KR100968080B1 - A novel antibiotic peptide hominicin and its derivatives from staphylococcus hominis mbbl 2-9 - Google Patents

Abstract

PURPOSE: A Staphylococcus hominis MBBL 2-9 strain is provided to ensure antibacterial activity to bacteria and to prevent gram positive/antibiotics resistant pathogen. CONSTITUTION: An antibacterial peptide hominicin which is isolated from Staphylococcus hominis MBBL 2-9 has an amino acid of sequence number 1. The antibacterial peptide is themal resistant, acid resistant, basic resistant, and non-cytotoxicity. The antibacterial peptide contains DmIle(N1, N1-dimethyl isoleucine), Dhb(dehydrobutyrine), and Dmp(N2, N2-dimethyl-1,2-propanediamine). A medicinal composition for preventing gram positive pathogen or antibiotics resistant pathogen contains the antibacterial peptide as an active ingredient.

Description

Novel antimicrobial peptide hominicins and derivatives produced from Staphylococcus hominis MBL2 -９ {A NOVEL ANTIBIOTIC PEPTIDE HOMINICIN AND ITS DERIVATIVES FROM STAPHYLOCOCCUS HOMINIS MBBL 2-9}

The invention according to the site of infection bacteremia, endocarditis, septic arthritis, pneumonia, osteomyelitis, skin infections, colitis, various Staphylococcus aureus causes diseases such as food poisoning (Staphylococcus aureus ) relates to a microbial strain having antimicrobial activity, a novel antimicrobial peptide they produce, and a drug for controlling Gram-positive pathogens containing the antimicrobial peptide as an active ingredient.

All living organisms on earth possess biological defense mechanisms that can protect or protect themselves from microorganisms such as viruses, bacteria and fungi, one of which is a non-selective immune system using antibiotics including antimicrobial peptides. (non-specific immunity). Since the first discovery of penicillin notatum, which inhibited the growth of the pathogenic bacterium Staphylococcus aureus by Fleming in 1929, various scientists Antibiotics have been steadily developed.

In particular, antimicrobial peptides are attracting attention in various fields such as biopesticides, feed probiotic, food preservatives, and pharmaceuticals due to some unique advantages. For the advantages of the antimicrobial peptides mentioned above, first, they have a strong antimicrobial activity against a wider range of microorganisms than previously reported antibiotics. Second, since the antimicrobial peptides are only active against pathogens invading from outside without destroying the host cell, the antimicrobial peptide is highly likely to be developed as a safe and useful antimicrobial material without adverse effects on humans, animals and plants. Third, conventional antibiotics are highly likely to cause microbial resistance due to excessive use, while antimicrobial peptides exhibit very low antimicrobial effects due to a different mechanism of activity than antibiotics.

Accordingly, several studies have been conducted with respect to antimicrobial peptides or microbial strains producing the same. New antimicrobial peptide isolated from Korean toad (registered patent number 10-0149278-0000), new antimicrobial peptide isolated from catfish (registered patent number 10-0330136-0000), antibacterial peptide derived from plant (registered patent number 10 -0346072-0000), antimicrobial peptide-producing microbial strains derived from Korean traditional salted fish (registered patent No. 10-0407074-0000), Lactobacillus plantarum IB 213 (KCTC 18106P), Lactobacillus fermentum IB 261 (KCTC 18107P) and Swine probiotics (registered patent number 10-0723843-0000) and antibacterial probiotics for fish farming (registered patent number 10-0685237-0000) using the same have been disclosed.

In light of the above, the present inventors have conducted research to find useful microorganisms producing antimicrobial peptides known to play an important role in protecting organisms by inhibiting the growth of external pathogenic microorganisms and novel antimicrobial peptides produced thereby. Thus, the present invention has been completed.

Microorganism type in producing the above-mentioned antimicrobial peptides mainly Bifidobacterium (Bifidobacterium), Bacillus (Bacillus), Lactobacillus bacteria (Lactobacillus), Lactococcus (Lactococcus), Phedi O Rhodococcus (Pediococcus) and Lu Pocono stock (Leuconostoc) The genus is mostly occupied. In contrast, the present inventors have isolated Staphylococcus hominis strains from humans, which have not been reported as antimicrobial peptide producing strains to date, and the antimicrobial peptides produced from the strains have extreme conditions (high temperature) compared to conventional antimicrobial peptides. , Strong acids / bases), no antimicrobial activity was lost, and no new antimicrobial peptides were found.

Therefore, provided in the present invention is Staphylococcus causing various diseases depending on the site of infection ( Staphylococcus) having an antifungal activity against Staphylococcus aureus) Caucus hoe Nice (Staphylococcus hominis ) MBBL 2-9 strains, as well as the novel peptide hominicin produced therefrom. The novel peptides of the invention in addition to Staphylococcus aureus Bacillus subtilis (Bacillus subtilis ), Micrococcus luteus ) and Lactobacillus It has strong antimicrobial activity against various Gram-positive bacteria, including reuteri . In addition, it is possible to control humans and animals from the pathogens using the microbial strain of the present invention or the antimicrobial peptides produced therefrom as a medicine.

The isolation and identification of the microbial strain Staphylococcus hominis MBBL 2-9 according to the present invention is as follows. Isolation of antimicrobial peptide-producing microorganisms from 300 healthy women from the Department of Obstetrics and Gynecology, Seoul National Hospital, separated 300 microorganisms according to morphological characteristics and culture conditions. Staphylococcus aureus ) showed the best activity of the MBBL 2-9 strains. Molecular identification of the cells of the selected highly active microorganisms was carried out using a polymerase chain reaction of 16S rDNA using Universal primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3 ') and 1492R (5'-TACGGTYACCTTGTTGTTACGACTT-3'). PCR). Identification and naming of the selected strains determined the 16S rDNA sequences and compared them with data from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov//BLAST) and the phylogenic tree. The strain was identified at the species level by Staphylococcus hominis ) showed more than 99% molecular identity with DSM 20328T (FIG. 1). In addition, on March 4, 2008, it was deposited with the Korea Agricultural Microbiological Resource Center in the Institute of Agricultural Biotechnology, which is a depository institution for Korean patent strains, and was assigned accession number KACC 91354P.

As discussed above, the Staphylococcus hominis of the present invention ( Staphylococcus) hominis) MBBL 2-9 strains and novel antimicrobial peptide produced therefrom are antibacterial test strains of Staphylococcus aureus (Staphylococcus aureus showed excellent antimicrobial activity, as well as Bacillus subtilis and Micrococcus luteus ) and also have excellent antimicrobial activity against Gram-positive bacteria. Thus, microbial strains and antimicrobial peptides can be used as medicines to effectively control Gram-positive / antibiotic resistant pathogens that cause disease in humans or animals. In the case of the microbial strain and the antimicrobial peptide of the present invention, it is environmentally friendly like other antimicrobial peptides that are commonly used, and has no advantage of no cytotoxicity and resistance to pathogens.

Hereinafter, the present invention will be described in detail by way of examples. These examples are only for illustrating the invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art to which the present invention pertains. Will be self explanatory.

Example 1

Cultivation and Antimicrobial Activity of Antimicrobial Peptide Producing Microorganisms

The selected microbial strains were shaken for 24 hours at 37 ° C with De Man Rogosa Sharpe (MRS) liquid medium and centrifuged to collect supernatants and cells. The antimicrobial activity was investigated by the size of inhibitory rings using the paper disk method. Staphylococcus aureus , 10 ⁶ cfu / ml) were preliminarily plated on Brain Heart Infusion (BHI) agar plate medium, and the culture supernatant and cells were dropped on paper disks, and then cultured at 37 ° C for 24 hours. It was. Inhibitory ring size showing antimicrobial activity was based on 6 mm or more. Microbial strains as well as unconcentrated culture supernatants showed excellent antimicrobial activity against pathogens (FIG. 2).

Example 2

Isolation and Purification of Antimicrobial Peptides

Star of the present invention Philo Lactococcus hoe varnish (Staphylococcus hominis ) To obtain pure antimicrobial peptides by separating and purifying antimicrobial peptides produced from MBBL 2-9 strains, the strains were cultured in MRS liquid medium for 24 hours and then centrifuged to obtain only culture supernatant. Chloroform (chloroform) corresponding to 1/2 of the supernatant was added thereto, mixed vigorously at room temperature for 30 minutes, and centrifuged at high speed to extract a crude extract including an antimicrobial peptide. The extract containing the antimicrobial peptide was evaporated to dryness and dissolved in sodium phosphate buffer (5 mM, pH 7.0), followed by ion-exchange chromatography. After separating lyophilized aliquots from ion exchange resin chromatography, the antibacterial activity of the desalted aliquots was verified using a hydrophobic cartridge. Aliquots showing antimicrobial activity were applied to reverse-phase HPLC to isolate pure antimicrobial peptides. Figure 3 shows that the antimicrobial peptides were correctly separated by a reversed phase column, Figure 4 shows that the separation is the main active material showing antibacterial activity. In addition, Tricine SDS-PAGE was performed to measure the purity and molecular weight of the purified antimicrobial peptide. As a result, a single peptide band estimated to have a very high molecular weight of 3,000 Da was obtained (FIG. 5). As a result of analyzing the sample containing the antimicrobial peptide by liquid chromatography-mass spectrometry (LC-MS), it was possible to obtain the main peak estimated as the antimicrobial peptide at the elution time of 44.5 minutes (Fig. 6A). Three peaks of / z 1020, 1237, and 1563 were observed (FIG. 6B). Among them, m / z 1237 and 1563 peaks were identified as fragments of antimicrobial peptides generated during ionization. As m / z 1020 was found to be a divalent cation, the exact molecular weight of the antimicrobial peptide was determined to be 2038.2 Da.

Example 3

Characterization and Inhibitory Activity of Antimicrobial Peptides

Crude extract comprising an antimicrobial peptide produced from Staphylococcus hominis MBBL 2-9 of the present invention prepared in Example 2 (crude extract) at 25, 37, 55, 70 ° C. 48 hours, 100 Treatment was carried out at 30 ° C. for 60 minutes at 15 ° C. and for 15 minutes at 121 ° C. In order to investigate the stability of the crude ion (pH) of the crude extract (pH) adjusted to pH 2.0-10.0 using 0.5 M HCl or 0.5 M NaOH and incubated for 2 hours at 37 ° C and neutralized Antimicrobial activity was verified. In addition, crude extracts were treated with pepsin, trypsin, alpha-chymotrypsin, proteinase K, and lipase, respectively. Was investigated. Finally, blood agar medium was used to investigate the cytotoxicity of bovine RBCs of purified antimicrobial peptides to bovine RBCs. The results of investigation of heat, pH stability and sensitivity of various degradation enzymes of crude extract including antimicrobial peptides are as follows. Crude extracts did not lose any antimicrobial activity even when heated at 121 ° C. for 15 minutes and showed excellent antimicrobial activity within all pH ranges (2.0-10.0) [Table 1]. In addition, other enzymes other than proteinase K exhibited antimicrobial effect without degradation [Table 1]. Moreover, the purified antimicrobial peptides at the concentration showing antimicrobial activity were found to have no cytotoxicity since no inhibitory ring was observed in the blood agar plate (FIG. 7). In addition, the Staphylococcus hominis of the present invention prepared in Example 2 ( Staphylococcus hominis ) A crude extract containing an antimicrobial peptide produced from MBBL 2-9, Micrococcus in the same way as in Staphylococcus aureus. luteus ) and Bacillus subtilis subtilis ), the antimicrobial activity was measured by measuring the size of the inhibitory ring, and showed excellent antimicrobial activity (FIG. 8). In addition, MRSA (methicillin-resistant Staphylococcus) aureus ) and VISA (vancomycin-intermediate Staphylococcus) As a result of measuring the inhibitory activity of the antimicrobial peptide against aureus ), it showed inhibitory activity from 62.5 μM concentration (FIGS. 9 and 10).

Example 4

Amino Acid Sequence Analysis of the Antimicrobial Peptides of the Present Invention

To identify the amino acid sequence of the antimicrobial peptide, proteinase K was mixed at a ratio of 9: 1 (vol / vol) to a bacteriocin sample purified using reverse-phase HPLC, followed by 90 ° at 37 ° C. Reacted in minutes. The enzyme-treated samples were applied again to the reversed phase column to obtain two fractions of 36 and 48 minutes, and then freeze-dried to perform nuclear magnetic resonance spectroscopy (NMR spectroscopy). The total amino acid sequence of bacteriocin was determined based on the molecular weights obtained by fragmentation of each of the two fractions, each of the two fractions obtained by nuclear magnetic resonance spectroscopy, and by the fractionation of each of the two fractions using a mass spectrometer. Is the same as In addition, the antimicrobial peptide is expected to exhibit antimicrobial activity against Gram-positive bacteria and antibiotic resistant pathogens by containing the non-natural amino acids DmIle, Dhb, and Dmp. In addition, the antimicrobial peptide is expected to exhibit antimicrobial activity against Gram-positive bacteria and antibiotic-resistant pathogens due to the composition ratio of more than 70% of Ile, Pro, Dhb, and Ala among the total amino acids. In addition, the antimicrobial peptides are expected to exhibit antimicrobial activity against Gram-positive bacteria and antibiotic-resistant pathogens because they contain one or more amino acid conserved sequence X-Dhb-Pro. On the other hand, it can be seen that the antimicrobial peptide of the present invention is novel by confirming that the amino acid sequence such as SEQ ID NO: 1 does not match the amino acid sequence of the bacteriocin reported so far.

FIG. 1 is Staphylococcus hoe Nice (Staphylococcus hominis ) is a phylogenic tree based on the 16S rDNA nucleotide sequence of MBBL 2-9.

FIG. 2 is Staphylococcus hoe Nice (Staphylococcus hominis) MBBL 2-9 strain and the culture supernatant of Staphylococcus aureus (Staphylococcus aureus ) shows antimicrobial activity.

3 shows the fraction of antimicrobial peptides using reversed phase columns.

4 shows antimicrobial activity of antimicrobial peptides.

5 shows Tricine SDS-PAGE of purified antibacterial peptides.

6 shows antimicrobial peptide analysis using liquid chromatography-mass spectrometry techniques.

7 shows cytotoxicity verification of antimicrobial peptides.

8 is a Lactococcus Lu No. mini God antimicrobial peptide micro Proteus (Micrococcus luteus) and Bacillus subtilis (Bacillus subtilis ).

9 shows the antimicrobial activity of MRSA (methicillin-resistant Staphylococcus aureus ) of the antimicrobial peptide hominicin.

Figure 10 shows the antimicrobial activity of vancomycin-intermediate Staphylococcus aureus (VISA) of the antimicrobial peptide hominicin.

<110> Seoul National University R & DB Foundation <120> A NOVEL ANTIBIOTIC PEPTIDE HOMINICIN AND ITS DERIVATIVES FROM          STAPHYLOCOCCUS HOMINIS MBBL 2-9 <130> KACC 91354P <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> PRT Staphylococcus hominis <220> <221> SITE <222> (1) DmIle (N1, N1-dimethyl isoleucine) <220> <221> SITE <222> (2) <223> dehydrobutyrine (Dhb) <220> <221> SITE <222> (5) <223> dehydrobutyrine (Dhb) <220> <221> SITE <222> (8) <223> dehydrobutyrine (Dhb) <220> <221> SITE <222> (15) <223> dehydrobutyrine (Dhb) <220> <221> SITE <222> (21) Dmp (N 2, N 2 -dimethyl-1, 2-propanediamine) <400> 1 Xaa Xaa Pro Ala Xaa Pro Phe Xaa Pro Ala Ile Thr Glu Ile Xaa Ala   1 5 10 15 Ala Val Ile Ala Xaa              20  

Claims (11)

delete delete delete delete delete Having the amino acid sequence of DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp Antibacterial peptides. 7. The antimicrobial peptide of claim 6 which is heat resistant, acid resistant, base resistant, and cytotoxic. 7. A non-natural amino acid DmIle ( N1 , N1- dimethyl isoleucine), Dhb (dehydrobutyrine), and Dmp ( N2 , N2- dimethyl-1,2-propanediamine), and Ile, Pro of all amino acids Antimicrobial peptide, characterized in that it has a control activity against Gram-positive pathogens or antibiotic-resistant pathogens by comprising, Dhb, and Ala in a composition ratio of 70% or more and 90% or less. delete The antimicrobial peptide according to claim 6, wherein the antimicrobial peptide has one or more amino acid conserved sequences, X-Dhb-Pro, which has a control activity against Gram-positive pathogens or antibiotic-resistant pathogens. An antimicrobial peptide that represents an unspecified amino acid and Pro represents proline. A pharmaceutical composition for controlling Gram-positive pathogens or antibiotic-resistant pathogens, comprising the antimicrobial peptide according to any one of claims 6 to 8 and 10 as an active ingredient.

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