatment group and the blank value (blank, medium and colorimetric reagent only) was reduced to less than 0.05. The experiment was carried out 3 times.

1.5. In vitro for serum stability of AMP ( in vitro ) analysis

AMP was dissolved in 50% (v/v) non-inactivated FBS (Hyclone ^™ ) and incubated at 37°C. A certain amount was aliquoted three times at 0, 60 and 120 minutes of incubation, and the antimicrobial activity against E. coli (ATCC 25922) was evaluated using the sample incubated as described above.

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™). It was incubated at 37° C. and 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 were used 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 3 times.

1.7. Double dye film breakdown analysis

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

_{The absorbance of each plate was measured in λ ex} 640 nm and λ _em 700 nm, DiOC ₂ (3) for TO-PRO-3 iodide using a fluorescent microplate reader (Gemini EM, Molecular Devices, Sunnyvale, CA, USA). For λ _ex 480 nm and λ _em 530 nm, λ _ex and λ _em denote 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 Poitiers Hospital has approved the use of human skin samples for research. All subjects provided written consent in accordance with the Helsinki Declaration. Normal abdominal or breast skin samples were obtained from patients undergoing plastic surgery, and after fat was removed, they were washed with a PBS phosphate buffered saline solution (PBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) free of calcium and magnesium. 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 a dispase solution (25 U/ml; Life Technologies, Carlsbad, CA, USA). The epidermal sheet was removed from the dermis, and keratinocytes were dissociated at 37° C. for 15 minutes by trypsin-EDTA (Gibco). Then, the cell suspension was filtered through a 280 μm sterile filter. DMEM medium (Gibco) supplemented with 10% (vol/vol) of FBS (Gibco) was added and the suspension was centrifuged at 25° C. and 300×g for 10 minutes. Bovine pituitary extract (25 μg/ml) and recombinant epidermal growth factor (EGF) (0.25 ng/ml; all purchased from Invitrogen, Carlsbad, CA, USA) supplemented keratinocyte-serum-free medium ( 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. The culture was incubated at 37° C. until 80% confluence in a humid atmosphere _{of 5% CO 2, 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. Then, the cells were placed only in K-SFM overnight before the experiment.

1.9. Evaluation of the 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% fusion, and were treated with various concentrations of ModoCath peptide for 24 hours. Cell viability was evaluated using a cell proliferation kit II (XTT; Roche, Basel, Switzerland) according to the manufacturer's protocol. The XTT labeling mixture was added after incubation for 24 hours in the presence or absence of the indicated concentration of peptide.

1.10. WNV strain production

A strain 1 of WNV isolated from the human brain during an epidemic in Tunisia in 1997 was described by Dr. Repark Gopard [Dr. I. Leparc Goffart (French National Reference Center on Arboviruses, Marseille, France). The virus stock was produced in clonal cells ( Aedes . albopictus clone C6/36 cell, ATCC® CRL-1660 ^™ ). Cells were fused 50% at 28° C. in Leibovitz's L-15 medium (Gibco) supplemented with 2% tryptose-phosphate (Gibco) and 5% FBS (Gibco) in a 75-cm ^{2 tissue culture flask.} After incubation until (confluence), it was infected for 72 hours with a multiplicity of infection (MOI) of 0.01. The cell supernatant of infected cells and uninfected C6/36 cells used as a control was separated by centrifugation in a 50 ml tube at 1500 × g for 15 minutes. Thereafter, the supernatant and virus suspension from the uninfected C6/36 suspension were ultrafiltered at 3000 xg for 5 minutes using an Amicon Ultra-4 centrifugal filter unit 100 kDa (Dutscher). The supernatant and virus suspension from the uninfected C6/36 suspension were frozen at -80° C. in a cryotube containing 500 μl of Levovitz L-15 medium supplemented with 0.5 M sucrose and 50 mM HEPES. ^{The final viral titer was 10 7.97} TCID50 (50% tissue culture infectious dose, 50% tissue culture) per milliliter (ml) as measured using a 10-fold serial dilution of the virus sample in a Vero cell monolayer (described below). culture infective dose).

1.11. Virus quantification through endpoint dilution analysis

On the day before titration, Vero cells were inoculated into 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 put into 6 wells in a row. Initial data were obtained after incubation at 37° C. for 120 hours at 5% CO _2. Wells containing cells exhibiting cytopathic effect were considered positive for viral infection. Then, the titer of the virus suspension was measured using the Karber method for evaluating TCID50.

1.12. Virus infection

Human primary keratinocyte culture (60-80% degree of fusion) obtained from 3 different patients was infected with an MOI of 0.1 and incubated for 24 hours at 37°C in _{5% CO 2 in K-SFM (Invitrogen) medium.} I did. As described below, cell culture supernatants and cell monolayers were collected for viral quantification by RT-qPCR and transcriptome analysis of inflammatory markers.

1.13. Antiviral evaluation

The antiviral properties of the ModoCath peptide were first evaluated by evaluating the effect on the growth kinetics of the virus 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 before adding WNV at an MOI of 0.1 TCID50 per cell. Uninfected cultures with and without peptides were used as controls. _{After incubation for 24 hours at 37° C. in 5% CO 2} in K-SFM (Invitrogen) medium, cell culture supernatant and cell monolayer were collected for virus quantification and transcriptome analysis of inflammatory markers by RT-qPCR. The viral killing properties of the ModoCath peptide directly against the virus were determined by pre-incubating with the peptide 0.1 ml of virus stock (described above) with the peptide at 37° C. for 1 hour before titration, as described above, by dilution of the endpoints using Vero cells. It was evaluated through analysis. The virus titer thus determined was compared to the value of the untreated virus suspension evaluated in the same way.

1.14. RNA extraction

In order to quantify viral RNA in the cell supernatant, 200 μl of total DNA/RNA of the keratinocyte supernatant was extracted using a NucliSENS easyMAG® automated system (bioMerieux, Marcy-l'Etoile, France) according to the manufacturer's instructions. In order to quantify intracellular viral RNA and evaluate the host inflammatory response, total RNA from the keratinocyte monolayer was extracted using a Nucleo-Spin RNA extraction kit according to the manufacturer's (Macherey-Nagel, Duren, Germany) instructions. 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 the prior literature [Garcia M, et al., 2018. Frontiers in Cellular and Infection Microbiology 8] using a 1-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 primers (5'-CACTAAGGTCCACACCATTCTC-3'), 0.25 μl (0.1 μM) of 5 'FAM and 3'Dark quencher probe (5'-AATGTGGGAAGCAGTGAAGGACGA-3'), 0.5 μl of SuperScript III reverse transcriptase (Invitrogen) and DNA polymerase platinum Taq (Invitrogen), 0.5 μl of RNase out (Invitrogen), And 5 μl of total RNA was added to the reaction mixture containing 5.25 μl water. The range of the calibration curve was determined using a transcript produced using a plasmid containing a WNV genome without a gene encoding a structural protein. Transcripts were diluted to obtain a range of calibration curves for quantifying viral load of ^{10 2} to 10 ^{7 RNA copies/ml.}

1.16. Transcriptome analysis of 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 the LightCycler 480 system (Roche). The reaction mixture contains 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 total volume. It is contained in 10 μl. PCR conditions were as follows: 40 amplification cycles including 5 minutes at 95°C, 20 seconds at 95°C, 15 seconds at 64°C, and 20 seconds at 72°C. Relative mRNA expression of the target gene was normalized to two independent control housekeeping genes (GAPDH and 28S rRNA genes) and recorded as fold change in ^{RNA: 2ΔΔ CT = 2 ΔCTsample-ΔCTreference using the ΔΔCT method.}

2. Results

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

In Figure 4 the strategy for in silico identification of AMP from the gray short-tailed opossum genome is described. 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 calycin, 21 cathelicidin analogs, 1 hepcidin and 1 LEAP-2 (e-value <0.001). Seven of the 21 cathelicidins have not been previously described, and the cathelicidin-related peptide Oh-Cath-like isoform X2 and secreted phosphoprotein 24, which did not meet the properties of functional cathelicidin, After it was excluded, it was designated as the presumed name ModoCath 13-19 (FIG. 11). Conservation of the CLD domain and cysteine motif among the 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 8 gray short-tailed opossum cathelicidins with antimicrobial activity

Antimicrobial Sequence Scanning System (AMPA, http://tcoffee.crg.cat/apps/ampa/do ), DBAASP (for 21 cathelicidin-like sequences of opossums) to predict the protein secondary structure and key regions of antimicrobial activity In silico using Database of Antimicrobial Activity and Structure of Peptides, https://dbaasp.org/prediction ) and protein sequence analysis workbench (PSIPRED, http://bioinf.cs.ucl.ac.uk/psipred/ ) databases Analyzed. In this assay, it was strongly predicted that 8 sequences including ΔModoCath1, 2, 4 to 7, 12 and 19 will 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 was 27 to 41 amino acids with a molecular weight of 3.04 to 4.85 kDa. Was found to be (Table 1). The ratio of hydrophobic moieties and net charge to the key region was 25 to 41% and +4 to +12, respectively. Their sequence similarity with 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 activity domains against 8 cathelicidins of gray short-tailed opossums selected according to their antimicrobial activity.

2.3. Differences in antimicrobial specificities of ΔModoCath1, 5 and 6

Of the eight ModoCath peptides predicted for antimicrobial activity, in the present invention, three kinds of peptides, ΔModoCath 1, 5 and 6, were chemically synthesized based on the amino acid properties of the core sequence and sequence homology to the remaining possum cathelicidin ( 6 and 7). The antimicrobial activity of the peptide was three Gram-negative strains [ Escherichia coli , Pseudomonas aeruginosa , and Salmonella typhimurium ] and three Gram-positive strains [ Staphylococcus aureus ), Bacillus cerebellum. A panel of bacteria including Bacillus cereus , and Enterococcus faecalis] was evaluated. All of the three peptides showed strong antibacterial activity and showed differences in bacterial strain specificity (Table 2). ΔModoCath1 exhibited the strongest activity against Gram-positive and Gram-negative bacteria with MIC of 0.75 to 3 μg/ml except B. cereus (30 μg/ml). ΔModoCath5 showed antimicrobial activity against Gram-positive strains with MIC of 1.5 to 6 μg/ml. ΔModoCath6 showed bactericidal activity only against E. coli among the tested bacteria panels.

Table 2 below shows the antimicrobial activity of the three types of opossum cathelicidin compared to conventional antibiotics with respect to the standard bacterial strain.

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. The peptide was incubated for 0 to 120 minutes in 50% fetal bovine serum (FBS; Hyclone ^™ , Logan, UT, USA), and the antimicrobial activity of the peptide against E. coli was evaluated over time (FIG. 8 ). The antimicrobial activity of ΔModoCath1 was not affected even after 120 minutes 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 the two cathelicidins ΔModoCath1 and 5, which have broad antimicrobial activity, are not very sensitive to inhibitory substances in serum.

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

Cathelicidin, which has potent antimicrobial activity, can negatively affect mammalian cells. Thus, 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 evaluated (Table 3). For all cells tested for ΔModoCath1 and 6, the cell viability at concentrations of 16 μg/ml or less was similar to or greater than 90%, indicating that the cells were minimally affected at these concentrations. However, ΔModoCath5 showed a change in cell viability of 67% to 96% at this concentration, showing 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 anti-tumor activity (Table 3).

Table 3 below shows the cytotoxicity of three types of possum cathelicidin against mammalian cells.

2.6. Destruction of bacterial membrane permeability by ModoCath

Fluorescent dyes were 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 the cytoplasm due to the damaged membrane and disruption of the membrane potential. Treatment of B. cereus , E. coli and S. aureus with ΔModoCath1, 5, and 6 resulted in an increase in fluorescence in cells incubated with dye regardless of cell type (FIG. 1). These results are the same as observed in the action of Nisin, a lantibiotic known to create pores that destroy membrane permeability. The lower level of effect on the membrane permeability of E. coli by nisin treatment than that of S. aureus and B. cereus is consistent with nisin's activity preference for gram-positive bacteria rather than gram-negative bacteria.

The destruction of the 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 bactericidal activity of ModoCath (Table 2). In contrast, vancomycin, which does not destroy cell membranes, did not show any change in the fluorescence results of any dye. Therefore, it was concluded that the antimicrobial activity of the three ModoCaths identified in the present invention was mediated by an increase in membrane permeability due to membrane destruction.

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 16 μg/ml, which is a concentration in which the cell viability exceeds 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 remarkably reduced, reducing virus production by about 500 times. In addition, in the cell monolayer, ΔModoCath5 treatment showed an approximately 1-log (93%) reduction in WNV viral load.

In the second step, the cellular innate immune response to WNV infection in the presence and absence of a peptide was identified in order to evaluate the potential immunomodulatory properties for the antiviral activity of ModoCath5. The expression profiles of participants involved in the antiviral response of cells were studied by transcriptome analysis. The mRNA expression levels of molecules such as type III interferon interleukin 28A (IL28A), chemokine CXCL10, and three interferon-stimulating genes (ISG: IFIT2, ISG20 and viperin), which are known to have antiviral activity, were monitored (FIG. 9 ). ). ΔModoCath5 treatment in keratinocytes during WNV infection did not significantly modulate cellular antiviral responses compared to in untreated infected cells. The presence of ΔModoCath5 reduced CXCL10, IFIT2 and IL28A mRNA levels, which may have been associated with reduced viral replication in keratinocytes treated with the peptide. Although viperin expression was not changed, 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 the WNV infectious titer after pre-incubation in the presence of these peptides at a final concentration of 16 μg/ml for 1 hour. The infectious titer measured in the presence of the peptide was compared to the infectious titer of the untreated virus suspension. No direct virus killing effect of the ModoCath peptide was 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 mechanism of activation requires further study.

3. Discussion

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

The antimicrobial activity of ModoCath4, a species of possum cathelicidin, was confirmed in a previous study [Peel E, et al., 2017. Microbiology 163:1457-1465.]. Determination of the active key sequence for cathelicidin propeptide depends on the predicted enzyme cleavage site, secondary structure, cationic charge and hydrophobicity. In the present invention, by adjusting the neutrophil elastase cleavage site, the core sequence of three types of opossum cathelicidin, previously thought to have no antimicrobial activity, was slightly modified. Interestingly, it was possible to detect antimicrobial activity against pathogenic bacteria 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 kinds of sequences not tested in Table 1 may also exhibit antimicrobial activity. Thus, along with ModoCath4 of the previous study, antimicrobial activity was confirmed against four types of opossum cathelicidin.

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

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

The antiviral properties of cathelicidin have been studied with less focus than their bactericidal effect, but several, including LL-37, protegrin-1, SMAP-29, BMAP-27, and frog temporin. Cathelicidin has shown antiviral activity against several pathogenic viruses in humans. In the present invention, it was first confirmed that ΔModoCath5 has a strong antiviral activity against WNV. In the present invention, only 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 cellular antiviral responses. However, in contrast to LL-37, ΔModoCath5 did not show a direct virus killing effect at the concentration tested (FIG. 10 ). In addition, ΔModoCath5 did not regulate the production of tomite 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 progress to date for the therapeutic use of endogenous AMP and the characterization of new AMPs, including studies on the efficacy and specificity, mechanism of action and cytotoxicity in mammals for pathogens, research on this has been Still limited. Animals such as opossums that carry a large catalog of cathelicidins could be an interesting model to study the systematic effects of cathelicidin. Given its broad bactericidal and antiviral activity, ΔModoCath5 could be an interesting candidate for therapeutic use.

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 maturation 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 is low to moderate and has been shown to be stable in serum. According to the analysis of the present invention, it was confirmed that the bactericidal and antiviral activities of ModoCath are exhibited by membrane destruction and inhibition of viral replication, respectively. ModoCath and its derivatives will be promising candidates for antibacterial and antiviral therapy.

4. Summary

An object of the present invention is to characterize cathelicidin of gray short-tailed opossum by in silico analysis, and experimentally verify their antimicrobial effects against various pathogenic bacteria and West Nile virus (WNV). It is to do. A genome-wide in silico analysis of the recent genome assembly of gray short-tailed opossums identified 56 antimicrobial peptides (AMPs) composed of 8 different families. Among the AMPs, in silico analysis was performed to predict the presence of antimicrobial domains for 19 cathelicidins, that is, ModoCath1 to 19. Three of these (ModoCath1, 5 and 6) were selected and experimentally evaluated for antimicrobial activity. All three peptides exhibited various antimicrobial activity spectra against a panel of Gram positive and negative bacterial strains. Cytotoxicity to mammalian cells was low to moderate and showed stability in serum. By dual-dye membrane disruption assay, it was confirmed that the bactericidal activity of ModoCath was caused by membrane disruption. In addition, ModoCath5 showed a strong antiviral activity against West Nile virus (WNV) by inhibiting viral replication, which is, considering the number of mammals, possum cathelicidin can develop an antimicrobial endogenous substance. It 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 may be promising candidates for controlling multiple drug-resistant pathogens and inhibiting viral replication, including WNV.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Antibacterial polypeptide from Monodelphis domestica with potent antibacterial activity against gram-negative bacteria and antibacterial composition comprising the same <130> P190754 <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 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 (13)

Antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 6. The antimicrobial polypeptide according to claim 1, wherein the amino acid sequence is derived from the genome of a gray short-tailed opossum. The antimicrobial polypeptide according to claim 1, characterized in that it has an amino acid sequence in which the hydrophobic residue is 37%. The antimicrobial polypeptide according to claim 1, which has an amino acid sequence having a net charge ratio of +6. An antibacterial pharmaceutical composition comprising the antimicrobial polypeptide of claim 1. The antimicrobial pharmaceutical composition according to claim 5, wherein the antimicrobial polypeptide has antibacterial activity against Gram-negative bacteria. The antimicrobial pharmaceutical composition according to claim 5, wherein the antimicrobial polypeptide has antibacterial activity against Escherichia coli. The antimicrobial pharmaceutical composition according to claim 5, wherein the antimicrobial polypeptide is MIC of 5 μg/ml or less for Escherichia coli. A natural preservative comprising the antimicrobial polypeptide of claim 1. A feed additive comprising the antimicrobial polypeptide of claim 1. A cosmetic composition comprising the antimicrobial polypeptide of claim 1. An antibacterial product for hygiene comprising the antimicrobial polypeptide of claim 1. A medical antibacterial product comprising the antimicrobial polypeptide of claim 1.

Images