Antibacterial proteins having bacteriolytic activity against streptococci

Abstract

The present invention relates to an antibacterial protein SBL2200 having a bacteriolytic activity specific to Streptococcus (S trept cocc i), and to a method for preparing the antibacterial protein SBL2200 having a bacteriolytic activity specific to Streptococcus (S trept cocc i). More particularly, the present invention relates to: an antibacterial protein SBL2200 specific for streptococcus, characterized by having an ability to specifically lyse streptococcus and having an amino acid sequence represented by SEQ ID NO: 1; and a pharmaceutical composition for treating infection induced by streptococcus, containing the antibacterial protein SBL2200 specific for streptococcus as an active ingredient and a compound.

Description

Technical Field

[0001] The present invention relates to an antimicrobial protein SBL2200 having bacteriolytic activity against streptococci, and more particularly to an antimicrobial protein SBL2200 specific for streptococci, characterized by having the ability to specifically lyse streptococci that may cause infectious diseases and having an amino acid sequence represented by sequence number 1, and a pharmaceutical composition for treating infectious diseases induced by streptococci, the pharmaceutical composition comprising the antimicrobial protein SBL2200 specific for streptococci as an active ingredient. Background Art

[0002] Streptococci are Gram-positive bacteria that grow in pairs or chains. They can be divided into α-hemolytic (partial hemolysis), β-hemolytic (complete hemolysis) and γ-hemolytic (no hemolysis, enterococcus) according to the degree of hemolysis induced. α-hemolytic can be further subdivided according to biochemical characteristics (Optochin susceptibility), while β-hemolytic can be further subdivided into groups A to V (serotype) according to the serological characteristics (Lancefield classification) of the C-polysaccharide produced during metabolism.

[0003] Streptococci are widely distributed in nature. Although some species belong to the normal flora of humans, it is known that Streptococcus pneumoniae (S.pneumoniae), Streptococcus pyogenes (S.pyogenes) and Streptococcus agalactiae (S.agalactiae) are serious pathogens. Infectious diseases induced by pathogenic streptococci as mentioned above include bacteremia, endocarditis, abscesses, dental caries, pneumonia, meningitis, sinusitis, otitis media, pharyngitis, scarlet fever, suppurative skin diseases, cellulitis, necrotizing fasciitis, rheumatic fever, glomerulonephritis, neonatal infections (meningitis, pneumonia, bacteremia), urinary tract infections, chorioamnionitis, endometritis and wound infections.

[0004] Specifically, it is known that the main pathogen of group A streptococcal infection is Streptococcus pyogenes (S. pyogenes), which mainly induces non-invasive infections such as pharyngitis, suppurative skin diseases and soft tissue inflammation through the throat and skin, and invasive infections such as rheumatic fever, acute glomerulonephritis, sepsis, scarlet fever and bacteremia. It has been reported that as of 2018, there were tens of millions of non-invasive group A streptococcal infections in the United States, and about 12,500 to 20,000 people were infected with invasive group A streptococci each year, of which about 1,250 to 1,900 died. Antibiotics used to treat group A streptococcal infections include penicillin, amoxicillin, erythromycin, clindamycin and macrolides. As of 2017, the antibiotic resistance rates to erythromycin, clindamycin and macrolides have shown an annual growth trend of approximately 20%.

[0005] It is known that the main pathogen of group B streptococcal infection is Streptococcus agalactiae (S. agalactiae), which mainly inhabits the intestines or female genitals. It can infect newborns during childbirth and induce diseases such as sepsis and meningitis. It can also induce bacteremia, pneumonia, bone and joint infections, skin and soft tissue infections in adults. It is reported that as of 2016, there were about 31,000 cases of serious group B streptococcal infection in the United States, of which about 1,700 people died. In addition, it has been reported that for the treatment of group B streptococcal infection as mentioned above, penicillin, clindamycin and erythromycin are mainly used, but as for clindamycin and erythromycin, as of 2016, about 40% to 60% of antibiotic resistance has been shown.

[0006] In order to effectively deal with streptococcal infections that have become resistant to existing antibiotics, it is necessary to develop new antibacterial substances, especially there is an urgent need to develop pharmaceutical preparations with new mechanisms of action. Summary of the invention

[0007] Therefore, as a solution to the problems caused by the use of existing antibiotics to treat harmful pathogenic streptococci, the present inventors intend to provide an antimicrobial protein SBL2200 that can specifically lyse streptococci, and provide a pharmaceutical composition comprising the antimicrobial protein as an active ingredient and which can be used to treat streptococcal infections, and further provide a method for effectively treating streptococcal infections using the pharmaceutical composition.

[0008] Therefore, an object of the present invention is to provide an antimicrobial protein SBL2200 having an amino acid sequence represented by SEQ ID NO: 1 and having antimicrobial activity capable of specifically lysing streptococci.

[0009] Another object of the present invention is to provide a method for effectively producing the antimicrobial protein SBL2200 having an antimicrobial activity that can specifically lyse streptococci and having an amino acid sequence represented by sequence number 1. In this regard, Escherichia coli AB-22 is provided as a production strain of the antimicrobial protein SBL2200. The production strain, i.e., Escherichia coli AB-22, was deposited in the Bioresource Center of the Korea Institute of Biotechnology on July 12, 2022 (deposit number KCTC 15033BP).

[0010] Another object of the present invention is to provide a pharmaceutical composition for the purpose of treating streptococcal infection, comprising the antimicrobial protein SBL2200 that can specifically lyse streptococci as an active ingredient.

[0011] In addition, another object of the present invention is to provide a method for treating streptococcal infection using a pharmaceutical composition comprising the antimicrobial protein SBL2200 as an active ingredient.

[0012] In order to achieve the above-mentioned objectives, the inventors of the present invention utilized the information of various antimicrobial proteins known to have antimicrobial activity against various bacterial species to maximize their own knowledge and professional capabilities and produced various antimicrobial protein candidates in the form of recombinant proteins, and screened out antimicrobial proteins with excellent lytic activity by detecting their lytic activity against streptococci, and further developed a method for effectively producing the antimicrobial proteins, and finally developed a pharmaceutical composition that contains the antimicrobial proteins as active ingredients and can be used for the purpose of treating streptococcal infections, thereby completing the present invention.

[0013] Therefore, in one aspect of the present invention, the present invention provides an amino acid sequence of an antimicrobial protein SBL2200 that can specifically lyse streptococci. Specifically, it is an amino acid sequence corresponding to sequence number 1. The antimicrobial protein SBL2200 that can specifically lyse streptococci is composed of 279 amino acid residues and has a molecular weight of approximately 31.3 kDa.

[0014] It is obvious that the amino acid sequence of sequence number 1 can be partially deformed by relevant practitioners using known techniques. The deformation includes partial substitution of the amino acid sequence, partial addition of the amino acid sequence, and partial deletion of the amino acid sequence. However, it is preferable to apply the amino acid sequence of sequence number 1 disclosed in the present invention.

[0015] In addition, the present invention provides a production strain, namely Escherichia coli AB-22, which can be used for producing the antimicrobial protein SBL2200 having the amino acid sequence of SEQ ID NO: 1. The production strain, namely Escherichia coli AB-22, is a strain that can produce SBL2200 by transforming Escherichia coli with a plasmid having the nucleic acid sequence (4,907 bp) of SEQ ID NO: 2 produced by the present inventors.

[0016] In another aspect of the present invention, the present invention provides a pharmaceutical composition that can be effectively used for treating streptococcal infection, comprising, as an active ingredient, an antimicrobial protein SBL2200 characterized by the amino acid sequence of SEQ ID NO: 1 and having a specific bacteriolytic ability against streptococci.

[0017] The antimicrobial protein SBL2200 characterized by the amino acid sequence of SEQ ID NO. 1 and having a specific bacteriolytic ability against streptococci contained in the pharmaceutical composition of the present invention can specifically bacteriolyze streptococci as described above, and thus has the effect of treating various diseases induced by streptococci. In addition, the pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, bactericide, and therapeutic agent, and is used to treat various diseases induced by streptococci.

[0018] Therefore, in another aspect of the present invention, the present invention provides a method for treating various diseases induced by streptococci by administering an antimicrobial protein SBL2200 characterized by having an amino acid sequence represented by SEQ ID NO: 1 and specific to streptococci to a subject infected with tennis ball.

[0019] In the present specification, "diseases induced by streptococci" refers to diseases induced by streptococcal infection, such as bacteremia, endocarditis, abscesses, dental caries, pneumonia, meningitis, sinusitis, otitis media, pharyngitis, scarlet fever, suppurative skin diseases, cellulitis, necrotizing fasciitis, rheumatic fever, glomerulonephritis, neonatal infections (meningitis, pneumonia, bacteremia), urinary tract infections, chorioamnionitis, endometritis and wound infections, but is not limited to these.

[0020] In this specification, streptococci may be sensitive to existing antibiotics or resistant to existing antibiotics. In other words, whether or not the streptococci are resistant to existing antibiotics has no effect.

[0021] The term "treatment" or "treatment" as used in this specification refers to all actions that can inhibit infection induced by Streptococcus and alleviate the pathological state of diseases induced by Streptococcus.

[0022] In order to improve the efficiency for the above-mentioned application purposes, antibacterial substances that can provide antibacterial activity against other bacterial species may be added to the pharmaceutical composition of the present invention.

[0023] The pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are substances commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, but are not limited thereto. In addition to the above ingredients, the pharmaceutical composition of the present invention may additionally include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives.

[0024] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenterally, it can be administered by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration. In addition, it can also be used by coating or spraying on the diseased area, but is not limited to this.

[0025] The pharmaceutical composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients in a method that can be easily implemented by a person having general knowledge of the technical field to which the present invention belongs, so as to be manufactured into a unit dosage form or a form to be loaded into a multi-dose container. In this case, the dosage form can be a solution, suspension or emulsion in an oily or aqueous medium, or a paste, powder, granule, tablet or capsule, and can also contain a dispersant or stabilizer.

[0026] In addition, the appropriate coating, spraying or administration amount of the pharmaceutical composition can vary depending on factors such as formulation method, administration method, age, body weight, sex, disease symptoms, diet, administration time, administration route, excretion rate and reaction sensitivity. Generally speaking, an experienced doctor or veterinarian can easily determine and prescribe an effective dosage according to the desired therapeutic effect.

[0027] The pharmaceutical composition of the present invention can be implemented as an antibiotic, disinfectant, bactericide, and therapeutic agent.

[0028] According to the method for treating infection of Streptococcus using a pharmaceutical composition containing the antimicrobial protein SBL2200 having the amino acid sequence represented by SEQ ID NO. 1 as an active ingredient, it is possible to effectively act on Streptococcus that is resistant to existing antibiotics or antimicrobial substances. In addition, the antimicrobial protein SBL2200 specific to Streptococcus of the present invention does not affect other normal commensal bacteria except Streptococcus, thereby minimizing the side effects caused by using the pharmaceutical composition containing the antimicrobial protein SBL2200 as an active ingredient. Existing antibiotics have the problem of inducing multiple side effects because they also have adverse effects on probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an electrophoresis photograph illustrating the results of producing the antibacterial protein SBL2200, which is characterized by the amino acid sequence represented by sequence number 1 and is specific to Streptococcus, in the form of a recombinant protein. Lane M is a protein size marker, and lane 1 is the chromatography eluate during secondary purification. DETAILED DESCRIPTION

[0030] Next, the present invention will be described in more detail with reference to examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[0031] Example 1: Preparation of antimicrobial protein SBL2200 specific to Streptococcus

[0032] Next, the production of the antimicrobial protein SBL2200 specific to Streptococcus will be described. In this example, Escherichia coli AB-22, a strain produced by transforming Escherichia coli with a plasmid having the nucleic acid sequence of SEQ ID NO: 2 produced by the present inventors, was used as a production strain.

[0033] After adding 20 μl of Escherichia coli AB-22 to 20 ml of LB medium (Tryptone 10 g / L, yeast extract 5 g / L and NaCl 10 g / L) containing 50 μg / ml kanamycin for inoculation, shaking culture was performed overnight at 37°C. The next day, the culture solution that had been cultured overnight was added to 1 L of LB medium containing 50 μg / ml kanamycin at a volume ratio of 1 / 100. Culture was performed at a temperature of 37°C with a stirring speed of 200 rpm. When the cell concentration reached 0.3 to 0.35 based on the absorbance at 600 nm, the culture temperature was changed to 25°C. Then, after about 20 minutes, when the cell concentration reached 0.5 based on the absorbance at 600 nm, L-arabinose was added to a final concentration of 0.2% to induce the expression of the antimicrobial protein SBL2200 having the amino acid sequence represented by sequence number 1, and the culture was continued for an additional 6 hours.

[0034] After the culture was completed, the cell culture solution was taken and centrifuged at 6,000 rpm for 10 minutes at 4°C to recover the cell precipitate, and the recovered cell precipitate was suspended in 40 ml of a buffer solution (50 mM potassium phosphate and 2 M NaCl, pH 7.5). The cell suspension prepared as described above was disrupted by ultrasonic disruption, which was repeated for 15 minutes under the condition of applying ultrasonic waves for 3 seconds and then stopping for 3 seconds, and was performed in an ice bath.

[0035] After the cells were pulverized, the supernatant obtained by centrifuging the cell lysate at 13,000 rpm at 4° C. for 20 minutes was purified by a general hydrophobic interaction chromatography and anion exchange chromatography purification process.

[0036] Next, the purification process will be briefly described. In this example, 5 ml of HiTrap was used as the hydrophobic interaction resin. TMPhenyl HP (GE Healthcare) and 5 ml of HiTrap were used as anion-exchange resin. TM Q HP (GE Healthcare). As the primary purification process, the hydrophobic interaction chromatography operation was performed after the chromatography column was equilibrated in advance with buffer A (50 mM potassium phosphate and 2 M NaCl, pH 7.5). After the sample was dripped onto the chromatography column, it was washed with 5 column volumes (CV, Column Volume) of buffer A at a flow rate of 5 ml / min. After the washing was completed, the primary purification process, i.e., the hydrophobic interaction chromatography operation, was performed at a flow rate of 5 ml / min with a concentration gradient (Gradient) from 0% to 100% from buffer A to buffer B. Next, the secondary purification process, i.e., the anion exchange resin chromatography operation, was performed using the obtained primary purification eluate. The cation exchange resin chromatography operation was performed after the chromatography column was equilibrated with buffer B (buffer B, 50mM potassium phosphate, pH 7.5) in advance. After the sample obtained in the initial purification process was dripped onto the chromatography column, it was washed at a flow rate of 5ml / min with 10 column volumes (CV) at 30% of buffer C (buffer C, 50mM potassium phosphate and 1M NaCl, pH 7.5) in buffer B. After the washing was completed, the chromatography operation was performed at a flow rate of 5ml / min with a concentration gradient from 30% to 100% from buffer B to buffer C. In the process, the desired antimicrobial protein SBL2200 with the amino acid sequence represented by sequence number 1 was eluted. Figure 1 The figure shows the electrophoresis analysis results of the antibacterial protein SBL2200 purified by the chromatography purification process.

[0037] The fractions containing the antimicrobial protein SBL2200 at a high concentration were collected from the purified fractions obtained, and the fractions were dialyzed against a buffer solution (50 mM potassium phosphate, pH 7.5) to perform medium exchange, thereby obtaining an antimicrobial protein SBL2200 solution having a purity of 90% or more.

[0038] The "SBL2200 pharmaceutical composition" was produced by buffer exchange of the SBL2200 solution concentrated as described above with a buffer (buffer, 50 mM potassium phosphate, 5% (w / v) sorbitol and 0.1% (w / v) poloxamer 188, pH 7.5).

[0039] Example 2: Antimicrobial activity detection of antimicrobial protein SBL2200

[0040] The antibacterial activity of the streptococcus-specific antibacterial protein SBL2200 produced in Example 1 was tested. The target strain information used in the antibacterial activity test is shown in Table 1.

[0041] Table 1

[0042] Antimicrobial activity test strains

[0043]

[0044]

[0045] In the antibacterial activity test, turbidity reduction assay and minimum inhibitory concentration (MIC) assay were used. Turbidity reduction assay was implemented as described below. The test object bacteria were suspended in physiological saline so that the absorbance at 600nm reached about 0.7, and then 0.1ml of antimicrobial protein SBL2200 solution (final concentration: 2.5μM) was added to 0.9ml of the suspension, and then the absorbance was measured at 600nm for 30 minutes. As a negative control group, a buffer (buffer, 50mM potassium phosphate, 5% (w / v) sorbitol and 0.1% (w / v) poloxamer 188, pH 7.5) that did not contain antimicrobial protein SBL2200 was used instead of the antimicrobial protein SBL2200 solution. The minimum inhibitory concentration (MIC) was performed and derived according to the general minimum inhibitory concentration (MIC) test method specified in the Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0046] The results showed that the antimicrobial protein SBL2200 only exhibited bacteriolytic activity against Streptococcus, and did not exhibit bacteriolytic activity against other test subject strains. The results are shown in Table 2.

[0047] Table 2

[0048] Antimicrobial activity test results

[0049]

[0050]

[0051] The above results confirmed that the antibacterial activity of the antibacterial protein SBL2200 of the present invention has very significant specificity against Streptococcus.

[0052] The above results confirm that the antimicrobial protein SBL2200 specific to streptococci of the present invention can kill streptococci by lysing them. The above characteristics indicate that the pharmaceutical composition containing the antimicrobial protein SBL2200 specific to streptococci can be used for the purpose of killing streptococci when infected by streptococci, and can also be used for the purpose of treating streptococcal infection in the same manner as general antibiotics.

[0053] Example 3: Detection of the therapeutic effect of the Streptococcus-specific antimicrobial protein SBL2200 on Streptococcus infection

[0054] The therapeutic effect of the antimicrobial protein SBL2200 on streptococcal infection was tested by an infection animal model test. Specifically, 5-week-old ICR mice weighing about 20 g were used as test animals. A total of 20 mice were divided into two groups (10 mice in each group), and each mouse was injected with 1×10 8 cfu of Streptococcus agalactiae CARM 4503 bacteria (i.e. 1×10 8 cfu / animal) to induce infection. For the animals in the treatment group, 0.2 ml of the pharmaceutical composition of the antimicrobial protein SBL2200 was intravenously injected at 30 minutes, 12 hours and 24 hours after the forced bacterial infection. For the animals in the control group, only the same volume of buffer (buffer, 50mM potassium phosphate, 5% (w / v) sorbitol and 0.1% (w / v) poloxamer 188, pH 7.5) was injected. The injection of buffer was performed at 30 minutes, 12 hours and 24 hours after the forced bacterial infection in the same manner as the injection of the pharmaceutical composition of the antimicrobial protein SBL2200. The number of deaths per day was detected within five days after the forced bacterial infection, and the occurrence of special phenomena was detected twice a day in the morning and afternoon.

[0055] As a result, a significant therapeutic effect was observed. The number of deaths is shown in Table 3 below. The injection of the pharmaceutical composition of the antimicrobial protein SBL2200 of the present invention provided a significant improvement in the survival rate of infected animals. In addition, compared with the situation in which various special reactions such as erythema of lid margin and decreased activity were observed in the control group, no special reactions as described above were observed in the animals in the treatment group injected with the pharmaceutical composition of the antimicrobial protein SBL2200.

[0056] Table 3 Treatment effect of streptococcal infection

[0057]

[0058] The above results confirm that the antimicrobial protein SBL2200 of the present invention can effectively treat streptococcal infection. The above characteristics indicate that the pharmaceutical composition containing the antimicrobial protein SBL2200 as an active ingredient can be used for the purpose of treating streptococcal infection.

[0059] In the above content, specific parts of the present invention are described in detail, but people with general knowledge of the industry should understand that these specific descriptions are only preferred implementation examples, and the scope of the present invention is not limited thereby. Therefore, the substantive scope of the present invention should be defined by the attached claims and their equivalents.

[0060] Name of depository institutionKCTC

[0061] Accession number: KCTC 15033BP

[0062] Collection date: 20220712

[0063]

Claims

An antibacterial protein SBL2200 having antibacterial activity specific to Streptococcus and having an amino acid sequence represented by SEQ ID NO:

1.

2. The antimicrobial protein SBL2200 according to claim 1, characterized in that: The antimicrobial protein SBL2200 consists of 279 amino acids and has a molecular weight of 31.3 kDa.

3. A pharmaceutical composition for treating streptococcal infection, comprising the antimicrobial protein SBL2200 according to claim 1 as an active ingredient.

4. The pharmaceutical composition for treating streptococcal infection according to claim 3, characterized in that: The pharmaceutical composition is used as an antibiotic, disinfectant, bactericide or therapeutic agent.

5. A method for producing the antimicrobial protein SBL2200 according to claim 1 using the production strain Escherichia coli AB-22 (Deposit No. KCTC 15033BP) produced by transforming Escherichia coli with the plasmid represented by the nucleic acid sequence of SEQ ID NO: 2.

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