KR20220151425A - Screening method for anti-virulentin against pathogenic Vibrio bacteria and anti-virulentin screened thereby - Google Patents

Abstract

The present invention provides: a method for screening pathogenic inhibitors for pathogenic Vibrio bacteria that can effectively prevent or treat diseases caused by deadly pathogenic bacteria by disrupting or suppressing the expression of pathogenic factors without affecting the growth of major pathogenic Vibrio species; and a pathogenic inhibitor screened by the same.

Description

Screening method for anti-virulentin against pathogenic Vibrio bacteria and anti-virulentin screened thereby}

The present invention relates to a screening method for pathogenicity inhibitors against pathogenic Vibrio bacteria, and more particularly, to a screening method for pathogenicity inhibitors against pathogenic Vibrio bacteria and pathogenicity inhibitors screened thereby.

Pathogenic Vibrio species cause fatal infectious diseases in humans and animals and cause great damage to mankind today (e.g., Vibrio cholerae - cholera, V. vulnificus - sepsis, V. haemolyticus - diarrheal disease, V. anguillarum ). - Fish bottles, etc.). Although some antibiotics are currently being used to treat these diseases, their effectiveness is not high, and the need to develop appropriate new antibiotics has emerged due to the problem of antibiotic resistance, which has recently become a problem. In particular, conventional antibiotics have an action mechanism that inhibits or kills the growth of bacteria. These characteristics act as a strong selection pressure that causes natural selection of individuals resistant to antibiotics through mutations occurring in pathogenic bacteria or acquisition of antibiotic resistance genes. Therefore, the bacterial species in the ecosystem gradually result in the replacement of dominant species by individuals resistant to human-developed antibiotics. As a strategy to overcome the problems of these existing antibiotics, the normal expression of pathogenic factors without affecting the growth of bacteria is achieved by disrupting or inhibiting the signal transduction system that regulates the expression of pathogenic factors specific to pathogenic Vibrio species. Research on how to eliminate the onset factor by suppressing it is required. In this regard, Korean Patent Registration No. 2171321 proposes a therapeutic agent using an Rtx toxin production inhibitor, a toxin specifically found in Vibrio sepsis, and Patent Registration No. 11243696 uses an inhibitor of SmcR, a signaling protein involved in communication. An anti-pathogenic agent is disclosed.

However, in the case of the prior art, the target protein is different from the present invention and relates to a proliferation inhibitory use applicable only to Vibrio septic bacteria, and the technology for pathogenic inhibitors capable of inhibiting the expression of pathogenic factors of various Vibrio bacteria is an unexplored field. to be.

The present invention is intended to solve various problems, including the above problems, by targeting the ToxR signal receptor membrane protein, which only all Vibrio bacteria have specifically, as a pathogenic factor without affecting the growth of major pathogenic Vibrio species. It is an object of the present invention to provide a pharmaceutical composition for inhibiting the expression of pathogenic factors of Vibrio bacteria, which can effectively prevent or treat diseases caused by deadly pathogenic bacteria by disturbing or inhibiting the expression of. It is also an object of the present invention to provide a screening method for pathogenicity inhibitors against pathogenic Vibrio bacteria and pathogenicity inhibitors screened thereby. However, these tasks are illustrative, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating Vibrio infections containing a compound having the structure of Formula 1 as an active ingredient:

(화학식 1).

(Formula 1).

According to another aspect of the present invention, there is provided a feed additive for preventing Vibrio infection, comprising as an active ingredient a compound having the structure of Formula 1 below:

(화학식 1).

(Formula 1).

According to another aspect of the present invention, there is provided a method for treating an infection caused by Vibrio bacteria, comprising administering to a subject a compound having the structure of Formula 1 below:

(화학식 1).

(Formula 1).

According to another aspect of the present invention, preparing a vector comprising a gene construct in which the luxAB gene encoding a bioluminescent protein is operably linked to the leuO gene; Preparing a reporter strain by transducing the vector into a Vibrio sepsis strain in which the leuO gene is deleted; processing a test substance to the reporter strain; Measuring the degree of bioluminescence from the strain treated with the test material using a luminometer; and selecting a material whose bioluminescence is significantly reduced compared to a control in which the test material is not treated.

The pharmaceutical composition for suppressing the expression of pathogenic factors of Vibrio bacteria of the present invention made as described above overcomes the mechanism of action of antibiotics that inhibit the growth of existing bacteria, and does not significantly affect the growth of bacteria, and the expression of pathogenic factors. By inhibiting it, it is possible to develop a composition having an antipathogenic effect. In addition, the screening method for pathogenicity inhibitors against pathogenic Vibrio of the present invention uses the composition to develop a therapeutic agent capable of suppressing the onset of sepsis caused by Vibrio and Vibrio cholera, and the pathogenicity of pathogenic Vibrio species having a cFP regulatory network. can be utilized for Therefore, it is possible to solve the problem of antibiotic-resistant bacteria due to misuse of antibiotics, the problem of antibiotics remaining in food, and the problem of a wide range of hosts. Of course, the scope of the present invention is not limited by these effects.

Figure 1 is a schematic diagram schematically showing the process of suppressing the signal transduction process of the Vibrio pathogenic factor expression cFP regulatory network.
Figure 2 is a schematic diagram showing the structure of a report system constructed to screen substances that inhibit the expression of LeuO, a key regulator of the lower end of ToxR, a signal receptor membrane protein in the cFP signaling network in septic Vibrio bacteria.
Figure 3 is a graph showing the results of analyzing the amount of leuO transcription of V. vulnificus according to the treatment with the compound SG0010 of the present invention.
4 is a graph showing the results of analysis of resistance to H ₂ O ₂ of V. vulnificus treated with the compound SG0010 of the present invention.
Figure 5 is a graph analyzing the results of the amount of vvhA transcription of V. vulnificus according to the treatment with the compound SG0010 of the present invention.
Figure 6 is a graph analyzing the transcription amount of ctx , an exotoxin-encoding gene of V. cholerae according to the treatment of the compound SG0010 of the present invention.
Figure 7 is a graph showing the results of analyzing the survival rate of mice infected with V. vulnificus according to the treatment of the compound SG0010 of the present invention.
8 is a graph analyzing the survival rate of human HaCat cells according to treatment with the compound SG0010 of the present invention.
9 is a photomicrograph showing the phenotype of human HaCat cells treated with the compound SG0010 of the present invention.
Figure 10 is a graph showing the results of analyzing the cytokine expression level of human Raw cells according to the treatment of the compound SG0010 of the present invention.

Definition of Terms:

The term " Vibrio " as used in this document has been known so far, and 34 species are Gram-negative bacteria, and the cell shape is rod-shaped and curved, and is characterized by being single or several connected to form a spiral. There is one or several flagella at one end of the body, and it moves by actively swimming with it. Wild species of Vibrio are widely distributed in the sea, but are also found in fresh water and soil. Among Vibrio, there are many species that are pathogenic to humans or fish and shellfish. Representative pathogenic species include Vibrio cholerae , Vibrio parahaemolyticus , Vibrio vulnificus , and Vibrio alginoli. Ticus ( Vibrio alginolyticus ).

DETAILED DESCRIPTION OF THE INVENTION:

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating Vibrio infections containing a compound having the structure of Formula 1 as an active ingredient:

(화학식 1).

(Formula 1).

In the pharmaceutical composition, the Vibrio bacteria are pathogenic such as Vibrio cholerae , Vibrio parahaemolyticus , Vibrio vulnificus and Vibrio alginolyticus . It can be selected from the group consisting of Vibrio species, and can inhibit the expression of pathogenic factors selected from the group consisting of leuO, VvhA, and ctxAB .

According to another aspect of the present invention, there is provided a feed additive for preventing Vibrio infection, comprising as an active ingredient a compound having the structure of Formula 1 below:

(화학식 1).

(Formula 1).

According to another aspect of the present invention, there is provided a method for treating an infection caused by Vibrio bacteria, comprising administering to a subject a compound having the structure of Formula 1 below:

(화학식 1).

(Formula 1).

The method for preventing or treating an infectious disease caused by Vibrio bacteria of the present invention may be performed by directly administering the pharmaceutical composition of the present invention to an individual, or mixing it with food or drinking water of the individual and ingesting it. In the method for preventing or treating infectious diseases caused by Vibrio bacteria of the present invention, the route of administration may be administered through various oral or parenteral routes as long as it can reach the target tissue, specifically oral, rectal, topical, intravenous, It can be administered in a conventional manner through intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, and the like.

According to another aspect of the present invention, preparing a vector comprising a gene construct in which the luxAB gene encoding a bioluminescent protein is operably linked to the leuO gene; Preparing a reporter strain by transducing the vector into a Vibrio sepsis strain in which the leuO gene is deleted; processing a test substance to the reporter strain; Measuring the degree of bioluminescence from the strain treated with the test material using a luminometer; and selecting a material whose bioluminescence is significantly reduced compared to a control group in which the test material is not treated.

In the screening method, the leuO gene may be a polynucleotide including a part of the leu0 coding sequence from 420 bp upstream from the leuO transcription initiation site, and a part of the leuO coding sequence is a nucleic acid sequence from the transcription initiation site to the lower part of 68 bp can be

It is obvious to those skilled in the art that an appropriate total daily amount of the pharmaceutical composition administered in the method of the present invention can be determined within the scope of sound medical judgment. The specific therapeutically effective amount for a specific subject is the type and degree of response to be achieved, the patient's age, weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, along with the specific composition It is preferable to apply differently according to various factors including drugs used or concurrently used and similar factors well known in the medical field.

In one embodiment of the present invention, diseases caused by Vibrio bacteria are preferably Vibrio enteritis food poisoning, cholera, Vibrio sepsis and Vibrio fish infections, but are not limited thereto.

The pharmaceutical composition of the present invention is formulated for oral administration or parenteral administration by mixing with pharmaceutically acceptable carriers, excipients, additives, and the like. Examples of pharmaceutically acceptable carriers or excipients include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cacao butter, ethylene glycol, and others commonly used. Examples of solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. In such a solid composition, the plant extract as an active ingredient is added with at least one inert diluent such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, magnesium aluminometasilicate, and the like. mixed The solid composition may contain additives other than an inert diluent according to a conventional method, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium cellulose glycolate, and a solubilizing agent such as glutamic acid or aspartic acid. Tablets or pills may, if necessary, be coated with a film made of a material such as sucrose, gelatin, hydroxypropyl methylcellulose phthalate, or the like, and in some cases may be coated with two or more layers. The liquid composition for oral administration includes pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, and the like, and may include a commonly used inert diluent such as purified water, ethanol, and the like. In addition to the inert diluent, the composition may contain auxiliary agents such as wetting agents and suspending agents, sweetening agents, flavoring agents, aromatic agents, preservatives, and the like.

Injections for parenteral administration include aseptic aqueous or non-aqueous solutions, suspensions, and emulsifiers. Aqueous solutions and suspensions include, for example, water for injection and physiological saline for injection. Non-aqueous solutions/suspensions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80 ^% . Such a composition may further contain adjuvants such as a preservative, a wetting agent, an emulsifier, a dispersing agent, a stabilizer (e.g., lactose), and a solubilizing agent (e.g., glutamic acid, aspartic acid). These can be sterilized by conventional sterilization methods, such as, for example, filter sterilization using a microfiltration membrane, heat sterilization such as high-pressure steam sterilization, or disinfectant formulation. The injection may be a solution preparation or a freeze-dried preparation that can be used by dissolving at the time of use. As an excipient for freeze-drying, for example, sugar alcohols or sugars such as mannitol and glucose may be used.

The pharmaceutical composition of the present invention is administered by oral administration or parenteral administration. Preferably, a parenteral administration method, for example, administration by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal administration, transmucosal administration (transrectal administration, etc.), transpulmonary administration, etc. to be. Of course, oral administration can also be used.

The dosage of the active ingredient of the pharmaceutical composition of the present invention can be determined depending on the severity of the disease, the age of the patient, etc., but is generally in the range of 0.005 μg/kg to 100 mg/kg, preferably 0.02 μg/kg to It is in the range of 5 mg/kg. However, since the dosage of the pharmaceutical composition of the present invention is determined in light of various related factors such as the route of administration, age, sex, weight, and severity of the patient, the dosage is not to limit the scope of the present invention in any aspect. should not be understood

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, drug It may be determined according to factors including sensitivity to, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitant drugs, generally 1 to 500 mg per 1 kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

Currently, some antibiotics are used to treat diseases caused by Vibrio sp., but they are not effective, and the development of appropriate and new antibiotics is required due to the problem of antibiotic resistance, which has recently become a problem. On the other hand, pathogenic microorganisms survive and proliferate in the host through the production of various virulence factors that are toxic to the host, and have a regulatory mechanism that allows various virulence factors to be expressed and acted as a whole during the pathogenesis process. have developed The regulatory mechanism of these toxic factors is very sophisticated, and the production of toxic factors can be suppressed by inhibiting the regulatory mechanism. Through this, the toxicity of pathogenic microorganisms can be weakened, and the toxicity of microorganisms can be easily inhibited by the host's immune response. Since inhibition of the production mechanism of toxic factors does not artificially inhibit the growth of microorganisms, it is very unlikely to cause antibiotic resistance, and has recently attracted attention among experts as one of the new treatment strategies for pathogenic microorganisms. However, until now, only studies limited to specific microorganisms have been actively conducted, and studies on the inhibition of toxic factor production control mechanisms for other pathogenic microorganisms are insufficient.

Accordingly, the present inventors have completely identified the signal transduction process of the pathogenic factor expression signal transduction system of pathogenic Vibrio bacteria in order to develop a substance that suppresses the pathogenicity of pathogenic Vibrio bacteria that cause diseases in humans, and searched for substances that inhibit this process. . Search for substances that inhibit the action of ToxR, a membrane receptor protein that receives signals in the cFP regulatory network, and find substances with such titer, among the substances, without affecting the growth of septic Vibrio bacteria, human cells It is non-toxic, and anti-pathogenic substances exhibiting an inhibitory effect on the expression of pathogenic factors of Vibrio bacteria were selected by searching and selecting substances having a significant level of inhibitory effect on the expression of pathogenic factors. That is, we found a pathogenic factor expression control network (cFP regulatory network) that appears specifically in Vibrio species, and the network plays a key role in regulating the expression of major pathogenic factors in Vibrio species, and the factors involved in the network and their The mechanism of action was identified (Park et al., J. Bacteriol. 188:2214-2221, 2006; Kim et al., Infect. Immun . 9:e00932-17, 2018; Park et al., Sci. Rep. 9 :20135, 2019; Park et al., Appl. Microbiol. Biotechnol. DOI 10.1007/s00253-020-01646-4, 2020; Park et al., J. Microbiol. Biotechnol. DOI 10.4014/jmb.2001.01021, 2020).

Specifically, the present inventors constructed a report system to screen for substances that inhibit the expression of LeuO, a key regulator of the lower end of ToxR, a signal receptor membrane protein in the cFP signaling network in Vibrio sepsis (FIG. 2). First, in order to quantitatively measure the expression of LeuO , the upper part of leuO was cloned into the upper part of the luxAB gene of the pHK0011 vector to construct a transcriptional fusion. ) was amplified using the PCR technique, and the product was cloned in the form of transcriptional conjugation at the top of the luxAB gene of the pHK0011 vector. The 442-bp region contains all of the following elements essential for leuO expression (Park et al., Scientific Reports , 9: 20135, 2019): 1) ToxR box for ToxR binding (light blue), 2) Promoter region, RNA polymerase binding site for transcription (underlined in blue), 3) AT-rich region required for bend formation in the upper region of leuO (region with high frequency of sequences A and T; underlined in italics). The 442-bp length is the minimum length including all of the essential elements described above. MO6 ΔleuO (pHK0011:: leuO ) strain was constructed by transforming the constructed pHK0011:: leuO into a MO6 ΔleuO strain, which is a mutant constructed by deleting the leuO gene, and used as an indicator strain.

The recombinant reporter strain prepared by the present inventors has the following differences compared to the leuO transcription conjugates of previously published papers: 1) lacZ is used as a reporter gene in the existing transcription conjugate, 2) the length of the upper region of the conjugated leuO is longer (about 540-bp), 3) unlike lacZ , the expression of the luxAB gene can be measured more accurately and quickly using a luminometer.

Briefly explaining the cFP regulatory network of the present invention, the diketopiparazine (DKP)-based cyclo (Phenylalanine-Proline) (cFP) substance, a signal substance produced and secreted by Vibrio bacterial species, is an intermembrane space region of ToxR, a membrane protein of Vibrio bacteria ( It binds to the periplasmic domain and initiates a signal. The ToxR induces the expression of the leuO gene, which is a positive regulator of cytoplasmic signal transduction, and the LeuO expressed by the leuO gene induces the expression of various toxic factors including OmpU, and at the same time, through the vHUαβ protein, the sigma factor RpoS. enhances the stability of mRNA, thereby enhancing the expression of a series of virulence factors (FIG. 1). The main pathogenic factors regulated by the signaling network include OmpU, KatG, VvpE, etc. in the case of Vibrio sepsis, and CtxAB in the case of Vibrio cholerae. It is understood that the network is present in pathogenic Vibrio species other than the cholera species, and it is expected that the expression of many major pathogenic factors is regulated by the network. Therefore, a substance capable of inhibiting the regulation process by the signaling network (compound SG0010) disrupts or inhibits the expression of pathogenic factors without affecting the growth of major pathogenic Vibrio species, thereby causing fatal pathogenic bacteria of the genus Vibrio such as cholera. It is thought to be able to prevent or treat infectious diseases caused by

The present inventors quantitatively and qualitatively measured the potency of the above substances that specifically inhibit Vibrio strains selected in the present invention as follows. 1) Effect on transcription of leuO , a master regulator of Vibrio sepsis, 2) Measurement of resistance to H ₂ O ₂ of Vibrio sepsis, 3) Effect on transcription of VvhA, a pathogenic factor of Vibrio sepsis 4) Effect on transcription of ctxAB (cholera toxin coding gene), a major pathogenic factor of cholera bacillus, 5) LD ₅₀ measurement in mice, 6) Quantitative measurement of effect on survival of human cells (MTT assay) and observation of cell morphology and measurement of cytokine production).

Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. It is provided to fully inform you.

Experiment method

reagent

In the present invention, LB (Difco, Detroit, MI), antibiotics (Sigma-Aldrich), methanol, n-decylaldehyde, DMSO, compound SG0010, PBS, Z-buffer, SDS, chloroform, ONPG, Na ₂ CO ₃ , H ₂ O ₂ , iron dextran, sodium bicarbonate, EZ-cytox, DMEM, and FBS (Gibco) were used.

bacterial culture

Escherichia coli , Vibrio parahaemolyticus , and Vibrio cholerae strains used in the present invention were cultured at 37 ° C and 230 rpm after treatment with appropriate antibiotics in Luria-Bertani (LB) medium, and Vibrio vulnificus strains were treated with appropriate antibiotics in LB medium. It was incubated at 30°C and 230 rpm. The concentrations of the above antibiotics were treated with ampicillin 50 μg/mL, kanamycin 25 μg/mL, tetracycline 10 μg/mL, and chloramphenicol 25 μg/mL in the case of E. coli , and in the case of Vibrio species, kanamycin 100 μg/mL, tetra It was treated with cyclin 1 μg/mL and chloramphenicol 2 μg/mL.

Laboratory animals and breeding

3-week-old C57BL/6 female mice with an average body weight of 11 g were purchased from DBL (Eumseong-gun, Chungcheongbuk-do, Republic of Korea) and used in the present invention after an acclimatization period of 7 days. Experimental animals were raised in groups of 4 to 6 per plastic cage at a temperature of 20 to 25 °C, humidity of 40 to 45%, and a 12-hour cycle of day/night cycles, and rodent pellet feed was fed during the acclimatization period.

cell culture

Human keratinocytes (HaCaT) used in the present invention were cultured in DMEM containing FBS at 37° C. and 5% CO ₂ in a saturated state.

report system

The present inventors constructed a report system to screen for substances that inhibit the expression of LeuO, a key regulator of the lower end of ToxR, a signal receptor membrane protein, in the cFP signaling network in septic Vibrio bacteria. In the above system, focusing on the fact that when ToxR is attached to a cFP signal substance, it enhances the expression of LeuO, a lower regulator, the nucleic acid sequence including the 442 bp region at the 5'-end of the LeuO gene and part of the coding sequence was converted into the pHK0011 vector (Jeong et al . al ., J. Biol. Chem. 276: 13875-13880, 2001), a reporter construct was prepared by connecting to the upper end of the luxAB gene, which is a bioluminescent gene (FIG. 2). A reporter strain (MO6 ΔleuO (pHK0011:: leuO )) was prepared by transduction into MO6 ΔleuO , a mutant septic vibrio strain in which the LeuO coding gene of the reporter construct was deleted. A total of 6,566 representative libraries provided by the Korea Compound Bank were used for the reporter strain. Each of the two compounds was treated and the Lux activity was quantitatively measured using a luminometer.At this time, DMSO was used for the negative control group and cFP (final concentration: 5 mM) diluted in DMSO was used for the positive control group. As a result, the value was less than 0.3. Substances that can be seen were first screened, and secondary screening was performed on the selected substances under the same conditions, and finally a substance showing significant activity Butyl2-(3-(2-chlorobenzyl)-2-imino-2,3- dihidro-1H-benzo[d]imidazol-1-yl)acetate, formula 1) was selected, and the compound of formula 1 was named “SG0010”. The structure of the compound SG0010 is shown in Formula 1 below:

(화학식 1)

(Formula 1)

Bioluminescence Assays

We inoculated V. vulnificus, V. cholerae , or V. parahaemolyticus grown overnight in LB medium to a fresh LB medium to an OD ₆₀₀ of 0.005. Before inoculation, the compound SG0010 of the present invention was treated at a concentration of 50 μM and DMSO was treated as a control. Then, 0.0006% (v/v) n-decylaldehyde dissolved in 50% methanol was put into a luminometer (Mithras LB 940; Berthold, Bad Wildbach, Germany), and bioluminescence was measured under various growth conditions using the luminometer. measured. Then, the amount of transcription was expressed by normalizing the luminescence unit to the cell concentration (relative light unit, RLU).

β-galactosidase assay

We inoculated V. vulnificus, V. cholerae , or V. parahaemolyticus grown overnight in LB medium to a fresh LB medium to an OD ₆₀₀ of 0.005. Before inoculation, the compound SG0010 of the present invention was treated at a concentration of 50 μM and DMSO was treated as a control. After that, 1 drop and 2 drops of 0.05% SDS and chloroform were treated in an E-tube containing 400 μl of Z buffer, and 100 μl of bacteria were added and reacted in a water bath at 30 ° C for 10 minutes. 100 μl was added and the time was measured until the color changed to yellow. Subsequently, 250 μl of Na ₂ CO ₃ was treated, and 100 μl of the supernatant was sprayed on a 96-well plate by centrifugation at 11000 rpm for 2 minutes. did

ROS tolerance measurement

We inoculated V. vulnificus grown overnight in LB medium into fresh LB to an OD ₆₀₀ of 0.005. Before inoculation, the compound SG0010 at a concentration of 50 mM was diluted to 50 μM and treated with DMSO as a control. When the OD ₆₀₀ reached approximately 0.1, the bacteria were divided in half and then treated with H ₂ O ₂ to 625 μM, and treated with H ₂ O as a control. After that, the bacteria were diluted and spread on LA plates, and the measured cell number was expressed as a ratio.

Mouse infection assay

We purchased 3-week-old C57BL/6 female mice with an average body weight of 11 g from DBL and used them in the present invention after an acclimatization period of 7 days. After removing all feed, inoculate iron dextran by intraperitoneal injection at a dose of 100 μg/g to administer iron, and then orally administer 8.5% (w/v) sodium bicarbonate solution (50 μl) to suppress the action of gastric acid to kill bacteria. increased the survival rate of Thereafter, 100 μl of the bacteria and PBS enriched at a concentration of 10 ¹⁰ cell/ml using PBS were fed, and 30 μl of the compound SG0010 50 mM and DMSO was fed, and the survival rate of the mice was confirmed for 24 hours.

Example 1: leuO transcriptional effects of

The present inventors measured the amount of transcription of leuO , a master regulator of Vibrio sepsis, according to the treatment with SG0010, the compound of the present invention. To this end, the reporter strain prepared as described above was treated with the compound SG0010 at various concentrations, and the luciferase activity was quantified. As a result, it was confirmed that the compound SG0010 of the present invention inhibits leuO transcription at a statistically significant level (FIG. 3).

Example 2: Resistance to hydrogen peroxide

The present inventors analyzed the resistance to H ₂ O ₂ of Vibrio sepsis treated with the compound SG0010 of the present invention. KatG, which exists at the bottom of the ToxR-LeuO-Vhuα-RpoS-KatG signal transduction system, is a hydrogen peroxide decomposition enzyme (catalase) that increases the resistance of Vibrio bacteria to ROS (reactive oxygen species). Therefore, since the expression of leuO decreased when the SG0010 of the present invention was treated, it was investigated whether resistance to H ₂ O ₂ was reduced by suppressing the expression of katG. As a result, it was confirmed that resistance to H ₂ O ₂ decreased when treated with 50 μM of SG0010 (FIG. 4).

Example 3: Effect on expression of exotoxin VvhA

The present inventors analyzed the effect of treatment with the compound SG0010 on the expression of VvhA of Vibrio sepsis. In a previous study, it was reported that the survival rate of mice infected with V. vulnificus increased about 4 to 5 times when the gene vvhA encoding hemolysin, a representative exotoxin of Vibrio sepsis, was removed. Previous studies have shown that ToxRS increases the transcription of vvhA. Therefore, since SG0010 of the present invention is involved in a series of signal transduction systems starting from ToxR, in order to examine whether the material also inhibits vvhA , a gene construct was prepared by transcribing the lacZ reporter gene at the lower end of vvhA , After transduction into V. vulnificus strains, they were treated with 50 μM of SG0010, respectively, and the activity of β-galatosidase was quantitatively measured. As a result, it was found that the compound SG0010 of the present invention inhibits the transcription of vvhA at a significant level (FIG. 5).

Example 4: Effect of CtxAB (choleratoxin) on transcription

The present inventors analyzed the effect of treatment with the compound SG0010 on the transcription of CtxAB (choleratoxin), a major pathogenic factor of Vibrio cholerae. Vibrio ( Vibrio ) In addition to septic Vibrio bacteria ( V. vulnificus ), there is also a representative pathogenic bacterium, cholera bacteria ( V. cholerae ), which secretes an exotoxin protein cholera toxin (cholera toxin) to cause fatal diarrhea in humans. The expression of ctx encoding cholera exotoxin is regulated by ToxR. Therefore, in order to investigate whether ToxR of V. cholerae , which has high homology to ToxR of V. vulnificus , is also affected by SG0010, the effect of SG0010 on the transcription amount of ctxAB in El Tor strain, a wild-type cholerae strain, was quantitatively measured. As a result, it was confirmed that the expression of ctxAB was inhibited by treatment with the compound SG0010 of the present invention (FIG. 6).

Example 5: Mouse survival rate analysis

V. vulnificus infects and kills mice, which are model experimental animals. As a method for examining the effect of the compound SG0010 of the present invention on the pathogenicity of V. vulnificus , mice were tested. After oral administration of the strain to the mice, the compound of the present invention, SG0010, was administered to the mice, and the survival rate was observed for 24 hours. As a result, it was found that the survival rate of the group administered with compound SG0010 was significantly increased compared to the control group (positive control group: MO6) (FIG. 7).

Example 6: Analysis of viability of human cells

The present inventors investigated the survival rate of human keratinocytes according to the compound SG0010 treatment. In order to utilize the compound SG0010 of the present invention as a pathogenic inhibitor, there should be no risk to host (human) cells. Accordingly, in order to confirm the toxicity of the material to human cells, human keratinocytes, HaCaT, were treated with 50 μM of the compound SG0010, MTT assay was performed, and the shape of the cells was observed through a microscope. As a result, it was found that the compound SG0010 had no effect on the survival and phenotype of HaCaT cells (FIGS. 8 and 9).

Example 7: Immune response

The present inventors investigated the effect of SG0010 of the present invention on the immune response of eukaryotic cells. To this end, raw cells, which are mouse monocytes, were activated with LPS and treated with 1 μM of the compound SG0010 to analyze the expression levels of IL-1β and IL-6. As a result, it was confirmed that both IL-1β and IL-6 expressions were suppressed in primitive cells (FIG. 10). The above results suggest that the compound SG0010, which is an antipathogenic composition selected according to an embodiment of the present invention, can prevent or treat diseases caused by pathogenic bacteria by disrupting or inhibiting the expression of pathogenic factors of Vibrio bacteria.

Although the present invention has been described with reference to the above-described embodiments, these are only examples, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

Claims (7)

A pharmaceutical composition for the treatment of Vibrio infections containing a compound having the structure of Formula 1 as an active ingredient:

(Formula 1). According to claim 1,
The Vibrio bacteria are selected from the group consisting of Vibrio cholerae , Vibrio parahaemolyticus , Vibrio vulnificus and Vibrio alginolyticus . composition. According to claim 1,
A pharmaceutical composition that inhibits the expression of a pathogenic factor selected from the group consisting of leuO, VvhA and ctxAB . Feed additive for preventing Vibrio infection, comprising as an active ingredient a compound having the structure of Formula 1 below:

(Formula 1). A method for treating an infection caused by Vibrio bacteria, comprising administering to a subject a compound having the structure of Formula 1 below:

(Formula ). preparing a vector comprising a gene construct in which a luxAB gene encoding a bioluminescent protein is operably linked to a leuO gene;
Preparing a reporter strain by transducing the vector into a Vibrio sepsis strain in which the leuO gene is deleted;
processing a test substance to the reporter strain;
Measuring the degree of bioluminescence from the strain treated with the test material using a luminometer; and
A method for screening a Vibrio pathogenicity inhibitor comprising the step of selecting a material that significantly reduces bioluminescence compared to a control group in which the test material is not treated. According to claim 6,
The leuO gene is a polynucleotide containing a part of the leu0 coding sequence from 420 bp upstream from the leuO transcription start site, screening method.

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