ANTIBACTERIAL PROTEIN CDL200 HAVING LYTIC ACTIVITY AGAINST CLOSTRIDIOIDES DIFFICILE

Abstract

The present invention relates to an antibacterial protein CDL200 with an antibacterial activity specific against Clostridioides difficile. More specifically, the present invention relates to an antibacterial protein CDL200 specific to Clostridioides difficile that may infect and cause disease in animals including humans, the antibacterial protein CDL200 being characterized by having the ability to specifically lyse the Clostridioides difficile and containing the amino acid sequence represented by SEQ ID NO: 1. In addition, the present invention relates to a pharmaceutical composition for treating an infection or disease caused by Clostridioides difficile, the composition containing the antibacterial protein CDL200 specific to Clostridioides difficile as an active ingredient.

Claims

1. A Clostridioides difficile-specific antibacterial protein CDL200 having specific antibacterial activity to Clostridioides difficile and comprising the amino acid sequence represented by SEQ ID NO: 1.

2. The Clostridioides difficile-specific antibacterial protein CDL200 of claim 1, characterized in that the antibacterial protein CDL200 comprises 160 amino acids and has a molecular weight of 17.6 kDa.

3. A pharmaceutical composition for treating a Clostridioides difficile infection, the composition comprising the Clostridioides difficile-specific antibacterial protein CDL200 of claim 1 as an active ingredient.

4. The pharmaceutical composition of claim 3, characterized in that the composition is used for food compositions, antibiotics, disinfectants, fungicides, or therapeutic uses.

5. A method of producing the Clostridioides difficile-specific antibacterial protein CDL200 of claim 1 using a strain produced by transforming Escherichia coli using a plasmid represented by the gene sequence of SEQ ID NO: 2.

Description

DESCRIPTION OF DRAWINGS

[0029] FIG. 1 is an electrophoretic photograph showing the results of preparing a Clostridioides difficile-specific antibacterial protein CDL200 characterized by containing an amino acid sequence represented by SEQ ID NO: 1, in the form of a recombinant protein, in which lane M is a protein size marker, lane 1 is a cell lysate, lane 2 is a chromatographic solution during purification, and lanes 3 through 9 are purified fractions.

[0030] FIG. 2 shows the results of a liquid lysis test of confirming the effect of an antibacterial protein CDL200 on Clostridioides difficile. A negative control used was a buffer solution itself that did not contain an antibacterial protein CDL200, meaning that a lower value indicates a better antibacterial activity. The horizontal axis represents the strain type and the vertical axis represents the absorbance at 600 nm.

[0031] FIG. 3 shows the results of a turbidity reduction assay of confirming the effect of an antibacterial protein CDL200 on Clostridioides difficile. A negative control used was a buffer solution itself that did not contain an antibacterial protein CDL200. The horizontal axis represents time in minutes and the vertical axis represents absorbance at 600 nm.

BEST MODE

[0032] Hereinafter, the present invention will be described in more detail with reference to Examples. However, the Examples are merely examples of the present invention, and the scope of the present invention is not limited to the Examples.

Example 1: Preparation of Clostridioides Difficile-Specific Antibacterial Protein DL200

[0033] A method of preparing a Clostridioides difficile-specific antibacterial protein CDL200 will be described below. In this example, TOP10-CDL200, which is a strain produced by transforming Escherichia coli using a plasmid having the gene sequence of SEQ ID NO: 2 produced by the inventors, was used as a production strain.

[0034] 20 ml of LB medium containing 50 μg/ml kanamycin (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) was inoculated with 20 μl of the TOP10-CDL200, followed by shaking culture at 37° C. for overnight. The next day, the overnight culture solution was added to 0.5 L of LB medium containing 50 μg/ml kanamycin in a 1/100 volume ratio. The culture was performed under conditions of a stirring speed of 220 rpm and a temperature of 37° C. When the cell concentration reached 0.5 on an absorbance basis at 600 nm, anhydrotetracycline was added so that the final concentration became 0.2 μg/ml. In this way, the expression of an antibacterial protein CDL200 of the amino acid sequence represented by SEQ ID NO: 1 was induced. Then, 3 hours of culturing was performed.

[0035] After the end of the culture, the cell culture was taken and centrifuged at 7,000 rpm and 4° C. for 15 minutes to recover a cell precipitate. The collected cell precipitate was floated in 30 ml of a buffer solution (50 mM K.sub.3PO.sub.4, pH 7.5). The cells in the cell suspension thus prepared underwent ultrasonic pulverization. The ultrasonic pulverization was performed in a manner that a cycle of three seconds of cell breaking and 3 seconds of resting was repeated for a total of 15 minutes in an ice bath.

[0036] After the cell breaking, the cell-broken solution was centrifuged at 7,000 rpm at 4° C. for 15 minutes to obtain a supernatant, and the supernatant was purified through a conventional cation-exchange chromatography purification process.

[0037] The purification process will be briefly described below. In the present example, 5 ml of HiTrap™ SP FF (GE Healthcare Company) was used as a cation-exchange resin. The chromatography was performed after pre-equilibrating the column with buffer A (50 mM tris-hydrochloric acid, pH 7.5). A sample was dropped on the column, and 10 column volume (CV) of the buffer A was forced to flow at a flow rate of 5 ml/min for washing. After the washing, chromatography was performed at a flow rate of 5 ml/min in a condition in which a concentration gradient from the buffer A to the buffer B (50 mM tris K.sub.3PO.sub.4 hydrochloric acid, 1 M NaCl, pH 7.5) became from 0% to 100% was obtained. In this process, the elution of the target antibacterial protein CDL200 having the amino acid sequence represented by SEQ ID NO: 1 was achieved. FIG. 1 shows the results of electrophoretic analysis of the purified antibacterial protein CDL200 using the chromatographic purification process.

[0038] Among the obtained purified fractions, fractions containing the antibacterial protein CDL200 at a high concentration, which correspond to lanes 3 to 9 in FIG. 1, were collected, and the collected fractions were subjected to dialysis using a buffer solution (20 mM K.sub.3PO.sub.4 hydrochloric acid, pH 7.0) for medium exchange. Thus, an antibacterial protein CDL200 solution having a purity of 90% or more was obtained.

Example 2: Investigation of Antibacterial Activity of Antibacterial Protein CDL200 Through Liquid Lysis Test

[0039] Antibacterial activity of the antibiotic resistant Clostridioides difficile of the Clostridioides difficile-specific antibacterial protein CDL200 prepared according to Example 1 was investigated. As the Clostridioides difficile used in the antibacterial activity investigation, eight strains of antibiotic resistant Clostridioides difficile were used. The details thereof are shown in Table 1.

TABLE-US-00001 TABLE 1 Species Strain Source Clostridioides CCARM 0184 Antibacterial difficile CCARM 0185 resistant bacteria CCARM 0186 bank CCARM 0187 CCARM 0188 CCARM 0189 CCARM 0190 CCARM 0348

[0040] Antibacterial activity investigations were performed using a liquid lysis test. Experimental methods for the liquid lysis test will be described below. The test strain was floated in a physiological saline solution to a concentration corresponding to an absorbance of 0.5 at 600 nm, and 0.05 ml of the CDL200 solution (20 μM) prepared in Example 1 was added to and mixed well with the solution (final concentration: 2 μM). The mixture was left still at 37° C. for 30 minutes, and then the absorbance was measured. In this case, a buffer solution (20 mM K.sub.3PO.sub.4, pH 7.0) that does not contain the protein CDL200 was added in place of the antibacterial protein CDL200 solution as a negative control. On the other hand, the antibacterial activity evaluation of bacteria other than Clostridioides difficile was performed using the same method as in the liquid lysis test. The evaluation was performed on 2-week Enterococcus faecalis 2, 2-week Enterococcus faecium, 2-week Staphylococcus aureus 2, 2-week Salmonella, and 2-week Escherichia coli 2.

[0041] As a result, the antibacterial protein CDL200 exhibited bacteriolytic activity only against Clostridioides difficile and did not exhibit the bacteriolytic activity against the other tested strains. The test results for Clostridioides difficile against which the bacteriolytic activity was confirmed are shown in FIG. 2.

[0042] On the basis of these results, it was confirmed that the antibacterial activity of the antibacterial protein of the present invention is very specific to Clostridioides difficile.

Example 3: Investigation of Antibacterial Activity of Antibacterial Protein CDL200 Through Turbidity Reduction Assay

[0043] The antibacterial activity of the antibacterial protein CDL200 against the eight strains of Clostridioides difficile tested in Example 2 was evaluated using a turbidity reduction assay. Experimental methods for the turbidity reduction assay will be described below. After the test bacteria were suspended in a physiological saline solution such that an absorbance was about 0.6 to 0.7 at 600 nm, 0.1 ml of the antibacterial protein CDL200 solution was added to the suspension so that the final concentration was 2.5 μg/ml. The absorbance at 600 nm was then measured for 30 minutes. A buffer solution (20 mM K.sub.3PO.sub.4, pH 7.0) containing no antibacterial protein CDL200 was used instead of the antibacterial protein CDL200 solution, as a negative control.

[0044] The experimental results for eight strains were similar. In the results, the antibacterial protein CDL200 rapidly lysed the tested bacteria so that the absorbance was reduced. This rapid lytic effect is characteristic of the antibacterial protein CDL200 according to the present invention, and no existing antibiotics have not yet provided such a good effect. The experimental results for CCARM 0190 strains are presented in FIG. 3.

[0045] The results described above clearly showed that the Clostridioides difficile-specific antibacterial protein CDL200 of the present invention could eventually kill Clostridioides difficile by lysis. This characteristic shows that a pharmaceutical composition containing the Clostridioides difficile-specific antibacterial protein CDL200 can be utilized for the purpose of killing Clostridioides difficile upon infection with Clostridioides difficile and can also be utilized in the same manner as conventional antibiotics for the purpose of treating Clostridioides difficile infections.

Example 4: Investigation of Therapeutic Effect of Clostridioides Difficile-Specific Antibacterial Protein DL200 on Clostridioides Difficile Infection

[0046] The therapeutic effect of the Clostridioides difficile-specific antibacterial protein CDL200 on Clostridioides difficile infections was investigated through infection animal model experiments.

[0047] Specifically, 6-week-old SD rats [of a specific pathogen-free (SPF) grade] were used as test animals. A total of 12 rats were given drinking water containing antibiotics (kanamycin 0.4 mg/ml, gentamycin 0.035 mg/ml, colistin 850 U/ml, metronidazole 0.215 mg/ml, and vancomycin 0.045 mg/ml) for 5 days. Sterilized drinking water was then provided for 2 days. Clindamycin (20 mg/kg) was injected intraperitoneally on the last day of the provision of the sterilized drinking water (i.e., one day before Clostridioides difficile infection). The day after the clindamycin injection, a Clostridioides difficile forced infection was performed. The Clostridioides difficile forced infection was performed by preparing Clostridioides difficile in a sporulated state, floating the prepared strain with sterilized physiological saline, and injecting 0.2 ml of the prepared fluid (corresponding to about 107 CFU/ml) into the gavage using a Sonde. In forty-eight hours after the forced infection, the rats were divided into two groups. A first group composed of six rats received no test treatment drug (control group), while a second group composed of the other 6 rats received the test treatment drug three times a day. The test treatment drug was forcedly administrated into the gavage using a Sonde. The test treatment drug administration was continued for 7 days. The test treatment drug was prepared by putting a powder which is a mixture of a lyophilizate of an antibacterial protein CDL200 solution and an excipient (sucrose) in conventional enteric capsules. The amount of the test treatment drug per capsule was 5 mg of the antibacterial protein CDL200. The survival of the animals in each group was investigated until Day 7 of test treatment. The results are presented in Table 2.

TABLE-US-00002 TABLE 2 Survival rate(%) D1 D2 D3 D4 D5 D6 D7 Control 100 83.3 50 16.7 0 0 0 Group Test 100 100 100 100 100 100 100 group

[0048] From the above results, it is confirmed that the antibacterial protein CDL200 of the present invention is effective in treating a Clostridioides difficile infection. This characteristic indicates that a pharmaceutical composition containing the antibacterial protein CDL200 of the present invention as an active ingredient can be utilized for treatment of fungal infections.

[0049] While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that the specific description is only a preferred embodiment, and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the claims appended hereto and their equivalents.