Recombinant endolysin for controlling clostridioides difficile infectious disease, and use thereof

Abstract

The present invention relates to a recombinant endolysin for controlling Clostridioides difficile infectious disease, and a use thereof, and, more specifically, to endolysin PHICD111_20024_EAD having excellent salt stability, and a use thereof. The endolysin PHICD111_20024_EAD, newly isolated in the present invention, exhibits specific activity against Clostridioides difficile, which is one of the causative bacteria of intestinal inflammation, and strains belonging to the genus Bacillus and Listeria monocytogenes, which are known as causative bacteria of food poisoning. The endolysin PHICD111_20024_EAD of the present invention does not affect humans and animals, and thus is safe and applicable to the pharmaceutical industry, food industry, biotechnology and the like, and also can effectively kill bacteria at a target site or target substance without problems related to antibacterial resistance.

Description

Recombinant endolysin for controlling Clostridioides difficile infection and its use

[0001] The present invention relates to a recombinant endolysin for controlling Clostridioides difficile infection and its use, and more specifically, to an endolysin PHICD111_20024_EAD having excellent salt stability and its use.

[0002] Clostridioides difficile is a Gram-positive, spore-forming, anaerobic microorganism known to be resistant to antibiotics and to cause Clostridioides difficile infection (CDI). It produces two types of toxins and induces inflammation in the intestines, leading to symptoms such as diarrhea. The number of patients with Clostridioides difficile has been steadily increasing, particularly in hospitals, and its spore-forming and antibiotic-resistant properties necessitate the development of effective methods to eradicate it.

[0003] Endolysin, a potential treatment for antibiotic-resistant bacteria, is a bacteriophage-derived antibiotic released by bacteriophages when they rupture bacteria and undergo a lytic life cycle. Endolysin, which kills bacteria by rupturing the peptidoglycan in their cell walls, is known to resist the development of resistance, unlike antibiotics.

[0004] The purpose of the present invention is to provide an endolysin protein PHICD111_20024_EAD having an amino acid sequence represented by sequence number 1 and having excellent salt stability.

[0005] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Clostridioides difficile infectious disease, which comprises the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0006] Another object of the present invention is to provide an antibiotic, disinfectant, food additive and feed additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0007] To achieve the above purpose, the present invention provides an endolysin protein PHICD111_20024_EAD having an amino acid sequence represented by sequence number 1 and having excellent salt stability.

[0008] In addition, the present invention provides a pharmaceutical composition for preventing or treating Clostridioides difficile infectious disease, comprising the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0009] In addition, the present invention provides an antibiotic for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which comprises the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0010] In addition, the present invention provides a disinfectant for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0011] In addition, the present invention provides a food additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0012] In addition, the present invention provides a feed additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0013] The present invention relates to a recombinant endolysin for controlling Clostridioides difficile infection and its use, and more particularly, to an endolysin PHICD111_20024_EAD having excellent salt stability and its use. The newly isolated endolysin PHICD111_20024_EAD of the present invention shows specific activity against Clostridioides difficile, one of the causative bacteria causing intestinal inflammation, and strains belonging to the genus Bacillus and Listeria monocytogenes, known as causative bacteria of food poisoning. The endolysin PHICD111_20024_EAD of the present invention is safe because it does not affect humans and animals, and can be applied to the pharmaceutical industry, food industry, biotechnology, etc., and has the advantage of effectively killing bacteria at a target site or target substance without the problem of antimicrobial resistance.

[0014] Figure 1 shows the domain structure of PHICD111_20024 and the similar domain structure of the endolysin CD27L. It appears to be composed of domains exhibiting the characteristics of N-acetylmuramoyl-L-alanine amidase.

[0015] Figure 2 shows a comparison of the antibacterial effect of the endolysin PHICD111_20024_EAD of the present invention with that of the control group, CD27L_EAD, under a buffer composition.

[0016] Figure 3 shows a comparison of the antibacterial effect of the endolysin PHICD111_20024_EAD of the present invention according to changes in salt, pH, and temperature.

[0017] Figure 4 shows a graph showing the change in antibacterial effect of the endolysin PHICD111_20024_EAD of the present invention in proportion to salt concentration.

[0018] Figure 5 shows the aspect of suppressing the antibacterial effect of the endolysin PHICD111_20024_EAD of the present invention during long-term culture in a medium composition.

[0019] Figure 6 shows a graph comparing the minimum inhibitory concentration values ​​of the endolysin PHICD111_20024_EAD of the present invention with the control group CD27L_EAD.

[0020] The present invention provides an endolysin protein PHICD111_20024_EAD having an amino acid sequence represented by sequence number 1 and having excellent salt stability.

[0021] Specifically, the gene encoding the endolysin protein may be composed of a base sequence represented by sequence number 2, but is not limited thereto.

[0022]

[0023] In addition, the present invention provides a pharmaceutical composition for preventing or treating Clostridioides difficile infectious disease, comprising the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0024] Specifically, the Clostridioides difficile infectious disease may be, but is not limited to, endocarditis, diarrhea, enteritis, inflammatory bowel disease (IBD), pseudomembranous colitis, toxic megacolon, gastrointestinal perforation, or sepsis.

[0025] When the composition of the present invention is a pharmaceutical composition, for administration, it may contain, in addition to the above-described active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. The carrier, excipient, and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0026] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions, according to conventional methods. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules. Such solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the above-mentioned active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. In addition to liquid paraffin for oral administration, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be added to prepare the formulation. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrosol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0027] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of the disease, the drug form, and the time, but can be appropriately selected by a person skilled in the art. The daily dosage of the composition is preferably 0.001 mg / kg to 50 mg / kg, and can be administered once or several times a day as needed.

[0028]

[0029] In addition, the present invention provides an antibiotic for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which comprises the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0030]

[0031] The term “antibiotic” as used herein refers to a general term for cosmetic or pharmaceutical preservatives, bactericides and antimicrobial agents.

[0032] In addition, the present invention provides a disinfectant for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0033] The disinfectant containing the PHICD111_20024_EAD endolysin protein of the present invention as an active ingredient can be usefully used as a disinfectant for hospitals and health care to prevent hospital infections, and can also be usefully used as a general household disinfectant, a disinfectant for food and cooking places and facilities, and a disinfectant for livestock sheds in the livestock industry.

[0034]

[0035] In addition, the present invention provides a food additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0036] The food additives of the present invention include preservatives, sterilizers, antioxidants, spices, seasonings, sweeteners, flavoring agents, leavening agents, reinforcing agents, improvers, emulsifiers, various nutrients, flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, coloring agents, thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, antifoaming agents, solvents, release agents, preservatives, quality improvers, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., which can be added to foods by immersion, spraying, or mixing.

[0037]

[0038] In addition, the present invention provides a feed additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, which contains the PHICD111_20024_EAD endolysin protein as an active ingredient.

[0039] The PHICD111_20024_EAD endolysin protein of the present invention can be used as an additive to livestock feed and livestock drinking water for the purpose of preventing or treating bacterial infection, and can improve or maintain animal feed intake, growth, feed efficiency, survival rate, feeding condition, productivity, etc.

[0040] Below, the present invention is described in detail using examples that do not limit the scope of the invention. The following examples are intended to concretize the invention and do not limit or restrict the scope of the invention. Therefore, anything that a specialist in the technical field can easily infer from the detailed description and examples of the invention is interpreted as falling within the scope of the invention.

[0041]

[0042] <Example 1> Confirmation of the host spectrum showing sensitivity to endolysin PHICD111_20024_EAD

[0043] Bacteria were grown in 20 mM Tris-HCl buffer at an OD 600 = 2.0, and each endolysin is also diluted in 20 mM Tris-HCl buffer to a concentration of 0.32 μM. Afterwards, cells and endolysin are mixed in a 1:1 ratio, and the final concentration is 0.16 μM, which is the cell population OD 600 =1.0. Measure from 0 to 60 minutes under aerobic conditions using a plate reader from the time of mixing. As a control, use a group containing a 1:1 mixture of Tris-HCl buffer and cells without endolysin. Measure OD at 37°C for 1 hour in a plate reader. 600 , and measure the OD reduction value (each OD 600 / Control's OD 600 )*100 (%), if the value is greater than 90, it is indicated as (-), if it is between 70 and 90, it is indicated as (+), if it is between 50 and 70, it is indicated as (++), and if it is less than 50, it is indicated as (+++).

[0044] Table 1 was designed to identify the host spectrum showing sensitivity to endolysin PHICD111_20024_EAD, and its activity level was shown based on Table 2.

[0045] The results of the experiment showed antibacterial activity against Bacillus and Listeria strains, and antibacterial activity was also confirmed against some Clostridium strains. This showed that similar susceptibility was shown to bacteria with similar peptidoglycan structures.

[0046] Bacterial strainsGram'smethodEADactivityClostridioidesdifficileATCC 9689++++ClostridioidesdifficileATCC 43599++++ClostridioidesdifficileATCC 43600++++ClostridioidesdifficileNCCP 11080++++ClostridioidesdifficileNCCP 11820++++ClostridioidesdifficileNCCP 11840++++Staphylococcusaureussubsp. Aureus ATCC 29213+-StaphylococcusepidermidisATCC 35983+-StreptococcuspyogenesATCC 19615+-EnterococcusfaecalisATCC 29212+-BifidobacteriumadolescentisATCC 15703+-BacilluscereusATCC 14579+-BacilluscereusNCCP 10623+-BacilluscereusNCCP10624+++BacillussubtilisATCC23857++++BacillussubtilisSRCM103551++++ClostridiumperfringensATCC13124++ClostridiumperfringensNCCP15911++ClostridiumsporogenesKCTC5654++++ClostridiumtertiumKCTC5897++PseudomonasaeruginosaATCC 27853--KlebsiellapneumoniaeMGH 78578ATCC 700721--CronobactersakazakiiATCC 29544--SalmonellaTyphimurium 14028s--AcinetobacterbaumanniiATCC19606--EscherichiacoliK-12 MG1655--VibriocholeraeATCC 14033--YersiniaenterocoliticaATCC 55075--Fusobacteriumnucleatumsubsp.NucleatumATCC 25586-+LactobacillusplantarumATCC 8014+-LactobacillusparacaseiKCTC 3510+-LactobacillusgasseriATCC 33323+-ListeriainnocuaKCTC 3586+++ListeriamonocytogenesKCTC 3569+++Listeriaivanoviisubsp.ivanoviiKCTC 3444+++antibiotics resistant strainsMethicillin ResistantStaphylococcusAureusKCTC 1928+-E.faecalisCCARM 5537+-E.faeciumCCARM 5202+-S.aureusCCARM 3689+-Klebsiellapneumoniae(IMI resistance)1366019--Klebsiellapneumoniae(IMI resistance) 140522-4113--Acinetobacter baumannii (IMI resistance) F-750--Salmonellatetracyclin resistance IVK B01009--ESBLEscherichiacoliO2 HID5005--EscherichiacoliK03-Bact-08-037--.

[0047]

[0048] 90< X-70< X<90+50< X<70++X< 50+++

[0049]

[0050] <실시예2>엔돌라이신PHICD111_20024_EAD및CD27L_EAD의항균효과 비교

[0051] 박테리아는 20 mM Tris-HCl buffer 상에 OD 600= 2.0, and each endolysin is also diluted in 20 mM Tris-HCl buffer to a concentration of 0.32 μM. Afterwards, cells and endolysin are mixed in a 1:1 ratio, and the final concentration is 0.16 μM, which is the cell population OD 600 =1.0 is the condition to meet. OD from 0 to 60 minutes under aerobic conditions from the mixing point 600 was measured. As a control, a group containing Tris-HCl buffer without endolysin and cells mixed 1:1 was used and designated as buffer only.

[0052] As a result of confirming the activity between CD27L_EAD and PHICD111_20024_EAD through the turbidity reduction test, CD27L_EAD showed a high effect in Tris-HCl buffer (pH=7.5) (Fig. 2).

[0053]

[0054] <Example 3> Comparison of antibacterial effects of endolysin PHICD111_20024_EAD according to changes in salt, pH, and temperature.

[0055] Salt and pH were adjusted to the appropriate salt / pH concentrations by using NaCl, HCl, and NaOH in 20 mM Tris-HCl buffer, and then 20 mM Tris-HCl buffer was prepared. Then, bacteria were grown on the 20 mM Tris-HCl buffer to an OD 600=2.0, Endolysin was also diluted to a concentration of 0.32 μM and mixed 1:1. Each Endolysin was incubated at each temperature for 30 minutes through a heat block and then mixed with bacteria. The OD reduction value at 15 minutes, 30 minutes and 60 minutes under aerobic conditions was measured in a plate reader, and {(OD of Control 600 -Each OD 600 ) / Control's OD 600}*100 (%) is expressed as.

[0056] The differences in the activities of CD27L_EAD and PHICD111_20024_EAD due to changes in salt, pH, and temperature were confirmed through a turbidity reduction test in Tris-HCl buffer. As a result, it was confirmed that the activity of PHICD111_20024_EAD was high at high salt concentrations (Fig. 3).

[0057]

[0058] <Example 4> Changes in the antibacterial effect of PHICD111_20024_EAD according to salt addition

[0059] The results of the Turbidity Reduction Test by Salt were confirmed by a concentration-dependent graph showing that activity was preserved even at high salt concentrations (Fig. 4).

[0060] The graph in Fig. 4 is a continuous graph that shows the change in slope according to the change in time from 0 to 60 minutes in the bar graph of Fig. 3 in which salt progressed.

[0061]

[0062] <Example 5> Long-term growth inhibition effect of PHICD111_20024_EAD according to medium composition

[0063] C. difficile ATCC 9689 strain pre-cultured in BHI broth was incubated in fresh BHI broth at OD 600 =0.01 and OD was measured at 1-hour intervals under anaerobic culture conditions. 600 was measured. At this time, the main culture was performed by adding CD27L_EAD and PHICD111_20024_EAD to BHI media at concentrations that were each reduced by half from 80 μM to 20 μM.

[0064] The results of the Turbidity Reduction Test in Buffer with High Salt Content were confirmed to have an inhibitory effect on growth while growing the bacteria on the medium. The inhibitory effect of PHICD111_20024_EAD, which maintained activity even at high salt concentrations during culture after addition from the beginning of bacterial growth, was proportional to the concentration (Fig. 5).

[0065]

[0066] <Example 6> Comparison of the minimum inhibitory concentration values ​​of PHICD111_20024 with the control group, CD27L_EAD

[0067] C. difficile ATCC 9689 strain pre-cultured in BHI broth was incubated in fresh BHI broth on a 96-well plate at OD 600 =0.1 was inoculated. At this time, CD27L_EAD and PHICD111_20024_EAD were added to BHI media at a concentration of 1280 μM to 10 μM, respectively, and cultured for 24 hours. OD 600 was measured. In the control group CD27L_EAD, a growth inhibition effect was observed when treated with 640 μM or more, but in PHICD111_20024_EAD, a growth inhibition effect was observed at a minimum of 160 μM or more, proving an excellent effect even in a high salt concentration environment (Fig. 6).

[0068]

[0069] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

PHICD111_20024_EAD endolysin protein having an amino acid sequence represented by sequence number 1 and excellent salt stability. In the first paragraph, the gene encoding the endolysin protein is characterized in that it consists of a base sequence represented by sequence number 2. PHICD111_20024_EAD endolysin protein. A pharmaceutical composition for preventing or treating an infectious disease caused by Clostridioides difficile, comprising the PHICD111_20024_EAD endolysin protein of claim 1 or 2 as an active ingredient. A pharmaceutical composition according to claim 3, wherein the Clostridioides difficile infectious disease is endocarditis, diarrhea, enteritis, inflammatory bowel disease (IBD), pseudomembranous colitis, toxic megacolon, gastrointestinal perforation, or sepsis. An antibiotic for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, comprising the PHICD111_20024_EAD endolysin protein of claim 1 or 2 as an active ingredient. A disinfectant for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, comprising the PHICD111_20024_EAD endolysin protein of claim 1 or 2 as an active ingredient. A food additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, comprising the PHICD111_20024_EAD endolysin protein of claim 1 or 2 as an active ingredient. A feed additive for killing one or more strains selected from the group consisting of Clostridioides difficile, Bacillus subtilis, Clostridium sporogenes, Listeria innocua, Listeria monocytogenes, and Listeria ivanovii, comprising the PHICD111_20024_EAD endolysin protein of claim 1 or 2 as an active ingredient.

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