KR101983668B1 - Antimicrobial polypeptide from hetercephalus glaber with strong antimicrobial activity against gram negative bacteria and pharmaceutical composition containing the same - Google Patents

Abstract

The present invention provides an antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 1. The antimicrobial polypeptide of the present invention is a novel cathelicidin protein derived from the Hetercephalus Glaber and exhibits strong antimicrobial activity against Gram-negative multi-drug resistant pathogens, but has low cytotoxicity and hemolysis, It may be used as an antibiotic.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an antimicrobial polypeptide derived from a nematode mouse having a strong antimicrobial activity against gram-negative bacteria, and to a pharmaceutical composition comprising the antimicrobial polypeptide and a pharmaceutical composition comprising the antimicrobial polypeptide and a pharmaceutical composition containing the antimicrobial polypeptide.

The present invention relates to a novel antimicrobial polypeptide derived from a Heterocephalus Glaber, and a pharmaceutical composition comprising the same. More particularly, the present invention relates to a novel antimicrobial polypeptide derived from a bacterium, And a pharmaceutical composition containing the same.

Due to the overuse of antibiotics, multiple drug resistance (MDR) pathogens have occurred and have become a global problem, reducing the efficacy of the major antimicrobials used to treat microbial infections. Antimicrobial peptide (AMP), known as host defense peptide (HDP), is an important component of congenital immunity and is a widely distributed endemic antibiotic. It has a rapid and broad range of antimicrobial activity, Lt; RTI ID = 0.0 > bacterial resistance, < / RTI > These are small, cationic, and amphipathic molecules that interact with microbial cell walls and then microbially die by temporarily puncturing or inhibiting cellular components.

Cathelicidin is a type of AMP found in most vertebrates including humans, monkeys, rats, mice, rabbits, cows, guinea pigs, goats, sheep, horses, birds and ducks. Having a cathelicidin-like domain (CLD), and having an antimicrobial domain in the C-terminal region, including a maturation sequence with a very variable length and biological activity. They are activated by protein cleavage to release CLD and C-terminal mature peptides, which are used for antimicrobial activity against bacterial, fungal and enveloped viruses, angiogenesis and wound healing, It plays an important role in.

Since the first AMP was discovered in frog skin mucosa in the 1960s, more than 5,500 AMPs have been updated in the antimicrobial peptide database (APD). AMP is a gene-encoding polypeptide that can be constitutively expressed or induced by a microbial infection, and thus they can be separated from natural sources such as the skin mucosa of reptiles and amphibians, which are the abundant sources of AMP, May be time consuming and laborious. As an alternative approach, the availability of genomic information from recent high-throughput sequencing can be exploited by combining the genome-wide analysis with its AMP database search and bioinformatics tools, Has become a useful and powerful means for identifying novel AMPs in the sequence.

Bullless mole rats are the longest-lived rodents of very long-lived mammals, with a delayed aging, high fertility until death, and resistance to both spontaneous and experimentally induced cancers. Thus, using its available genomic information, it is very advantageous to investigate the role of AMP, but the function of AMP in the immunity of naked mole rats has not been studied yet.

In this study, in silico analysis of whole genomes of naked mole rats was performed to identify new AMPs to select novel catecholysidine analogues and to compare antimicrobial activity domains predicted from this sequence and 2 Based on the difference structure, the maturation sequence of katelicidin was chemically synthesized to confirm its biological activity. The novel cathelicidin, Hg-CATH, showed very strong activity against gram negative bacteria, but the hemolysis and cytotoxicity were relatively low.

The present invention provides an antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the amino acid sequence may be derived from the genome of a naked mole rats.

In one embodiment, the polypeptide has an alpha-helical structure and may include hydrophobic and positively charged amino acids on the outer surface.

The present invention also provides a pharmaceutical composition for antimicrobial use comprising said antimicrobial polypeptide.

In one embodiment, the polypeptide may be one having antimicrobial activity against gram-negative bacteria.

In one embodiment, the pharmaceutical composition may be one having antimicrobial activity against Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, and S. typhimurium.

In one embodiment, the pharmaceutical composition may comprise the antimicrobial polypeptide at a concentration of 1 to 1,000 μg / ml.

In one embodiment, the pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier selected from the group consisting of a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, a diluent and an excipient.

The present invention also provides a food preservative comprising the antimicrobial polypeptide.

The present invention also provides a feed additive comprising the antimicrobial polypeptide.

The antimicrobial peptides derived from the naked mole rats according to the present invention have low hemolytic and cytotoxic activity and have a strong antimicrobial activity against gram negative bacteria.

Degree 1 shows the secondary structure predicted in the mature peptide region of naked mole rats (Fig. 1A), and helical wheel modeling (Fig. 1B) of the synthesized cathelicidin. The red cylindrical region in Fig. 1A is the? -Helical structure, and the yellow arrow region is the? -Shaped structure. In addition, Fig. 1B shows that the blue circle of the present invention represents a positively charged amino acid, and the yellow color circle represents a hydrophobic amino acid.
From this, the antimicrobial peptide of the present invention has positively charged and hydrophobic amino acids on the outer surface, and it is possible that the intracellular bacteria bind to the cell membrane and cell death through cell membrane penetration is possible.
2 shows the result of analysis of the synthetic antimicrobial peptide (SEQ ID NO: 1) of the present invention using APD3, which is a database of antimicrobial peptides (http://aps.unmc.edu/AP/alignment/align.php? Se2 = SKFFRKARKKLGKGLQKIKNVLRKY) FIG. 2A shows the number and distribution of hydrophobic amino acid residues, FIG. 2B shows the similarity of amino acid sequences compared with other known antimicrobial peptides, and FIG. 2C shows the net charge values.
As can be seen from the above data, hydrophobic residues were placed on the outer surface of the polypeptide of the present invention and could form an alpha-helical structure with a maximum similarity of 45.9% to other species.
Figure 3 shows the results of multi-sequence alignment of the full-length sequence of 14 Kethelicidins from Hg-CATH and 11 mammalian species. The gap is represented by a dash (-), and the same amino acid as the reference Hg-CATH (top) is represented by a dot (.). The four cysteines conserved in the cathelin-like domain (CLD) are shown as star (*). The sequence of the segment at which the mature peptide is active is indicated by an inverted triangle ().
Degree 4 shows phylogenetic analysis results of CLD analysis of 19 keltelicidin from 13 vertebrate animals. 102 amino acid sequences of CLD were collected from each cathelicidin. The name of the species is shown in parentheses. The numbers in each branch represent the bootstrap values to support each branch (1000 replication).
The accession numbers of the sequences are PMAP-36 (NP_001123437.1), PR-39 (NP_999615.1), PMAP-23 (NP_001123448.1), Bac7 (NP_001009301.1), Bac5 NP_777257.1), BMAP-28 (NP_776935.1), CAP18 (NP_001075774.1), rhLL-37 (NP_001028681.1), LL-37 (NP_004336.3), CRAMP (NP_034051.2), rCRAMP (NP_001094194. 1), fowlicidin-1 (NP_001001605.1), follicidin-2 (NP_001020001.2), poulridin-3 (NP_001298106.1), Na-CATH (EU622892) -CATH (EU622894), Bf-CATH (EU622893), and Hg-CATH (XP_004834996.1).
Degree 5 shows the stability of Hg-CATH peptide in serum. A Hg-CATH peptide and 50% fetal bovine serum (FBS, v / v) in after incubation for 0, 30, 60 and 100 minutes for Escherichia coli (E. coli) Antimicrobial activity was investigated. At all time points above, the culture showed a definite antimicrobial activity at 5 / / ml.
Degree 6 shows the cytotoxicity of sHg-CATH peptide to mammalian cell lines, and the effect of sHg-CATH on the 5 mammalian cell lines was evaluated at different concentrations. As a result, cytotoxicity of sHg-CATH was found to be low up to 20 μg / ml.
Degree 7 is the Hg-CATH treated E. coli (SEM) photograph of ATCC 25922. FIG. 7A shows an enlarged image (10,000-fold enlargement) of a control (peptide-untreated), and FIG. 7B shows an enlarged image (enlarged 20,000-fold) of a peptide-treated strain. This is the result of treatment of Hg-CATH in 2x MIC for 2 h in the log phase of bacteria.

The present invention provides an antimicrobial polypeptide having the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the antimicrobial polypeptide may be a polypeptide isolated from a species, expressed by gene cloning, or produced by chemical synthesis, but is not limited thereto.

In one embodiment, the amino acid sequence may be derived from the genome of a naked mole rats. Bullworm mice are the longest-lived rodents, which are characterized by delayed senescence, high fertility until death, resistance to both spontaneous and experimentally induced cancers, so that specific immune proteins different from other organisms can be detected It was expected to be.

In one embodiment, the antimicrobial polypeptide is characterized by having the amino acid sequence of the mature peptide at the 3'end of the cathelicidin protein identified in naked mole rats.

Kettelicidin is an antibacterial protein composed of 10 to 90 amino acids. Structural features include a conserved catecholysin-like domain (CLD) at the 5 'end and a differentially differentiated antimicrobial mature peptide sequence at the 3' end. Particularly, the 3 'end is released to the mature peptide by protein cleavage So that the antimicrobial action can be performed.

In one embodiment, the amino acid sequence of SEQ ID NO: 1 has a length of 15 to 40 amino acids, preferably 20 to 30 amino acids, most preferably 25 amino acids.

In one embodiment, the amino acid sequence of SEQ ID NO: 1 may have a homology of 60% or less, preferably 50% or less, with the amino acid sequence of a conventional antimicrobial polypeptide.

In the present invention, as a result of investigation of hydrophobicity and net charge, and sequence similarity with existing antimicrobial polypeptides using APD3, a database of antimicrobial peptides, the mature peptide has an α-helical structure having a positively charged and hydrophobic amino acid on its outer side .

In one embodiment, the polypeptide has an alpha-helical structure and may include hydrophobic and positively charged amino acids on the outer surface. The distribution of hydrophobic and positively charged amino acids on the outer surface of the polypeptide improves the binding of the target intrinsic microbes, especially Gram-negative microbes, to a new boll wall with a negative (-) charge, It is believed to help to penetrate the cell membrane.

In one embodiment, the antimicrobial polypeptide has an antimicrobial activity against gram negative and gram positive bacteria, preferably a strong antimicrobial activity against gram negative bacteria, and most preferably, Escherichia coli, But not limited to, one or more Gram-negative bacteria selected from the group consisting of Pseudomonas aeruginosa and Salmonella typhimurium.

Gram-negative bacteria are bacteria that are stained red when stained with Gram stain. The cell walls are thin (about 10 nm), and most of the cell membranes contain a large amount of lipopolysaccharide (LPS) on the outside. And causes other diseases including sepsis.

Examples of the gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Staphylococcus aureus, Streptococcus pneumoniae, Such as Shigella spp., Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella typhimurium, Clostridium difficile, Shigella, Yersinia pestis, Pseudomonas aeruginosa Pseudomonas aeruginosa, Enterobacteriaceae, Haemophilus, Brucella, Legionella, Burkholderia cepacia, Vibrio, Stenophropomonas maltophilia Stenotrophomonas maltophilia, and Neisseria.

"Gram-positive bacteria" are stained purple when stained with Gram stain and have thick cell walls of peptidoglycan layers. Examples of gram-positive bacteria include Staphylococci, Streptococci, Enterococci, Bacillus, Corynabacterium, Listeria, Lactobacillus, , Actinomyces, and the like, but are not limited thereto.

Normally, Gram-negative bacteria have a lower infection rate than Gram-positive bacteria, but have a higher mortality rate in infected patients. Gram-positive bacteria such as Staphylococcus aureus and Streptococcus such as Escherichia coli, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Acinetobacter baumannii, and the like are increasing in infection rate in hospitals, The mortality rate after infection is higher because it is infected mainly in terminal cancer patients, hematologic cancer patients and elderly people who have weakened immune system.

The present invention also provides a pharmaceutical composition for antimicrobial use comprising said antimicrobial polypeptide.

In one embodiment, the pharmaceutical composition may comprise the antimicrobial polypeptide at a concentration of 1 to 1,000 μg / ml, preferably 1 to 700 μg / ml, but is not limited to the weight, age, sex, Condition, diet, time of administration, method of administration, excretion rate, severity of disease, and the like. The antimicrobial pharmaceutical composition of the present invention may be administered orally or parenterally in accordance with the intended method.

In one embodiment, the pharmaceutical composition can be used for the treatment of diseases caused by Gram-negative bacteria.

Recently, antibiotic resistance of Gram-negative bacteria has become a problem, and it is urgent to develop new antibiotics to replace them. For example, the monitored pathogens having antibiotic resistance include Escherichia coli, Pseudomonas aeruginosa, Pneumoniae bacillus, Acinetobacter bacteria, Staphylococcus aureus, Streptococcus pneumoniae, Salmonella, Bacterial isolate, and Gonococcus, a single resistance to antibiotics including ciprofloxacin, cefotaxime, ceftazidime, meropenem, imipenem, penicillin, carfenem and the like. Or have multidrug resistance, which is a problem in the treatment of the diseases caused by them.

For example, the diseases caused by them are as follows.

Multidrug-resistant Genus Acinetobacter is present in the human respiratory and gastrointestinal tract, but it causes infections in patients with chronic lung disease, diabetics, long-term inpatients, and resuscitators.

In addition, multiple drug resistant Pseudomonas aeruginosa may be infected by injuries such as burns or surgery, respiratory or eye diseases, and especially in the case of cystic fibrosis and immunodeficiency, lethal sepsis, endocarditis , Peritonitis, malignant otitis externa, chronic purulent otitis media, and spinal osteomyelitis.

In the case of multiple drug resistant enterobacteriaceae (CRE), the major causative agents of enteropathy and food poisoning in humans include E. coli, Salmonella, and Enterobacteriaceae. Enterobacteriaceae is one of the major causative bacteria of nosocomial infection, and it causes mainly pneumonia and may cause death due to sepsis.

Carbapenem-resistant Klebsiella pneumoniae is an intra-oral and intestinal intestinal bacterium that is a causative agent of intracerebral and opportunistic infections. It is mainly caused by respiratory infections causing gastroenteritis, urinary tract infection, meningitis, sepsis and acute pneumonia.

In addition to the above, Neisseria gonorrhoeae, Neisseria meningitidis and Helicobacter pylori are also included.

In one embodiment, the pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier selected from the group consisting of a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, a diluent and an excipient.

The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added.

In addition, the pharmaceutical composition of the present invention can be manufactured in various formulations such as an aqueous solution, a suspension, an emulsion, etc., in the form of injection, pill, capsule, granule, ointment, oral administration agent or tablet.

The present invention also provides a food preservative comprising the antimicrobial polypeptide. The food preservative may be a food fungicide or a food preservative, but is not limited thereto.

Examples of the food preservative include food acceptable additives such as dihydroacetic acid, potassium sorbate, calcium sorbate, sodium benzoate, potassium benzoate, calcium benzoate, methyl paraoxybenzoate, propylparabenzoate, sodium propionate and calcium propionate But are not limited thereto.

The present invention also provides a feed additive comprising the antimicrobial polypeptide. The feed additives may include, but are not limited to, those commonly used in the manufacture of feeds such as glucose, peptone, yeast extract, dibasic phosphorus, magnesium sulfate, cysteine and the like.

Hereinafter, the present invention will be described in more detail with reference to examples. The examples illustrate the invention and do not limit the scope of the invention.

<Examples>

Materials and methods

1. Identification of in silico of AMP from genome of naked mole rats

To identify AMP in naked mole rats, a sequence of 2,488 antimicrobial peptides from UniProtKB / Swiss-Prot was downloaded. The query used was "antimicrobial peptide AND reviewed: yes." We then manually screened 310 sequences consisting of eight representative AMP families from vertebrate animals and identified the genome of the naked mole (taxid: 10181) And non-spontaneous protein sequence data from NCBI (http://blast.ncbi.nlm.nih.gov). As a result, we found that sequences that match the genome of the naked mole rats (E-value &lt; 0.001), and ten catecholysidine-like sequences were obtained for further analysis.

2. In silico analysis of catecholysin-like sequences

To determine the conserved cathelin-like domain (CLD) and mature peptide regions, 10 putative corticillin-like sequences of naked mole rats HMMER (https: // www.ebi.ac.uk/Tools/hmmer/) and AMPA (http://tcoffee.crg.cat/apps/ampa/do). Analysis of the secondary structure of the catecholysidine-like sequence was followed by the PSIPRED protein sequence analysis workbench (http://bioinf.cs.ucl.ac.uk/psipred/), and HeliQuest (http: // heliquest.ipmc.cnrs.fr/). We also determined mature peptide regions in the candidate and named Hg-CATH, where "Hg" refers to the naked mole and "CATH" refers to the cathelicidin. In addition, APD3, which is a representative antimicrobial peptide database (http://aps.unmc.edu/AP/prediction/prediction_main.php), was used to analyze the hydrophobicity, net charge and sequence similarity for AMP present in Hg-CATH .

3. Multiple sequence alignment and phylogenetic analysis

Multiple Sequence Alignment (MSA) analysis by full length sequences of 14 cathelicidins from 11 mammalian species and sequencing of 19 conserved canterin-like domains (CLD) from 13 different species were performed using MUSCLE &Lt; / RTI &gt; Information on the CLD region of each catecholysin was obtained from the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP /) and HMMER. The phylogenetic tree was created using the maximum likelihood method using the Mega program version 7.0 (www.megasoftware.net).

4. Synthesis of sHg-CATH

Based on the secondary structural predictions, we chose the N-terminal region in the Hg-CATH, excluding the nine amino acids at the end of the mature peptide. (N-SKFFRKARKKLGKGLQKIKNVLRKY-C) truncated at the end of Hg-CATH was chemically synthesized by solid phase peptide synthesis and purified by high performance liquid chromatography (HPLC) of GenScript to give sHg-CATH Respectively.

5. Antimicrobial activity test

(A15-CF-027-1S-1), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 29213 and Bacillus cereus ATCC 10876, Streptococcus iniana KCTC 3657, (Synthetic Hg-CATH) antibiotic resistant strains were isolated from 8 strains of Gram-negative and Gram-positive bacteria consisting of Gram-negative and Gram-positive bacteria consisting of Gram-negative and Gram-positive bacteria, and clinically isolated pneumococci (KBN06903599) Activity tests were carried out. Antibacterial activity tests were carried out on all strains except Streptococcus inia KCTC 3657 using a colorimetric method using a cell survival assay kit-WST (Dojindo). According to the manufacturer's protocol, a single colony-derived pre-inoculum of each bacteria in 5 ml of LB medium was prepared by incubating overnight at 37 占 폚. Overnight cultures were washed in 96-well plates with sterile saline (0.9% NaCl) containing 180 μl / well of fresh Mueller-Hinton (MH) and then inoculated at 10 ⁵ cfu / well. SHg-CATH at different, continuously diluted concentrations (0.5-70 [mu] g / ml) were diluted in 10 [mu] l of MH medium and added to each well and incubated at 37 [deg.] C for 6 hours. In addition, 10 占 퐇 of the coloring reagent was added, followed by incubation at 37 占 폚 for another 2 hours. Plates were read at 450 nm UV using a microplate spectrophotometer (Biorad). Since the Streptococcus inei KCTC 3657 grows extremely slowly in the liquid medium, the antimicrobial activity was evaluated using the disk diffusion method. The pre-inoculum of Streptococcus inia KCTC 3657 from a single colony grown in Brain heart infusion broth (BD Diagnostic Systems) was inoculated with sterile saline (0.9%) at OD ₆₀₀ to 0.5 McFarland standard. NaCl), and then plated on cation-regulated MH agar plates containing 5% of horse blood (Hanil Komed).

We added sHg-CATH of different series concentrations (1-64 [mu] g / ml) to a paper disc (Sigma Aldrich), dried and placed on an agar plate, Agar plates were incubated overnight at 37 ° C.

6. In vitro stability test in serum

A single colony of Escherichia coli ATCC 25922 was inoculated into 5 ml of LB medium and incubated overnight at 37 占 폚. The pre-inoculum of E. coli was washed with sterile 0.9% physiological saline and inoculated at 10 &lt; ⁵ &gt; cfu / well in a 96-well plate with 180 [mu] l / well of MH medium. SHg-CATH at various concentrations (1-5 μg / ml) were dissolved in 50% (v / v) fetal bovine serum (FBS; Hyclone ™) and incubated at 37 ° C. for 0, 30, 60 and 100 minutes, respectively . The viability of the cultured peptides against E. coli ATCC 25922 was tested as described above and repeated twice.

7. Hemolytic activity test

Freshly prepared chicken erythrocytes were washed 3 times with phosphate-buffered saline (PBS) and resuspended to 2% (v / v) in PBS. 100 占 퐇 of the suspension was added to a 96-well microtiter plate containing the same volume of sHg-CATH so that the final concentration of the peptide contained the above ranges. PBS and 1% (v / v) Triton-X 100 were used as 0% and 100% hemolytic controls, respectively. The plates were left at 37 占 폚 for 1 hour. The plate was then centrifuged and the supernatant transferred to a new plate. The outflow of hemoglobin in the supernatant was monitored by absorbance at 550 nm using a microplate reader (Perkin-Elmer Victor X4). Results were collected from three separate experiments. The hemolysis rate was defined as: &lt; RTI ID = 0.0 &gt;

Hemolysis rate (%) = 100 × (OD _peptide OD _{negative control} ) / (OD _{positive control} - OD _{negative control} )

8. In vitro cytotoxicity test

(Human fetal kidney cells), HaCaT (human keratinocytes), PK15 (porcine kidney cells), ST ATCC CRL-1746 (pig testicular fibroblasts), and 3D4 (human embryonic kidney cells) in order to confirm the effect of sHg- / 2 (macrophages of swine lung) were used. The cells were isolated by the addition of Accutase (Innovative Cell Technologies) and incubated in Dulbecco's medium containing 5% CO ₂ and 10% FBS (Hyclone ™) and 1% penicillin / streptomycin (Hyclone ^™ ) After culturing to 80% density in Dulbeco's modified Eagle's medium (DMEM; Hyclone ™), 1-4 × 10 ⁴ cells were dispensed into each well of a 96-well plate (Sigma Aldrich) . SHg-CATH, diluted differently from each other to a final concentration of 5, 10, 20, 25, 30 and 64 / / ml, was added to each well of the plate and incubated for 24 hours at 5% CO ₂ and 37 캜. For positive control and negative control for 100% cell hemolysis, wells treated with Triton-X 100 (Sigma Aldrich) and untreated wells were used, respectively. After the culture, the cell proliferation reagent as a color-developing reagent 10㎕ wst-1; was added ^{(Cell proliferation reagent wst-1 ™} Sigma Aldrich Co.) to each well of the plate and the plate for 4 hours, 5% CO ₂ and 37 ℃ Lt; / RTI &gt; according to the protocol of the manufacturer. All experiments were performed three times. To calculate cell viability, the absorbance at 450 nm and 650 nm of each well of the plate was measured using a microplate reader (xMark ™ spectrophotometer; Bio-Rad) and the absorbance results were collected from three separate tests , Cell viability (%) = 100 x (OD _{450 nm} of target sample - OD _{650 nm of} blank) / (OD _{450 nm} of negative control - OD _{650 nm of} blank).

9. Scanning electron microscope (SEM)

In order to study the sterilization mechanism of sHg-CATH against microorganisms, E. coli ATCC 25922 cells of 0.2 OD ₆₀₀ were inoculated in LB medium containing 2 x MIC (1.5 mu g / ml) of sHg-CATH, And cultured for 2 hours. The cultured cells were harvested by centrifugation at 4,500 rpm for 5 minutes, and 1 x PBS was added to the cell pellet and washed. The cells were then treated with 2% paraformaldehyde and 2.5% glutaraldehyde (Sigma Aldrich) in 1X PBS and allowed to stand overnight at 4 ° C to fix. Cells were washed with 1 x PBS and treated with 1% Osmium tetroxide (Sigma Aldrich) in 1 x PBS for 1 hr 30 min at 4 ° C as a post-fixation step. Cells were rinsed three times with sterile water to remove depletion steps with several grades (50%, 70%, 90% and 100%) of acetone to remove 1 × PBS and other chemicals added for fixation I have. The dehydrated samples were then dried with hexamethyldisilazane for 15 minutes for observation by field emission scanning electron microscopy (FE-SEM). Samples were analyzed before being imaged by JEOL JSM-7500F FE-SEM (Welwyn Garden City)  And sputter-coated with platinum using a Cressington sputter coater (sputter coater 108 auto, Cressington).

<Result>

1. Identification of 60 AMP-like proteins by an overall analysis of the naked mole genome

Using the AMPA and APD3 databases, non-redundant sequences of 2,488 AMPs downloaded from UniProtKB / Swiss-Prot were analyzed. Of these, 29 alpha -dipenesin, 166 beta -diphencines, 63 catecholizines, 2 galenines, 1 granulin, 11 heptadines, 6 LEAP-2 and 32 snakes 310 AMPs from vertebrate animals including snake waprin were obtained. BLAST analysis of these vertebrate AMPs compared to genomes of naked mole rats showed that four α-defensin, 28 β-dipenesin, 10 katelicidin, two galanin, , 60 kinds of AMP-like proteins derived from eight subunits consisting of two granulin, two LEAP-2, one hepcidin and 11 snake warfarin were identified (e - value < 0.001).

2. In silico assay for the characterization of the estimated catecholizidine-like peptides of naked mole rats

The conserved catelin-like domain (CLD) and the antimicrobial domain of the 10 predicted catecholysidine-like sequences of naked mole rats were determined by analysis using the HMMER and AMPA databases, respectively. Only two of the ten predicted catecholysidine-like sequences contained the conserved CLD and the antimicrobial domain at the same time. In that conclusion, one cathelicidin candidate was identified by analysis of the antimicrobial domain in the two catecholysidine-like sequences, and it was found that it contained an estimated antimicrobial domain with more than 30 amino acids. To evaluate its secondary structure, a PSIPRED protein sequence analysis workbench was used. The results show that the antimicrobial domain contains an a-helical structure, which is one of the most common characteristics for the structure of AMP (Fig. 1). The length, hydrophobicity and net charge of the a-helical structure-containing region (sHg-CATH) were 25 amino acids, 32 hydrophobic, and +11 respectively (see Tables 1 and 2b). The alignment result of sHg-CATH for the existing AMP revealed a maximum sequence similarity of 45.9% for pobRL-37, a typical cationic AMP and cercopithecidae, representative catechirididine (see Fig. 2a).

3. Protein of catechol-tyrosine-related protein in naked mole rats Genetic and evolutionary relationships

Further characterization and validation of the assay was performed for one of the ten catecholizidine-like sequences, the cathelicidin candidate (Hg-CATH). Hg-CATH and 11 mammalian species (Rattus norvegicus, Mus musculus, Canis lupus familiaris, Bos taurus, Ovis aries, Sus scrofa, Oryctolagus, length sequence of 14 already identified catecholacids derived from cucumbers, cuniculus, Macaca mlatta, Pongo pygmaeus, Pan troglodytes, and Homo sapiens. As a result, The CLD of individual catechirizidine was found to be preserved at a high level. However, the different catecholysids in different mammalian species have a relatively low degree of conservation of the CLD region (Figure 3). As predicted, all of the catecholcidines containing Hg-CATH have four cysteines in the CLD region as conserved motifs, interestingly, Hg-CATH has a unique feature of having longer CLDs than others 2). Since only one cathelicidin was identified in the present invention and there was no previously available nomenclature, the present inventors temporarily named Hg-CATH for the cathelicidin of the naked mole rats. At the C-terminus of the full-length sequence of the cathelicidin, as expected, the mature peptide was more diverse than in the N-terminal sequence containing CLD (Figure 3).

In order to study the evolutionary relationship between Hg-CATH and the other catecholizidins from various species, 19 CLDs from 13 vertebrate animals, including 9 mammals, 3 birds, and 3 snakes A phylogenetic tree was created by sequence (Fig. 4). Consistent with evolutionary relationships among the species, the CLDs of whales (Artiodactyla) were clustered together and the CLDs of reptiles and amphibians were distinct from those of mammalian CLDs. Interestingly, Hg-CATH may have evolved distinctly from primates and Rodentia, which is the result of comparing the full-length amino acid sequences of Hg-CATH with other mammalian cathelicidins as shown in Figure 3 Match.

4. Evaluation of antimicrobial activity of sHg-CATH

The antimicrobial activity of chemically synthesized Hg-CATH (sHg-CATH) was confirmed against a panel of reference bacteria. In the present invention, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus And a minimal inhibitory concentration (MIC) test on the bacterial panel consisting of streptococci inoculum. sHg-CATH exhibits very strong activity only against Gram-negative strains Escherichia coli, P. aeruginosa and Salmonella typhimurium among the bacterial panel, and the MIC is 0.75 ~ 5 / / ㎖, which is the highest value reported for AMP so far, Even at a concentration of 64 [mu] g / ml, Gram-positive strains (Staphylococcus aureus, Bacillus cereus And Streptococcus sp.) Were very weak or absent (Table 3). Taken together, sHg-CATH may have antimicrobial activity specifically for Gram-negative bacteria. Finally, the peptides were tested against clinically isolated antibiotic resistant strains Escherichia coli and pneumoniae, which showed complete resistance to ampicillin and low resistance to gentamicin. Interestingly, sHg-CATH showed relatively strong efficacy against these pathogens at 32 and 4 ug / ml, respectively.

Antibacterial activity of Hg-CATH and conventional antibiotics on standard bacterial strains and clinical isolates
Strain name MIC ([mu] g / ml) sHg-CATH Ampicillin ^a Gentamicin ^a Gram-negative bacteria Escherichia coli ATCC 25922 0.75 One 0.5 Pseudomonas aeruginosa ATCC 27853 One > 128 0.25 Salmonella typhimurium ATCC 14028 5 One 0.375 Gram-positive bacteria Staphylococcus aureus
ATCC 29213 75 0.1 0.1 Bacillus aureus ATCC 10876 > 64 16 0.25 Streptococcus inia KCTC 3657 > 64 One One Clinical isolate E. coli ^b (A15-CF-027-1S-1) 32 > 64 > 64 Pneumonia bacterium ^b (KBN06903599) 4 32 16

^a antibiotic for control

^b Clinical isolate

5. Confirmation of stability test in serum

We have found that the stability of AMP in serum is one of the most important features in applying AMP to novel clinical and therapeutic agents, so that the antimicrobial activity of Hg-CATH at various concentrations over different incubation times (0 min to 1h 40min) Was assessed. The results showed that the antimicrobial effect of sHg-CATH on the growth of E. coli decreased with the passage of time. However, it even sterilized the bacteria at a concentration of 5 μg / ml even after 100 minutes of culture (FIG. 5).

6. Low hemolytic activity of sHg-CATH

Hemolytic activity of sHg-CATH on chicken erythrocytes was calculated as a measure of cytotoxicity (Table 3). As shown in Table 3, sHg-CATH showed low hemolytic activity as compared with melitin, an AMP derived from bee venom as a positive control. The hemolysis rate of sHg-CATH was 11% at 50 μg / ml, but melittin showed complete hemolysis (100%) at 12.5 μg / ml.

7. sHg - Of CATH  Low cytotoxicity

In general, cytotoxicity of AMP is a major problem when applying AMP to pharmaceutical preparations. In order to confirm the cytotoxicity of sHg-CATHM, we used five mammalian cell lines treated with the peptides at different concentrations (5-20 .mu.g / ml), namely HEK293T (human fetal kidney cells), HaCaT ), PK15 (pig kidney cells), ST ATCC CRL-1746 (pig testicular fibroblasts), and 3D4 / 2 (macrophages of porcine lung). As a result, no cytotoxicity was detected for most of the cells until the peptide concentration reached 20 占 퐂 / ml (Fig. 6).

8. sHg - Of CATH  Effect of hole formation on bacterial membranes

To investigate the antimicrobial mechanism of the action of Hg-CATH, bacterial cells after sHg-CATH treatment were observed using a scanning electron microscope (SEM). As a result, E. coli having sHg-CATH The form of ATCC 25922 was modified by cell membrane disruption and pore formation (Fig. 6B), whereas control cells without the peptide showed cell membrane integrity and cell division as evidence of bacterial growth (Fig. 7).

<110> Konkuk University Industrial Cooperation Corp <120> Hg-CATH <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 25 <212> PRT <213> Heterocephalus glaber <400> 1 Ser Lys Phe Phe Arg Lys Ala Arg Lys Lys Leu Gly Lys Gly Leu Gln   1 5 10 15 Lys Ile Lys Asn Val Leu Arg Lys Tyr              20 25

Claims (10)

delete delete delete A pharmaceutical composition for antibiosis against gram-negative bacteria comprising an antimicrobial polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. delete The antimicrobial polypeptide according to claim 4, wherein the antimicrobial polypeptide has an antimicrobial activity against at least one gram-negative bacterium selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium &Lt; / RTI &gt; The pharmaceutical composition according to claim 4, wherein the antimicrobial polypeptide is contained at a concentration of 1 to 1000 μg / ml. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier selected from the group consisting of a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, a diluent and an excipient A pharmaceutical composition. A food preservative comprising an antimicrobial polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. A feed additive comprising an antimicrobial polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

Images