Patent Publication Number: US-2023159599-A1

Title: Antibacterial protein cdl200 having lytic activity against clostridioides difficile

Description:
TECHNICAL FIELD 
     The present invention relates to an antibacterial protein CDL200 having bacteriolytic activity against Clostridioides  difficile . More particularly, the present invention relates to a Clostridioides  difficile -specific antibacterial protein CDL200 having the ability to specifically lyse Clostridioides  difficile  that may cause diseases by infecting animals including humans and having an amino acid sequence represented by SEQ ID NO: 1 and to a pharmaceutical composition containing the Clostridioides  difficile -specific antibacterial protein CDL200 as an active ingredient for preventing or treating a Clostridioides  difficile  infection and diseases caused by Clostridioides  difficile.    
     BACKGROUND ART 
     Clostridioides  difficile  is a Gram-positive, spore-forming, anaerobic bacterium resident in in the intestinal tract of humans and animals and is known to cause disease by opportunistic infections. As with Methicillin-resistant  Staphylococcus aureus  (MRSA) and Vancomycin-Resistant Enterococci (VRE), Clostridioides  difficile  is one of the major causative species of pathogenic pathogens. When the balance of the normal intestinal microflora is disrupted due to antibiotic treatment, antibiotic resistant Clostridioides  difficile  dominates the intestine and secretes enterotoxin (toxin A) and cytotoxin (toxin B), thereby causing intestinal inflammation and necrosis. In addition, Clostridioides  difficile  causes diarrhea and pseudomembranous colitis, and if not properly treated, the disease aggravates into sepsis or toxic megacolon and, in severe cases, into death. 
     In 2008, 700 cases of Clostridioides  difficile  infection were reported in Korea, but in 2011, the number of cases reported increased to 2,521, which was an increase of about 3.6 times, and the mortality rate increased by about 2.5 times. In addition, the economic cost of Clostridioides  difficile  infections was about 270 million won in 2008 and increased to about 1.85 billion won corresponding to an increase of 6.45 times in 2011. It is now estimated that the figure has increased further. In recent years, not only nosocomial infections, but also community infections, such as recurring diseases after an infected person in a hospital comes out to the community after treatment, or spreading pathogens to others from an infected person, causing infection, have become a problem. In Korea, it is estimated that about 10% of Clostridioides  difficile  infections are community infections. A recent report prepared by the US Centers for Disease Control and Prevention (CDC) identifies antibiotic-resistant  Acinetobacter , Clostridioides  difficile , and Enterobacteriaceae as the most pressing threats. 
     Antibiotics have been widely used for the treatment of these Clostridioides  difficile  infections but, recently, Clostridioides  difficile  bacteria have continued to acquire resistance to antibiotics, resulting in a serious decrease in the therapeutic effect of antibiotics. In addition, secondary problems such as breaking the balance of the intestinal microbial community are caused. 
     In order to effectively cope with infection with Clostridioides  difficile , which has acquired resistance to existing antibiotics, it is necessary to develop new antibiotic/antibacterial substances. In particular, it is highly urgently required to develop a pharmaceutical formulation that can provide a rapid therapeutic effect. 
     DISCLOSURE 
     Technical Problem 
     Accordingly, as a solution to the problem arising from the use of existing antibiotics against the harmful pathogenic bacterium Clostridioides  difficile , the inventors of the present invention provide an antibacterial protein capable of specifically lysing Clostridioides  difficile , provides a pharmaceutical composition that contains the antibacterial protein as an active ingredient and which can be used to treat Clostridioides  difficile  infections, and provides a method of effectively treating infections or diseases caused by Clostridioides  difficile  with the use of the pharmaceutical composition. 
     Accordingly, an objective of the present invention is to provide an antibacterial protein CDL200 including an amino acid sequence represented by SEQ ID NO: 1 and having activity to specifically lyse Clostridioides  difficile.    
     Another objective of the present invention is to provide a method of efficiently producing an antibacterial protein CDL200 that has specific bacteriolytic activity against Clostridioides  difficile  and includes an amino acid sequence represented by SEQ ID NO: 1. 
     A further objective of the present invention is to provide a pharmaceutical composition for treating Clostridioides  difficile  injections, the composition containing, as an active ingredient, an antibacterial protein CDL200 capable of specifically lysing Clostridioides  difficile.    
     A yet further objective of the present invention is to provide a method of treating an infection or disease caused by Clostridioides  difficile  using a pharmaceutical composition containing the antibacterial protein CDL200 as an active ingredient. 
     A yet further objective of the present invention is to provide a food composition containing the antibacterial protein CDL200 as an active ingredient and to provide a method of alleviating an infection or disease caused by Clostridioides  difficile  using the same. 
     Technical Solution 
     In order to accomplish the above objectives, the inventors of the present application have completed the present invention produced various antibacterial protein candidates in the form of recombinant proteins by using information on various antibacterial proteins known to have antibacterial activity against various bacterial strains and then investigated their bacteriolytic activity against Clostridioides  difficile  to select antibacterial proteins having excellent bacteriolytic activity. In addition, the inventors developed a method that can efficiently prepare the selected antimicrobial protein, and finally developed a pharmaceutical composition that contains the protein as an active ingredient and thus can be used to treat Clostridioides  difficile  infections. 
     Therefore, according to an aspect of the present invention, the present invention provides an amino acid sequence of an antibacterial protein CDL200 having the bacteriolytic ability specifically to Clostridioides  difficile . Specifically, the amino acid sequence may correspond to an amino acid sequence represented by SEQ ID NO: 1. The antibacterial protein CDL200, which can specifically lyse Clostridioides  difficile , consists of 160 amino acid residues and has a molecular weight of about 17.6 kDa. 
     It is apparent that the amino acid sequence represented by SEQ ID NO: 1 can be partially modified by a person skilled in the art using known techniques. Such modifications include partial substitutions of amino acid sequences, partial addition of amino acid sequences, and partial deletion of amino acid sequences. However, it is most preferable to apply the amino acid sequence represented by SEQ ID NO: 1 as disclosed in the present invention. 
     Further, the present invention provides Top10-CDL200 which is a strain usable to produce an antibacterial protein CDL200 having an amino acid sequence represented by SEQ ID NO: 1. The strain Top10-CDL200 is a strain made to transform  Escherichia coli  to produce CDL200 by using a plasmid having the gene sequence of SEQ ID NO: 2 produced by the inventors. 
     According to another aspect of the present invention, the present invention provides a pharmaceutical composition that contains, as an active ingredient, an antibacterial protein CDL200 having an amino acid sequence represented by SEQ ID NO: 1 and a bacteriolytic ability specific to Clostridioides  difficile , thereby being effectively used for prevention or treatment of Clostridioides  difficile  infections or diseases. 
     The antimicrobial protein CDL200 according to the present invention, which is characterized by the amino acid sequence represented by SEQ ID NO: 1, has a specific biolytic ability to Clostridioides  difficile , and is contained in the pharmaceutical composition of the present invention, specifically lyses Clostridioides  difficile  strains as described above, thereby exhibiting an effect in the treatment of various diseases caused by Clostridioides  difficile . The pharmaceutical composition of the invention can thus be utilized for the treatment of diseases caused by Clostridioides  difficile . In addition, the pharmaceutical composition of the present invention can be embodied as antibiotics, disinfectants, bactericides, therapeutics, and the like and can be used for the treatment of various diseases caused by Clostridioides  difficile.    
     According to a further aspect of the invention, the present invention provides a method of treating various diseases caused by Clostridioides  difficile , by administering an antibacterial protein CDL200 that is specific to Clostridioides  difficile  and is characterized by having an amino acid sequence represented by SEQ ID NO: 1 to animals other than humans. 
     The term “disease caused by Clostridioides  difficile ” herein refers to a disease caused by a Clostridioides  difficile  infection and also collectively refers to, but is not limited to, symptoms involving inflammation and necrosis of the intestines, diarrhea, gastrointestinal colitis, gastrointestinal perforation, sepsis, addictive macrocolosis, and the like. 
     The Clostridioides  difficile  in this specification is irrespective of whether it is sensitive to conventional antibiotics or not or whether it is resistant to conventional antibiotics. That is, it does not matter whether or not the Clostridioides  difficile  has resistance to conventional antibiotics. 
     In this description, the term “treatment” or “therapy” indicates all actions that inhibit infections or diseases caused by Clostridioides  difficile  and alleviate the pathological condition of the diseases caused by Clostridioides  difficile.    
     In order to improve the effectiveness of the pharmaceutical composition in terms of such utilization purposes, antibacterial materials that confer antibacterial activity against other bacterial species may be added to the pharmaceutical composition of the present invention. 
     The pharmaceutically acceptable carrier included in the pharmaceutical composition according to the present invention is one that is generally used for the preparation of a pharmaceutical formulation, and examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil but are not limited thereto. The pharmaceutical composition according to the present invention may additionally include lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, and preservatives, in addition to the above ingredients. 
     The pharmaceutical composition according to the present invention may be administered orally or via parenteral administration. In the case of parenteral administration, the pharmaceutical composition may be administered using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration. Aside from the administration methods, the pharmaceutical composition may also be used in a manner to be applied or sprayed onto a disease site. 
     The pharmaceutical composition according to the present invention can be formulated according to a method that can be easily performed by those who are ordinarily skilled in the art to which the present invention pertains, using a pharmaceutically acceptable carrier and/or excipient, in the form of a unit dose or in a multi-dose container. The formulation may be in the form of a solution, suspension, or emulsion in oil or a water-soluble medium, extract, powder, granule, tablet, or capsule. A dispersing agent or stabilizer may be additionally included. 
     The appropriate application, spray, and dosage of the pharmaceutical composition may depend on factors such as the formulation method, the mode of administration, the age, weight, gender, and diseased condition of the subject animal or person, diet, administration time, administration route, excretion rate, and responsiveness. The ordinarily skilled physician or veterinarian may readily determine and prescribe dosages effective for the desired treatment. 
     The pharmaceutical composition according to the invention may be embodied as antibiotics, disinfectants, bactericides, therapeutics, and the like. In addition, the pharmaceutical composition according to the present invention may also be embodied in the form of a food composition. 
     Advantageous Effects 
     The method of treating Clostridioides  difficile  infections using the pharmaceutical composition containing the antibacterial protein CDL200 having the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, according to the present invention, can effectively act not only against common Clostridioides  difficile  bacterial but also against Clostridioides  difficile  bacteria which have acquired resistance to existing antibiotics or antibiotic materials. On the other hand, the Clostridioides  difficile -specific antibacterial protein CDL200 of the present invention affects only Clostridioides  difficile  but does not affect normal flora resident in the body, thereby minimizing the side effects of use of the pharmaceutical composition containing the antibacterial protein CDL200 as an effective component. In the case of existing antibiotics, they negatively affect event the beneficial bacteria in the body, thereby causing various side effects. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         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. 
         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. 
         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 
     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 
     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. 
     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. 
     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 3 PO 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. 
     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. 
     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 3 PO 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. 
     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 3 PO 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 
     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 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 
               
               
                   
                   
               
            
           
         
       
     
     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 3 PO 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. 
     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   . 
     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 
     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 3 PO 4 , pH 7.0) containing no antibacterial protein CDL200 was used instead of the antibacterial protein CDL200 solution, as a negative control. 
     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   . 
     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 
     The therapeutic effect of the Clostridioides  difficile -specific antibacterial protein CDL200 on Clostridioides  difficile  infections was investigated through infection animal model experiments. 
     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 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 
               
               
                   
                   
               
            
           
         
       
     
     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. 
     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.