KR102543966B1 - Peptide inhibiting adhesion of Enterotoxigenic Escherichia Coli Toxin - Google Patents

Abstract

A peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT) is disclosed. The peptide is a GM1a-like peptide that selectively binds to the B subunit of heat-heat toxin (LT) to inhibit the attachment of heat-heat toxin (LT) to GM1a, thereby preventing subsequent physiological consequences of heat-heat toxin (LT) Thus, it is possible to prevent or treat enterotoxin E. coli heat-febrile toxin (LT) infection and can be used as a new therapeutic agent.

Description

Peptide inhibiting adhesion of Enterotoxigenic Escherichia Coli Toxin}

The present invention provides a peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT), a nucleic acid molecule encoding the same, a recombinant expression vector containing the nucleic acid molecule, a transformant transformed with the recombinant expression vector, It relates to a pharmaceutical composition for preventing or treating enterotoxigenic Escherichia coli infection comprising the peptide.

Enterotoxigenic E. coli (ETEC) is responsible for the production of heat labile enterotoxin (LT). Enterotoxigenic E. coli is associated with more than 50,000 deaths and millions of cases of diarrhea each year. .Despite advances in sanitation, the mortality rate of the disease has not fallen, but neither has the morbidity.Infection with enterotoxigenic E. coli often causes long-term health problems, such as developmental disabilities and cognitive decline, which can set off a vicious cycle of poverty. In addition, travelers visiting endemic areas, particularly medical and military professionals, are susceptible to the disease, which is also associated with chronic conditions such as irritable bowel syndrome, watery diarrhea caused by enterotoxins, exacerbated by infection. It helps spread the disease through the fecal-oral route.

Enterotoxic Escherichia coli simultaneously produces one or both toxins, heat labile enterotoxin (LT) and heat stable enterotoxin (ST). Heterophilic toxin is similar to cholera toxin in its antigenic structure or mechanism of action. It consists of one A subunit (molecular weight: 25,000) and five B subunits (molecular weight: 11,500) in protein structure, and is synthesized with 18 and 21 signal peptides, respectively. do. The catalytically active A-subunit is bound to the torus-shaped core of the B-pentamer responsible for binding to epithelial cells. These two components together constitute a protein complex. Heterophilic toxin (LT) is generally more irregular than cholera toxin (CT) and binds to a variety of glycosphingolipids. These include glycosphingolipids such as disialogangliosides GD2 and GM1 and lacto-N-neotetraosylceramide (LNnT-Cer; paragloboside) and enteric polyglycosylceramides and glycosphingos containing sialic acid. included The LT-B subunit can enter host cells by binding to GM1a, a monosialoganglioside found on the surface of the plasma membrane of animal epithelial cells.

On the other hand, for the treatment of most infectious diseases and diseases caused by bacteria, including pathogenic Escherichia coli, there is no choice but to rely on chemotherapeutic agents, and most of them are treated with antibiotics.

However, due to the indiscriminate abuse and misuse of these antibiotics, many types of bacteria have now acquired resistance to antibiotics, and the frequency of appearance of resistant bacteria is gradually increasing.

Unlike other bacterial diseases, diseases caused by Escherichia coli have the characteristic of continuously occurring, and in order to prevent this, thorough disinfection and maintaining a hygienic environment to prevent exposure to pathogens is the top priority, but this is practically impossible.

Therefore, a substance that selectively binds to the B subunit of thermophilic toxin (LT) to inhibit the attachment of thermophilic toxin (LT) to GM1a on the cell membrane surface prevents the subsequent physiological consequences of thermophilic toxin (LT). , it is possible to prevent or treat enterotoxin E. coli heat-febrile toxin (LT) infection, so it can be used as a new therapeutic agent.

An object of the present invention is to provide a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT).

Another object of the present invention is to provide a nucleic acid molecule encoding the amino acid sequence of the aforementioned peptide.

Another object of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule.

Another object of the present invention is to provide a transformant transformed with an expression vector.

Another object of the present invention is a pharmaceutical for preventing or treating enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT) as an active ingredient to provide a composition.

Another object of the present invention is to provide a food composition for preventing or improving enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT) is to provide

In order to achieve the above object, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT).

In addition, the present invention provides a nucleic acid molecule encoding the amino acid sequence of the aforementioned peptide.

In addition, the present invention provides a recombinant expression vector containing the nucleic acid molecule.

In addition, the present invention provides a transformant transformed with the expression vector.

In addition, the present invention provides a pharmaceutical composition for preventing or treating enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT).

In addition, the present invention provides a food composition for preventing or improving enterotoxinic E. coli infection comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxin heat-labile toxin (LT).

The peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT) according to the present invention is a GM1a-like peptide, and selectively heat-heat toxin (LT) to suppress the attachment of heat-heat toxin (LT) to GM1a By binding to the B subunit of LT), it prevents the subsequent physiological consequences of heat-labile toxin (LT), enabling prevention or treatment of enterotoxin E. coli heat-heat toxin (LT) infection, and thus can be used as a new therapeutic agent.

Figure 1 is a schematic showing that immunotherapy against LT-B can be used to prevent and treat infectious disorders by determining the involvement of host GM1a glycan peptides in the immune response to pathogens.
Figure 2 shows the results of revealing the LT-B lectin factor related to GM1a ganglioside through plaque lift analysis in one embodiment of the present invention.
Figure 3 is a docking simulation with LT-B, a lectin candidate that binds to GM1a glycan in one embodiment of the present invention, to determine the binding affinity, and three ligand binding domains (LBD) found at the expected binding site ) is a schematic diagram showing.
4A is a graph showing the binding affinity of the GM1a-replicating peptide P2 to LT-B in an embodiment of the present invention, and B is a graph showing the inhibition rate according to the concentration of the GM1a-replicating peptide P2 (*P < 0.05 and **P < 0.01)).
Figure 5 shows the production levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α using ELISA to detect the effect of P2 after HCT-8 challenge infection of intestinal epithelial cells in one embodiment of the present invention. This is a graph showing the comparison results.

The present inventors developed a GM1a-like peptide for the purpose of inhibiting the attachment of enterotoxin E. coli heat-labile toxin (LT) to GM1a, a monosialoganglioside found on the plasma membrane surface of animal epithelial cells. The LT-B subunit was used to select LT-B binding peptides structurally similar to GM1a from the phage display library. Biopanning was used to identify three GM1a-like peptides based on LT-B recognition. We demonstrated that a GM1a-like peptide can mimic GM1a by blocking the binding of LT-B to GM1a. Using the phage-visible peptide library technology displayed on the envelope protein, we were able to obtain three peptides that successfully inhibited the binding of LT-B to GM1a. These GM1a-like peptides had a functional activity similar to that of GM1a, and had the effect of inhibiting the binding of GM1a to LT-B. In addition, the binding ability, functional inhibitory activity, and in vitro effect of GM1a-like peptides were evaluated using HCT-8 cells, a human intestinal epithelial cell line. It was confirmed that it can act as a new therapeutic agent to prevent subsequent physiological consequences caused by E. coli heat-labile toxin (LT).

As one embodiment for achieving the above object, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT).

The peptide may be prepared by a chemical peptide synthesis method known in the art, or by amplifying a gene encoding the peptide by PCR (polymerase chain reaction) or synthesizing it by a known method, and then cloning it into an expression vector and expressing it. there is.

In one embodiment of the present invention, in the adhesion test using LT-B, the GM1a-like peptide P2 corresponding to SEQ ID NO: 1 showed a significantly higher level of binding to LT-B than the control group.

Biological functional equivalents that can be included in the range of peptides that specifically bind to the B subunit of enterotoxin E. coli heat-labile toxin (LT) of the present invention are mutations in amino acid sequences that exhibit biological activity equivalent to those of the peptides of the present invention. It is clear to those skilled in the art that the inclusion will be limited to.

As another aspect, the present invention provides a nucleic acid molecule encoding the peptide. The nucleic acid molecule may be mutated by substitution, deletion, insertion, or a combination of one or more bases thereof. When preparing a nucleotide sequence by chemical synthesis, a synthesis method well known in the art, for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other auto-primer methods, and oligonucleotide synthesis methods on solid supports.

As another aspect, the present invention provides an expression vector containing the nucleic acid molecule and a transformant containing the expression vector.

The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a desired peptide in a desired host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert. The expression vector may be a mammalian cell (eg, human, monkey, rabbit, rat, hamster, mouse cell, etc.), a plant cell, a yeast cell, an insect cell, or a eukaryotic or bacterial cell (eg, E. coli, etc.) It can be a vector capable of replicating and/or expressing the nucleic acid molecule in a prokaryotic cell, preferably operably linked to an appropriate promoter so that the nucleic acid molecule can be expressed in a host cell, and comprising at least one selectable marker. It can be a vector that contains

The host cell into which the expression vector provided in the present invention can be introduced is not particularly limited as long as it can produce the peptide by expressing the nucleic acid molecule, and is preferably a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella typhimurium. ; yeast cells; fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or a plant cell, more preferably a mammalian animal cell.

In another aspect, the present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT) as an active ingredient. Or it provides a pharmaceutical composition for treatment.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to a peptide that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT). Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.

The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .

When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the peptide included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 μg/ml, toxicity to the human body may be exhibited.

The peptide according to the present invention may exhibit preventive and therapeutic effects of enterotoxin E. coli infection. Infectious diseases caused by the enterotoxin E. coli may be acute gastroenteritis due to enterotoxin E. coli infection, but is not limited thereto.

In another aspect, the present invention provides a food composition for preventing or ameliorating enterotoxic E. coli infection comprising a peptide that specifically binds to the B subunit of the enterotoxin heat-labile toxin (LT) as an active ingredient. can

The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions, in addition to containing peptides as active ingredients.

Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the above-mentioned flavors, natural flavors (thaumatin), stevia extracts, and synthetic flavors (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is

In addition, the food composition, in addition to extracts as active ingredients, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.

The food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving enterotoxinic E. coli infection. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions. The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example One

T7 page display

All operations including biopanning and phage propagation were performed according to the instructions provided by the manufacturer. Differential biopanning was performed with both negative and positive selection using polyclonal antibodies according to the instructions provided by the manufacturer (TB178 T7Select®System; Novagen). After rinsing with PBS (phosphate-buffered saline) and blocking in 1% bovine serum albumin for 1 hour, 25 liters of protein G+ agarose beads were treated (BSA). The beads were washed three times with PBS and then re-incubated with the T7 phage display cDNA library for an additional 2 hours. This process was repeated twice. It is necessary to use this subtractive biopanning strategy to remove proteins that react with IgG prior to immunization. Then, the supernatant of the T7 phage cDNA library was treated with polyclonal antibody immobilized on protein G+ agarose beads, and the mixture was left to incubate at 4 degrees Celsius for 24 hours. The bound T7 phage cDNA library was washed three times with 1 PBS and then eluted with 1% sodium dodecyl sulfate. During subsequent cycles of biopanning, the eluate was amplified using E. coli strain EDL933. After four rounds of biopanning, the selected phage library was subjected to immunological screening.

plaque lift assay

Approximately 1000 well-dispersed individual phage clones from agar plates were transferred to nitrocellulose membranes using the plaque lift technique. A nitrocellulose membrane (82 mm diameter HATF filter, Millipore Corp.) was placed over the agar and incubated for 10-15 minutes at room temperature. The nitrocellulose membrane was carefully lifted without disturbing the agar and plaque. The immunostaining procedure included an initial incubation with combined glycan (GM1a) LT-B for 1–2 h. The membrane was then rinsed and detection reagent applied. Detection reagents included horseradish peroxidase conjugated goat anti-mouse IgG followed by color development with 3,3-diaminobenzidine (DAB) and 0.01% H ₂ O ₂ .

docking simulation

This method allowed them to decipher the pathogen's lectin structure and identify the site where it attaches to the host's receptor. A three-dimensional (3D) structure of LT (PDB ID: 1LTS) was generated using the homology model approach, SWISS-MODEL, using the GM1a (PDB ID: 6LF2) 3D structure. Docking simulation was performed using the 3D structure of GM1a. The Chem-office application (http://www.cambridgesoft.com, version: 7.0) was also used to reduce energy use. Autodock Vina 1.1.2 was used for docking simulation. Based on the docking simulation results, possible hydrogen bonding and hydrophobic interactions were identified using HBPLUS and nonbonding contact parameters with default settings in LigPlot.

Solid phase peptide synthesis

The initial step in peptide synthesis on resin was to link the C-terminal amino acid to the resin. Temporary protecting groups protect the alpha amino group and reactive side chains from polymerization. Next, the resin was filtered and washed to remove by-products and excess reagents. The N-alpha protecting group was then removed and the resin washed again to remove byproducts and excess reagent. Then, the next amino acid was added until the peptide sequence was complete. The peptide was then released from the resin after rinsing to remove the protecting group. As in the above process, peptide synthesis was performed with the same protocol for P2. A GM1a-like peptide, P2 (SYTESMAGKRE: SEQ ID NO: 1), was similarly synthesized.

peptide Binding and inhibition assays

P2 peptide was coated on each well of the plate at a concentration of 25 ng per well. After blocking, each well was filled with LT-B for 2 hours at room temperature and washed 5 times with 1% BSA. Next, the plates were incubated at 4° C. for 15-16 hours. After another wash, the plates were stained with fluorescein isothiocyanate-conjugated staining reagent at a 1:10,000 dilution. After 2 hours at room temperature, the plate was washed and fluorescence was detected using a micro reader.

using T7 phage display GM1a with ganglioside Related LT Results of lectin receptor identification of -B

mRNA was isolated from ETEC so that it could be used for T7 phage display. After mRNA was extracted, cDNA was generated and a phage library was assembled. cDNA synthesis using oligo(dT), random or matched subunit specific primers produced equal amounts of transcript. Immune screening methods are performed in a manner consistent with industry requirements. Plaque lift testing revealed lectin factors associated with GM1a glycans. Thick patches were selected for selection and sequencing, and the results investigated. As a direct result of this, LT-B was mostly confirmed, and the following tests were conducted using LT-B (see FIG. 2).

LT The ligand binding domain of -B ( LBD ) of determination

The ligand binding domain (LBD) of LT-B that binds to GM1a ganglioside binding affinity was determined using docking simulations with LT-B, a lectin candidate that binds to GM1a ganglioside. In order to find the ligand-binding domain (LBD) of LT-B, hydrogen bonding, ionic bonding, and affinity for the ligand-interacting LT-B residue were investigated. This was done for all ligands. As a direct result of this, three LBDs were found at the expected binding sites (see Fig. 3).

to inhibit adhesion LT -B LBD Comparison of peptide ability and adhesion affinity

After the investigation was completed, they found three ligand-binding domains (LBDs). The peptide sequence SYTESMAGKRE (SEQ ID NO: 1) was a potential candidate for the binding site, and this sequence was named P2. Adhesion was tested using LT-B. The results are shown in FIG. 4 .

Referring to A of FIG. 4 , P2 had a significantly higher level of ability to bind to LT-B when compared to the control group. Control refers to the binding affinity of the matrix without peptide. Compared to the control group, the P2 peptide showed a statistically significant change.

Referring to FIG. 4B , P2 was treated at concentrations of 0, 10, 100, and 1000 nM, and in vitro treatment with peptide P2 inhibited LT-B activity in a concentration-dependent manner.

peptide In HCT-8 cells by P2 LT Inhibition of -B cytokine production

After challenge infection with intestinal epithelial cells HCT-8, cytokine levels were measured using ELISA to detect the effect of P2. As a result of measuring the levels of TNF-a, IL-6, and IL-1b, which are major inflammatory factors after infection, it was found that all three inflammatory markers were reduced (see FIG. 5). These findings provide conclusive evidence that P2 can inhibit LT-capable B interacting with GM1a on the cell surface and neutralize it when present in the culture medium.

Claims (6)

A peptide consisting of the amino acid sequence of SEQ ID NO: 1 that specifically binds to the B subunit of enterotoxigenic E. coli heat-labile toxin (LT). A nucleic acid molecule encoding the amino acid sequence of the peptide of claim 1. A recombinant expression vector comprising the nucleic acid molecule of claim 2. A transformant, other than a human, transformed with the recombinant expression vector of claim 3. A pharmaceutical composition for preventing or treating enterotoxigenic Escherichia coli infection comprising the peptide according to claim 1 as an active ingredient. A food composition for preventing or improving enterotoxigenic Escherichia coli infection comprising the peptide according to claim 1 as an active ingredient.

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