WO2017010845A1 - Composition for preventing or treating staphylococcal infectious diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating staphylococcal infectious diseases. Specifically, the soluble WTA-PGN represented by general formula 1 according to the present invention increases the production of IL-17A in the body of mice when administered to the mice, and thus, the soluble WTA-PGN can be favorably used in the prevention and treatment of staphylococcus.

Description

Composition for preventing or treating staphylococcal infection disease

The present invention relates to a pharmaceutical composition for the prevention or treatment of Staphylococcal infection disease.

Staph (Staphylococcus aureus) is a human skin, soft tissue (soft tissue) and can lead to severe bloodstream infection (Lowy FD, The New England Journal of Medicine, 339: 520-532, 1998). In addition, Staphylococcus aureus may be transformed into methicillin-resistant strain (methsaillin-resistant S. aureus , MRSA) resistant to beta-lactam family of methicillin. MRSA infections are difficult to treat and have a poor prognosis, which is a social problem. In particular, community-associated MRSA strains (CA-MRSA) have appeared in children who have never been admitted to the hospital, and the treatment of the treatment with conventional hospital-associated MRSA strains (HA-MRSA) It is adding to the difficulties. Recently, the USA300 MRSA strain, which is spreading in the United States, causes serious diseases in children or those with reduced immune function. Therefore, new vaccines or treatments having a prophylactic and therapeutic effect on MRSA infection are required.

Previously, researchers who have tried to develop staphylococcal vaccine candidates have failed in clinical stages despite the good prognosis in preclinical experiments. Until recently, no clinically useful staphylococcus vaccines have been developed. There is a situation.

Recent studies show that both humoral and cellular immunity are required to function as a staphylococcal vaccine and promote phagocytic cell effector function by T cell activation. It has been suggested that it should be done. At this time, humoral immunity promotes opsonophagocytosis by generating serum antibodies specific for the vaccine substance, and cellular immunity produces T cell-mediated IL-17A in the early stages of MRSA infection, leading to neutrophil recruitment and Promotes phagocytosis.

In addition, foreign researchers reported that immune responses occur due to an increase in the number of memory γδ-T cells in a mouse model infected with Staphylococcus aureus ( J. Immunol. , 192 (8): 3697-708, 2014). . The findings suggest that the memory response of γδ-T cells is induced in a manner similar to αβ-T cells upon exposure to Staphylococcus, and that IL-17A produced by γδ-T cells has a potent staphylococcal treatment effect. do.

Unlike Gram-negative bacteria, the cell walls of Gram-positive bacteria, such as staphylococci, are four components, including peptidoglycan (PGN), wall teichoic acid (WTA), lipoteicoic acid (LTA), and capsular polysaccharides (CP) It consists of. Targeting these cell wall components can be developed to prevent or treat bacterial infections, but the ligand material of bacteria recognized by the host's immune system has not been clearly identified, which is a problem.

In particular, the cell wall component of staphylococci is a glycopolymer, and its structure is very complicated, and it is difficult to separate and purify each as a single substance. In addition, various cell wall components are exposed to the outside, which makes it difficult to identify which component acts as a ligand in the host's immune system.

Therefore, the present inventors have diligently studied which component of the cell wall component of Staphylococcus aureus acts as a ligand in the host's immune system, and thus isolates the wall teichoic acid-attached peptidoglycan (WTA-PGN) having a specific structural formula. The present invention was completed by confirming that WTA-PGN is useful for preventing or treating Staphylococcal infection disease.

Accordingly, it is an object of the present invention to provide a composition for the prevention or treatment of Staphylococcal infection disease.

Another object of the present invention to provide a method for preventing or treating staphylococcal infection disease using the composition.

Still another object of the present invention is to provide a method for preparing soluble dumpeichoic acid-attached peptidoglycan (WTA-PGN) which can be used as an active ingredient in the composition.

In order to achieve the above object, the present invention is a composition for the prevention or treatment of staphylococcal infection disease, which contains the wall teichoic acid-attached peptidoglycan (WTA-PGN) represented by the general formula 1 as an active ingredient to provide:

[Formula 1]

In Formula 1, n is an integer of 10 to 50, m is an integer of 1 to 3, A is N-acetylmannosamine (ManNAc), B is N-acetylglucosamine (GlcNAc), O is An integer from 1 to 5, R ₁ and R ₂ are each independently hydroxy, tetrapeptide or pentapeptide, and R ₃ is hydroxy or N-acetylmuramic acid (MurNAc).

The present invention also provides a method for preventing or treating Staphylococcal infection disease, comprising administering the composition to a subject in need thereof.

Furthermore, the present invention provides: 1) to obtain a double-mutant strain is lgt (lipoprotein diacylglycerol transferase) and oatA (O-acetyl transferase) gene deletions from the wild type Staphylococcus aureus; 2) heating the double mutant strain to a temperature of 40 to 70 ° C; 3) adding β-lytic enzyme to the heated strain; 4) obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3); 5) adding lysozyme or mutanolysin to the soluble WTA-PGN containing fractions; And 6) obtaining a soluble WTA-PGN from the enzyme additive of step 5).

Soluble WTA-PGN represented by Formula 1 according to the present invention increases the production of IL-17A in the mouse body when administered to mice, the soluble WTA-PGN can be usefully used for the prevention and treatment of staphylococci have.

1 is a graph showing the elution pattern of S. aureus T384 strain ( ΔlgtΔoatA ) prepared in one embodiment of the present invention after treatment with β-lytic enzyme and HPLC separation.

Figure 2 is a graph showing the elution pattern separated by HPLC treatment of lysozyme to each fraction obtained by treatment with insoluble β-lytic enzyme to the heat-treated S. aureus T384 strain and separated by HPLC:

A: an elution pattern separated by HPLC after treating lysozyme to the A fraction;

B: an elution pattern separated by HPLC after treating lysozyme to the B fraction;

C: an elution pattern separated by HPLC after treating lysozyme to the C fraction;

D: an elution pattern separated by HPLC after treatment with lysozyme in the D fraction;

E: an elution pattern separated by HPLC after treatment of lysozyme to the E fraction; And

F: An elution pattern separated by HPLC after treating lysozyme to the F fraction.

Figure 3 shows the results of the production of IL-17A after injection into the mouse each peak fraction obtained after the enzyme treatment:

1: B peak treated with lysozyme and separated by HPLC with C fraction separated by HPLC;

2: B peak treated by lysozyme and separated by HPLC for the D fraction separated by HPLC;

3: C peak treated by lysozyme with HPLC fractionated and separated by HPLC; And

4: A mixture of the C peak of the E fraction and the B peak of the F fraction, each of which was treated with lysozyme and separated by HPLC for each of the E and F fractions separated by HPLC.

4 is a graph showing an elution pattern of Sephacryl-B-TSK-B fractions separated by a C18 reversed phase column.

FIG. 5 is a graph comparing the amount of IL-17A induced when mice were injected with each of the six fractions in which Sephacryl-B-TSK-B fractions were separated by a C18 reversed phase column (Con: Sephacryl-B-TSK). -B fraction, A: A fraction, B: B fraction, C: C fraction, D: D fraction, E: E fraction, F: F fraction).

FIG. 6 is a graph showing an elution pattern in which the C to E fractions separated from the C18 reverse phase column in Example 5 were re-separated into a C18 reverse phase column.

Figure 7 is a graph comparing the production of IL-17A induced when mice were injected with C-E fractions re-separated in a C18 reversed phase column (C: C fraction, D: D fraction, E: E fraction).

Hereinafter, terms used in the present invention are defined.

As used herein, the term "wall teichoic acid (WTA)" is one of the cell wall components of S. aureus , combined with a glycerol phosphate repeat unit and a ribitol phosphate repeat unit (N-acetyl Refers to a glycopolymer consisting of mannosamine)-(β-1,3)-(N-acetylglucosamine).

As used herein, the term "peptidoglycan (PGN)" refers to a repeat glycopolymer of N-acetylmuriamic acid (MurNAc) and N-acetylglucosamine (GlcNAc) linked by stem-peptide bonds.

As used herein, the term "wall teichoic acid-attached peptidoglycan (WTA-PGN)" refers to a structure in which the wall teichoic acid and peptidoglycan are covalently bonded.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the prevention or treatment of Staphylococcal infection disease, which comprises aste active acid-attached peptidoglycan (WTA-PGN) represented by the following general formula (1):

[Formula 1]

In Formula 1, n is an integer of 10 to 50, m is an integer of 1 to 3, A is N-acetylmannosamine (ManNAc), B is N-acetylglucosamine (GlcNAc), O is An integer from 1 to 5, R ₁ and R ₂ are each independently hydroxy, tetrapeptide or pentapeptide, and R ₃ is hydroxy or N-acetylmuramic acid (MurNAc).

In another embodiment of the present invention, in formula 1, n is an integer from 35 to 45, m is 3, A is ManNAc, B is GlcNAc, O is an integer from 1 to 5, R ₁ and Each R ₂ is independently hydroxy, tetrapeptide or pentapeptide and R ₃ is hydroxy or MurNAc.

In another embodiment of the present invention, in formula 1, n is 40, m is 3, A is ManNAc, B is GlcNAc, O is an integer from 1 to 5, R ₁ and R ₂ is Each independently is a tetrapeptide or pentapeptide and R ₃ is hydroxy or MurNAc.

In Formula 1, A and B may be linked to a β-position to each other. In addition, in the general formula 1, the tetrapeptide is -X ₁ -X ₂ -X ₃ -X ₄ (SEQ ID NO: 1), and the pentapeptide is

(SEQ ID NO: 2). Here, X ₁ may be Ala or Gly, X ₂ may be Glu or Asp, X ₃ may be Lys, Arg, or His, and X ₄ may be Ala or Gly. In one embodiment of the present invention, the tetrapeptide is-(L-Ala)-(D-Glu)-(L-Lys)-(D-Ala) (SEQ ID NO: 3), and the pentapeptide is

(SEQ ID NO: 4).

In one embodiment of the present invention, the PGN structure of Formula 1 may be represented by the following formula (1).

[Equation 1]

In a further embodiment of the invention, the R ₁ or R ₂ of the WTA-PGN may form the R cross-linking with each other and any one of ₁ or R ₂ of other WTA-PGN, The WTA-PGN along the two WTA-PGN may also be present in the form of a dimer combined with it.

The staphylococcus may be methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA) or pathogenic staphylococcus. In addition, the infectious diseases caused by Staphylococcus aureus include soft tissue infection, purulent arthritis, purulent osteomyelitis, otitis media, pneumonia, sepsis, acute respiratory tract infection, infection due to the use of a catheter, postoperative wound infection, bacteremia, Endocarditis and food poisoning.

In one example, the composition according to the present invention may further comprise a pharmaceutically acceptable carrier, diluent and adjuvant in addition to the WTA-PGN.

Carriers used in the compositions according to the invention are selected based on the method and route of administration, and standard drug compositions, and include, for example, carrier proteins (ie, bovine serum albumin (BSA), egg white albumin (OVA), human serum albumin). (HSA) and keyhole limpet hemocyanin (KLH)), solubilizers (ie ethanol, polysorbate and Cremophor EL ™), isotonic agents, preservatives, antioxidants, excipients (ie lactose, starch, crystalline cellulose, mannitol) , Maltose, calcium hydrogen phosphate, light anhydrous silicic acid and calcium carbonate), binder (ie starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose and gum arabic), lubricant (ie magnesium stearate) , Talc, and cured oils, and the like) and stabilizers (ie, lactose, mannitol, maltose, polysorbate, macrosol, polyoxyethylene cured castor oil). If desired, glycerin, dimethylacetamide, 70% sodium lactate, surfactants or basic substances (ie, sodium hydroxide, ethylenediamine, ethanol amine, sodium bicarbonate, arginine, meglumine or trisaminomethane) and the like can be included. have. Specifically, the composition according to the present invention can be combined with a known KLH solution (Calbiotec, dissolving 125 mg per ml of 50% glycerol solution) as a carrier protein to enhance antigenicity.

Diluents used in the compositions according to the invention can be selected based on the method and route of administration and the actual standard drug composition. Examples of diluents include water, physiological saline, phosphate buffered physiological saline, and bicarbonate solutions.

Adjuvants used in the compositions according to the invention may be selected based on the method and route of administration and the actual standard drug composition. Examples of adjuvant include cholera toxin, E. coli dipyrotoxin (LT), liposomes and immune stimulatory complex (ISCOM).

The present invention also provides a method for preventing or treating Staphylococcal infection disease, comprising administering the composition to a subject in need thereof.

The subject may be a mammal, specifically a human.

The method can prevent or treat staphylococcal infections by simultaneously inducing opsonophagocytosis and phagocytosis in a subject. The method may also increase the amount of IL-17A produced within 24 hours after administration of the composition according to the invention in a subject.

The administration may vary depending on the age, weight, sex and general state of health of the subject. Routes for such administration include oral and parenteral administration (eg, intravenous administration, arterial administration and topical administration), preferably parenteral administration.

Formulations for oral and parenteral administration and methods for their preparation are known to those skilled in the art. Formulations for oral and parenteral administration may be prepared by conventional procedures, eg, by admixing with the aforementioned pharmaceutically acceptable carriers. Examples of formulations for oral administration include solid or liquid formulations such as solvents, tablets, granules, powders or capsules. Examples of formulations for parenteral administration include solvents, suspensions, ointments, creams, suppositories, eye drops, nasal drops and ear drops. For the preparation of sustained-release tablets of the present formulations, biodegradable polymers (eg, poly-D, L-lactide-co-glycoside or polyglycoside) can be added to the buck base (eg, US patents). 5,417,986, 4,675,381 and 4,450,150). For oral administration, flavors and colorings may be added. Suitable pharmaceutical carriers, diluents and pharmaceutically necessary materials for their use are described in Remington's Pharmaceutical Sciences.

The dosage of the composition according to the present invention is determined based on the type of adjuvant, the method and frequency of administration, and the desired effect and may generally be from 1 μg to 100 mg of WTA-PGN in a single adult dose. When adjuvant is added to the composition of the present invention, the dosage may generally be from 1 μg to 1 mg of WTA-PGN in a single adult dose. The administration can be administered several times if necessary. For example, the composition may be administered again by supplementing three times after the composition has been initially administered at regular intervals. Optionally, the compositions for the first and second reinforcements may be administered 8-12 weeks and 16-20 weeks after the first administration using the same formulation, respectively.

Furthermore, the present invention provides: 1) to obtain a double-mutant strain is lgt (lipoprotein diacylglycerol transferase) and oatA (O-acetyl transferase) gene deletions from the wild type Staphylococcus aureus; 2) heating the double mutant strain to a temperature of 40 to 70 ° C; 3) adding β-lytic enzyme to the heated strain; 4) obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3); 5) adding lysozyme or mutanolysin to the soluble WTA-PGN containing fractions; And 6) obtaining a soluble WTA-PGN from the enzyme additive of step 5).

Hereinafter, a method of manufacturing WTA-PGN represented by the general formula 1 according to the present invention will be described in detail.

Step 1) there is provided a method comprising obtaining a double-mutant strains and oatA lgt gene deletion from the wild-type staphylococci.

Double mutant strain (ΔlgtΔoatA obtained in the above step 1)) whereby the lgt gene deletion not likely to be contaminated by the lipoprotein can be obtained pure WTA-PGN. In addition, the deletion of the oatA gene eliminates the acetyl group in the MurNAc residue of the PGN, and the resulting WTA-PGN can be easily degraded by the β-lytic enzyme of step 2).

Such double mutant strains can be obtained by known methods from wild type staphylococci, for example methicillin-resistant staphylococci (MRSA), methicillin-sensitive staphylococci (MSSA) or pathogenic staphylococci. For example, the double mutant strain T363 strain of the lgt gene while having a resistance to play Oh, my God (phleomycin) defect (Nakayama M et al, Journal of Immunology 189:. 5903-591, 2012) and resistant to erythromycin T0003 strain (Park KH et al. , Journal of Biological Chemistry 285, 27167-27175, 2010) with oatA gene deficient can be prepared by transduction through phage 80.

Step 2) provides a step of heating the double mutant strain to a temperature of 40 to 70 ℃.

The temperature may be 45 to 68 ℃, 50 to 65 ℃ or 55 to 63 ℃, in one embodiment of the present invention the temperature may be 60 ℃.

For example, step 2) may comprise the step of culturing the double mutant strain obtained in step 1), followed by heat treatment.

Step 3) provides a step of treating β-lytic enzyme on the heated strain.

The β-lytic enzyme plays a role in degrading pentaglycine ((Gly) 5) linkages linking the stem peptides present at the MurNAc residue of the insoluble WTA-PGN obtained in step 2), thereby insoluble WTA contained in the heated strain. Change PGN to soluble WTA-PGN.

Such β-lytic enzymes are commercially available or described in Li et al. , Journal of Biochemitry 122, 772-778, 1997, can be isolated and purified. An example of a commercially available β-lytic enzyme is lysostaphin.

Step 3) may be carried out by suspending the strain heated in step 2) in a buffer, and then adding β-lytic enzyme and reacting with stirring at 30 to 40 ° C. for 10 to 14 hours.

Step 4) provides a step of obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3).

This step can be carried out by passing the β-lytic enzyme additive through HPLC to obtain a fraction, and then selecting the fraction containing soluble WTA-PGN in the fraction. At this time, the fraction containing WTA-PGN can be confirmed by PAGE or silver nitrate staining method.

The column used in the HPLC purification may be HiTrap Q FF (GE Healthcare), which is an anion exchange resin that binds with the anion of WTA ribitol phosphate.

Step 5) provides the step of adding lysozyme or mutanolysine to the soluble WTA-PGN containing fractions.

The lysozyme or mutanolysine transforms the polymeric PGN into an oligomeric PGN by breaking down the bond between MurNAc and GlcNAc of the PGN.

Step 5) may be carried out by suspending the soluble WTA-PGN obtained in step 4) in a buffer, and then adding lysozyme or mutanolysine for 10 to 14 hours with stirring at 30 to 40 ° C.

Step 6) provides a step of obtaining soluble WTA-PGN from the enzyme additive of step 5).

The step can be carried out by passing lysozyme or mutanolysine enzyme additive through HPLC to obtain a fraction, and then selecting the fraction containing soluble WTA-PGN from the fraction. At this time, the fraction containing WTA-PGN can be confirmed by PAGE or silver nitrate staining method.

The column used for HPLC purification may be HiTrap-Q (GE Healthcare).

The method according to the invention may further comprise further purifying the WTA-PGN after step 6).

Said further purification can be carried out by gel filtration chromatography or reverse phase liquid chromatography.

In one embodiment of the invention, the soluble WTA-PGN prepared in step 6) can be further purified via gel filtration chromatography using Sephacryl S-200 HR column or reverse phase liquid chromatography using Symmetry Shield ^™ RP18 column. have.

In another embodiment of the present invention, the soluble WTA-PGN prepared in step 6) is further purified by gel filtration chromatography using Sephacryl S-200 HR column and reverse phase liquid chromatography using two Symmetry Shield ^™ RP18 columns. Can be.

The active fraction can be selected based on the amount of IL-17A produced after injection of each fraction obtained in the chromatography into the mouse abdominal cavity.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

Example 1 Preparation of Strains for Obtaining WTA Derivatives

To isolate WTA-PGN, Kazue Takahashi et al. , Plos One 8: e69739, 2013], S. aureus T384 strain (double mutation of RN4220 ΔlgtΔoatA ) was prepared.

The S. aureus strains T384 player Oh, my God while having a resistance to (phleomycin) lipoprotein diacylglycerol transferase dehydratase (lipoprotein diacylglycerol transferase, lgt) T363 strain of the deficient gene (Nakayama M et al., Journal of Immunology 189 : 5903-591, 2012) and T0003 strains resistant to erythromycin and lacking the O-acetyl transferase ( oatA ) gene (Park KH et al. , Journal of Biological Chemistry 285, 27167-). 27175, 2010) each gene was prepared by transducing the phage 80. The strain can be used to isolate WTA, WTA-PGN and PGN without contamination by lipoproteins by deletion of the lgt gene. In addition, due to the lack of the oatA gene, the PGN MurNAc residue 6 position oxygen does not have an acetyl group, so that the isolated PGN can be easily degraded by lysozyme.

Example 2. Heat Treatment of Strains

From the S. aureus T384 strain prepared in Example 1 (Park KH et al. , Journal of Biological Chemistry 285, 27167-27175, 2010; Jung DJ et al. , Journal of Immunology 189: 4951-4959, 2012) The method was modified to give an insoluble WTA-PGN.

Specifically, S. aureus T384 strain of Example 1 was inoculated in LB10 liquid medium (pH 7.0 to 7.2) and incubated for 12 hours at 180 rpm, 37 ℃ conditions. 1 ml of the cultured strain was dispensed into 500 ml LB10 liquid medium and incubated for 4 hours under the same conditions as above in a shaker, and then absorbance was measured at 600 nm every hour. When the OD _600nm = 0.6 was incubated and the culture was stopped at 4 ℃.

The stored strain was centrifuged at 6,000 rpm at 4 ° C. for 30 minutes to obtain pellets. 20 ml of saline was added to resuspend the pellet, which was centrifuged for 5 minutes at 3,000 × g, 4 ° C. to obtain pellet. The obtained pellet was resuspended in 20 ml of saline, which was diluted in 980 ml of saline at 60 ° C. and heat-treated for 30 minutes to a final OD _{600 nm} = 0.3. The flask was stirred every 5 minutes during the heat treatment process so that the strain was uniformly heat treated. After the heat treatment, the diluted solution of the strain was cooled to 30 ° C., which was centrifuged at 3,000 × g, 20 ° C. for 10 minutes to obtain pellets. The pellet was suspended in 10 ml saline and centrifuged for 5 minutes under the same conditions as above to obtain the pellet. The obtained pellet was washed three times with sterile distilled water, and the pellet was suspended in 3 ml of distilled water and lyophilized.

Example 3 Treatment of β-lytic Enzymes and Lysozyme

<3-1> β- lytic  Preparation of Enzymes

Β-lytic enzymes required to prepare soluble WTA-PGN are described in Li et al. , Journal of Biochemistry 122, 772-778, 1997].

First, 5 g of crude achromopeptidase was dissolved in 500 ml of sodium citrate buffer (pH 6.0), and then centrifuged for 15 minutes at 15,000 rpm and 4 ° C. The supernatant was loaded into a CM-Sepharose fast flow column (3 cm x 18 cm) equilibrated with 10 mM sodium citrate buffer (pH 6.0) and eluted by performing a prior gradient from 0 M to 0.5 M NaCl concentration. The eluted solution was measured for absorbance at 280 nm, and the fractions showing lytic activity were collected and concentrated. The concentrated sample was subjected to size exclusion chromatography on a Sephacryl S-100 column (1.6 cm x 87 cm) using 10 mM sodium citrate buffer (pH 6.0) containing 200 mM NaCl. The eluate was measured for absorbance at 280 nm, and the fractions showing lytic activity among the fractions with high absorbance were collected and concentrated again. Li et al. Based on the results of (1997), β-lytic enzyme fractions were selected from the concentrated fractions. The selected fractions were subjected to size exclusion chromatography on a Superdex-75 column (1 cm × 30 cm) using 10 mM sodium citrate buffer (pH 6.0) containing 200 mM NaCl. As a result, β-lytic enzyme was finally obtained.

The obtained β-lytic enzyme was incubated with PGN suspension derived from Micrococcus luteus (ATCC 9341) or insoluble staphylococcus, and the lytic activity or lytic activity of the enzyme was confirmed. In addition, enzymes obtained by Procise® Protein sequencer (Cat. # 491-0, Applied Biosystems, USA) or electrophoresis were identified. As a result, it was found that the obtained enzyme was a β-lytic enzyme by confirming that the N-terminal sequence was S-P-N-G-L-L-Q-F-P-F (SEQ ID NO: 5) and the size was about 20 kDa.

<3-2> β- lytic  Processing of enzymes

The process of suspending in 10 ml of 20 mM Tris-HCl buffer (pH 7.0) per 100 mg of the strain heat treated in Example 2 and centrifugation to remove the supernatant was repeated three times. It was confirmed that the pH of the supernatant was 7.0, and about 355.57 μg of β-lytic enzyme was added per 100 mg of the heat-treated S. aureus T384 strain. It was reacted with stirring at 180 rpm and 37 ° C. for 12 hours. Thereafter, the reaction solution was placed in a constant temperature water bath at 60 ° C. for 10 minutes to deactivate the enzyme, and centrifuged at 15,000 rpm and 4 ° C. for 10 minutes to obtain a supernatant. The supernatant obtained was filtered with a filter having a pore size of 0.45 μm and the filtrate was HPLC (805 MANOMETRIC MODULE, 811C) equipped with a HiTrap Q FF column equilibrated with 20 mM Tris-HCl buffer (pH 7.0) (buffer A). DYNAMIC MIXER, 305 PUMP, 306 PUMP, 151 UV / VIS Detector, Gilson, USA). Thereafter, 20 mM Tris-HCl buffer (pH 7.0) (buffer B) containing 1 M NaCl was eluted by performing a linear gradient from 0 M to 1 M NaCl concentration. The concentration gradient of the buffer B was performed for 100 minutes in a gradient up to 80%, 1 minute in a gradient up to 100%, and then eluted at 100% for 10 minutes.

As a result of the elution, a total of 6 fractions were obtained as shown in FIG. 1. Each fraction was named A, B, C, D, E and F, and each of these fractions was precipitated with acetone and then lyophilized.

<3-3> Lysozyme  process

Each lyophilized A to F fraction was suspended in 20 mM Tris-HCl buffer (pH 7.0). 12.5 μg of lysozyme (Cat. # 62970, Sigma-Aldrich Co. LLC., USA) was added to 1 mg of the lyophilized fraction and reacted with stirring at 180 rpm and 37 ° C. for 12 hours. The subsequent procedure was performed under the same conditions and methods as in Example <3-2>, except that the HiTrap Q column was used.

As a result of the elution, as shown in FIG. 2, one to three peaks were identified according to each fraction.

Each of the peaks identified above were named as AL-A for the A peak obtained in the A fraction, and the same as the BL-A for the A peak obtained in the B fraction, and AL-A, BL-A, CL-B, and DL. The -B, DL-C, EL-C and FL-B peaks were respectively precipitated with acetone and then lyophilized.

Example 4. Confirmation of IL-17A Production Increase Effect by Peak

It was confirmed the effect of increasing the yield of IL-17A for each peak obtained in Example <3-3>.

Specifically, among the obtained peaks, the mixed peaks (EL-C + FL-B) in which each of the CL-B, DL-B and DL-C peaks and the EL-C and FL-B peaks were mixed were prepared by lyophilization, respectively. It was. Meanwhile, C57BL / 6J female mice weighing 15 ± 0.5 g at 5 weeks of age were purchased from Korea Research Institute of Bioscience and Biomedical Research Center (Ochang Campus, Korea) and were commercially available solid feeds (Cat. # 5L79, Orient Bio, Inc.). Korea) was adapted to the environment (20-25 ° C., humidity 55%) for one week while feeding. Six to twelve animals per group were divided into six groups per group in a completely randomized design to provide diet and drinking water by ad libitum. Body weight and dietary intake of each mouse were measured once daily and the lights were turned on and off at 12 hour intervals.

200 μg of each of the prepared lyophilized peak fractions was suspended in LPS-free water at a concentration of 10 mg / ml, and 20 μl of the suspension was added to 80 μl of PBS (Cat. # 17-516Q, LONZA, USA). A total of 100 μl of diluent was injected into the mouse intraperitoneal by diluting. Six hours after the injection, the mice were sacrificed to separate the peritoneal leachate from the mice. To this end, the first 2 ㎖ of Ca ^{2 +} / Mg ^{2 +} as free-PBS after washing the abdominal cavity of the mouse, collecting the supernatant by centrifugation for 10 minutes in the abdominal cavity leachate (peritoneal lavage fluid) 200 xg, 4 ℃ conditions Store at -80 ° C. The amount of IL-17A is ELISA Ready-SET-Go! The kit (Cat. # 88-7371-88, eBioscience, USA) was used to measure according to the manufacturer's protocol. As a result, a value obtained by correcting the absorbance value measured at 450 nm to the absorbance value measured at 550 nm was determined as the amount of final IL-17A.

As a result, as shown in Figure 3, it was confirmed that significantly high levels of IL-17A are produced in mice injected with the abdominal cavity by lyophilizing the DL-B peak.

Example 5 Purification of Soluble WTA-PGN— (1)

The lyophilized fractions in Example <3-3> were further purified using various columns.

First, 50 mg of CL-B peak fraction was suspended in 400 μl of distilled water, and the suspension was injected into an HPLC apparatus (805 MANOMETRIC MODULE, 811C DYNAMIC MIXER, 305 PUMP, 306 PUMP, 151 UV / VIS Detector, Gilson, USA). . Before injection, Sephacryl S-200 HR columns (Cat. # 17-0584-01, GE Healthcare Life Sciences, UK) were connected and washed with distilled water and equilibrated with sterile distilled water. In addition, the solvent was allowed to flow until the UV detector was stabilized under conditions of a flow rate of 0.3 ml / min, a sensitivity of 2, a peak width of 10.0 sec, and a UV absorbance of 220 nm so that there was no error due to impurity inflow during the experiment. After the UV values had stabilized, the injector was converted to the loaded state to inject the suspended peak fractions. The eluent was obtained by converting the injector into the injection state before elution, and the elution conditions were the same as the equilibration conditions. The obtained eluate was named 'Sepacryl-B fraction' and stored by lyophilization.

Thereafter, the Sephacryl-B fraction was further purified using a TSK G2000SW column. Specifically, 3.5 mg of the lyophilized Sephacryl-B fraction was suspended in 5 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl to prepare a sample. On the other hand, the column was prepared by washing and equilibrating with the buffer. Equilibration was performed until the UV detector stabilized under conditions of flow rate 3 ml / min, sensitivity 0.5, peak width 10 seconds and UV absorbance 202 nm. After equilibration, the inlet was changed to a rod state to slowly inject the sample, and when the elution started, the inlet was changed to infusion to obtain an eluate. In this case, 5 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl was used as the elution buffer.

After confirming the IL-17A production amount of the obtained fraction by the same conditions and methods as in Example 4, the B fraction which most significantly increased the production of IL-17A was named 'Sepacryl-B-TSK-B fraction'. Lyophilized and stored.

A C18 reversed phase column was used to further purify the lyophilized Sephacryl-B-TSK-B fraction.

Specifically, 3 mg of lyophilized Sephacryl-B-TSK-B fraction was added to 0.05% (v / v) of trifluoroacetic acid (TFA, Cat. # 204-1771, Wako, Japan) buffer (1 liter of distilled water). Samples were prepared by suspending in 200 μl of 0.5 mL TFA). Flow rate 1 ml / min, sensitivity 1, column temperature 40 ° C. and UV absorbance were stabilized at 202 nm under UV detector, followed by elution. The concentration gradient of the mobile phase for elution was performed for 10 minutes at 0%, 25 minutes with gradients up to 41.7% and 2 minutes with gradients up to 100%, followed by elution at 100% for 10 minutes. At this time, 0.05% (v / v) of trifluoroacetic acid / 40% (v / v) of acetonitrile (Cat. # AH015-4, Burdick & Jackson, USA) buffer (600 mL of distilled water, acetonitrile) as a mobile phase solvent 400 ml and 0.5 ml TFA) were used.

As a result, as shown in FIG. 4, a fraction separated into six peaks of A to F was confirmed.

Example 6 Confirmation of the Effect of IL-17A Production Increase by Peak

Each fraction obtained in Example 5 was used to confirm IL-17A inducing activity in mice under the same conditions and methods as in Example 4, and the results are shown in FIG. 5.

As shown in FIG. 5, it was confirmed that significantly high levels of IL-17A were produced in mice injected into the abdominal cavity by lyophilizing the fraction of the C peak.

Example 7. Purification of Soluble WTA-PGN— (2)

Using a fraction of 2 mg of the lyophilized C to E peak obtained in Example 5, a C18 reversed phase column was used in the same conditions and methods as in Example 5 to purify it more purely.

As a result, as shown in FIG. 6, fractions separated into three peaks of C, D, and E were identified, and IL-17A-induced activity in mice was observed under the same conditions and methods as in Example 4 using these fractions. As a result, as shown in Figure 7, it was confirmed that the highest amount of IL-17A produced in the C fraction.

Example 8 Confirmation of Structure of PGN

<8-1> Of WTA  remove

In Example 7, WTA was first removed to confirm the structure of Fraction C, which showed a production increase activity of IL-17A.

Specifically, 3 mg of lyophilized C fraction was dissolved in 50 μl of sterile distilled water. 50 μl of 25% TFA was added thereto and incubated with stirring at a temperature of 37 ° C. for 12 hours. After incubation, 100 μl of sterile distilled water was added to prepare a final 6.25% concentration of TFA. Thereafter, the Symmetry Shield ™ RP18 column was used in the same conditions and methods as in Example 5 to purify only PGN free of WTA. At this time, the flow rate was carried out under the conditions of 1 mL / min, a sensitivity of 0.5, a column temperature of 40 ° C., and a UV absorbance of 203 nm. The concentration gradient of the mobile phase for elution was performed for 10 minutes at 0%, 50 minutes at gradient to 43.8%, 2 minutes at gradient to 100%, and then eluted at 100% for 10 minutes.

<8-2> Of PGN  Check structure

The eluate obtained in Example <8-1> was loaded onto the column via an autoinjector in a Nano-flow (n) LC-MS / MS system.

nLC-MS / MS systems are high resolution hybrid masses with nLC (EASY-nLC 1000, Thermo Fisher Scientific, USA) with fritless electrospray (ESI) columns (150 μm × 100 mm, Nikkyo Technos Co. Ltd, Japan) It consists of a spectrometer (Q-Exactive mass spectrometer, Thermo Fisher Scientific, USA).

nLC separation was eluted using a 32% (v / v) acetonitrile solution containing 0.1% (v / v) formic acid as elution buffer for 30 minutes at a flow rate of 300 nL / min. The eluate was sprayed online with a mass spectrometer at a pressure of 2.0 kPa in the bipolar state, and the analyzer was operated in data-dependent mode to automatically switch between MS and MS / MS acquisition. As a result, a mass resolution of 35,000 was obtained at m / z 400 through full-scan mass spectra (m / z 300 to 2,000) measurements. The ten strongest mass peaks with an intensity of 10,000 counts / s or more were identified in the survey scan, and each peak was separated within 3 m / z window to fractionate. Mass peaks were fractionated by high-energy collisional dissociation at corrected collision energy 15. For MS / MS measurements, a mass resolution of 17,500 at m / z 400 was used.

As a result, PGN having a structure represented by the following formula 1 was confirmed. It had five monosaccharide chains represented by GlcNAc-MurNAc-GlcNAc-MurNAc-GlcNAc and had one phosphate group bound to the fourth MurNAc residue. The PGN of the C fraction had a molecular weight of 2167.91.

[Equation 1]

Claims (14)

1. A composition for the prevention or treatment of Staphylococcal infection disease, which comprises a wall teichoic acid-attached peptidoglycan (WTA-PGN) represented by Formula 1 as an active ingredient:

   [Formula 1]

   

   In Formula 1, n is an integer of 10 to 50, m is an integer of 1 to 3, A is N-acetylmannosamine (ManNAc), B is N-acetylglucosamine (GlcNAc),

   O is an integer from 1 to 5, R ₁ and R ₂ are each independently hydroxy, tetrapeptide or pentapeptide, and R ₃ is hydroxy or N-acetylmuramic acid (MurNAc).
2. According to claim 1, wherein in Formula 1 A and B are linked to each other in the β-position, composition for the prevention or treatment of staphylococcal infection disease.
3. The compound of claim 1, wherein in Formula 1, n is an integer of 35 to 45, m is 3, A is ManNAc, B is GlcNAc, O is an integer of 1 to 5, R ₁ and R ₂ Are each independently hydroxy, tetrapeptide or pentapeptide, R ₃ is hydroxy or MurNAc, composition for the prevention or treatment of Staphylococcal infection disease.
4. The method of claim 3, wherein in Formula 1, n is 40, m is 3, A is ManNAc, B is GlcNAc, O is an integer of 1 to 5, R ₁ and R ₂ are each independently Tetrapeptide or pentapeptide, R ₃ is hydroxy or MurNAc, composition for the prevention or treatment of staphylococcal infection disease.
5. The method of claim 4, wherein the tetrapeptide is -X ₁ -X ₂ -X ₃ -X ₄ , wherein X ₁ is Ala or Gly, X ₂ is Glu or Asp, and X ₃ is Lys, Arg or His , X ₄ is Ala or Gly, composition for the prevention or treatment of staphylococcal infection disease.
6. The composition of claim 5, wherein the tetrapeptide is-(L-Ala)-(D-Glu)-(L-Lys)-(D-Ala).
7. The method according to claim 4, wherein the pentapeptide is

   

   Wherein X ₁ is Ala or Gly, X ₂ is Glu or Asp, X ₃ is Lys, Arg or His, and X ₄ is Ala or Gly.
8. The method of claim 7, wherein the pentapeptide is

   

   Phosphorus, staphylococcal infection disease prevention or treatment composition.
9. The method of claim 1, wherein the staphylococcus is Staphylococcus aureus (methicillin-resistant Staphylococcus aureus , MRSA), methicillin-sensitive Staphylococcus aureus (MSSA) or Staphylococcus aureus Composition for preventing or treating a disease.
10. The method of claim 1, wherein the staphylococcal infection disease is soft tissue infection, purulent arthritis, purulent osteomyelitis, otitis media, pneumonia, sepsis, acute respiratory tract infection, infection due to the use of a catheter, wound infection after surgery, Composition for the prevention or treatment of staphylococcal infection disease, selected from the group consisting of bacteremia, endocarditis and food poisoning.
11. A method for preventing or treating a Staphylococcal infection disease, comprising administering the composition of any one of claims 1 to 10 to a subject in need thereof.
12. The method of claim 11, wherein the method induces opsonophagocytosis and phagocytosis at the same time, the composition for preventing or treating staphylococcal infection disease.
13. The composition of claim 11, wherein the method increases IL-17A production in the subject within 24 hours after administration of the composition.
14. 1) from wild-type staphylococcal lgt (lipoprotein diacylglycerol transferase) and oatA (O-acetyl transferase) gene comprising: obtaining a double deletion mutant strain;

    2) heating the double mutant strain to a temperature of 40 to 70 ° C;

    3) adding β-lytic enzyme to the heated strain;

    4) obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3);

    5) adding lysozyme or mutanolysin to the soluble WTA-PGN containing fractions; And

    6) A method for preparing WTA-PGN represented by the general formula 1, comprising the step of obtaining soluble WTA-PGN from the enzyme additive of step 5).

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