Use of taurocholate and / or taurodeoxycholate in the preparation of a product for inhibiting enterohemorrhagic e. coli virulence

Abstract

The present application relates to the field of biological medicine, and particularly relates to application of taurine cholate and / or taurine deoxycholate in preparation of products for inhibiting enterohemorrhagic E. coli virulence. Researches prove that taurine cholate and / or taurine deoxycholate can significantly down-regulate expression of key genes of LEE virulence island, inhibit synthesis and secretion of virulence factor EspB, and reduce adhesion ability of bacteria to host cells; in vivo, the taurine cholate and / or taurine deoxycholate can effectively inhibit expression of virulence genes, reduce intestinal colonization, improve survival rate of infected mice, and reduce intestinal pathological damage. The application exerts pharmacological effects at sub-inhibitory concentration, does not inhibit bacterial growth, and is expected to avoid the risk of SOS response and Shiga toxin release induced by antibiotic treatment. The present application further provides application of the above-mentioned compounds in preparation of medicines for preventing and / or treating enterohemorrhagic E. coli infection, and a composition comprising the active ingredient, which can be flexibly prepared into a pharmaceutical preparation, a feed additive or a food preservative.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and particularly to the use of taurocholate and / or taurodeoxycholate in the preparation of products for inhibiting the virulence of enterohemorrhagic Escherichia coli, in the preparation of medicaments for the prevention and / or treatment of enterohemorrhagic Escherichia coli infection, and in compositions comprising taurocholate and / or taurodeoxycholate. Background Technology

[0002] Enterohemorrhagic Escherichia coli (EHEC) is a major pathogen causing foodborne illness outbreaks worldwide, with serotype O157:H7 being the most representative pathogenic type. Infection with this bacterium can lead to hemorrhagic colitis, and in severe cases, hemolytic uremic syndrome (HUS), posing a significant threat, especially to children and the elderly. Because antibiotic treatment may induce a bacterial SOS response, leading to a massive release of Shiga toxin, exacerbating the condition and even increasing the risk of death, routine antibiotic use is currently not recommended for EHEC infections in clinical practice. Developing novel and safe infection intervention methods has become an urgent technical challenge.

[0003] The pathogenicity of EHEC is highly dependent on its encoded Type III Secretion System (T3SS) and its related effector proteins. The core components of this system are encoded by virulence islands at the Locus of Enterocyte Effacement (LEE), including the regulatory factor Ler, the secretory device protein EscV, the secretory effector protein EspB, and the adhesion factor Intimin (encoded by the eae gene). These virulence factors work synergistically to mediate bacterial adhesion to host intestinal epithelial cells and the formation of attachment and efffacing (A / E) damage, which is a key step in EHEC pathogenicity. Therefore, specifically inhibiting LEE virulence island expression or T3SS function, without affecting bacterial survival—i.e., antiviral methods—is considered an ideal direction for controlling EHEC infection, avoiding the selective pressure and toxin release risks associated with traditional antibiotics.

[0004] In recent years, researchers have attempted to screen active substances that can inhibit the toxicity of EHEC from natural products and small molecule compound libraries. However, the existing antiviral candidates have the following shortcomings: (1) some compounds still have antibacterial activity at high concentrations, making it difficult to achieve precise regulation that only inhibits toxicity without affecting growth; (2) most studies only focus on in vitro morphological observations and lack a complete chain of evidence from molecular mechanisms to in vivo protective effects; (3) the safety or in vivo efficacy of some compounds has not been verified, and their prospects for clinical translation are unclear.

[0005] Therefore, developing a novel antiviral agent that can specifically inhibit the virulence of EHEC and has a clear protective effect in vivo is of great significance for filling the gap in safe treatment methods for EHEC. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the lack of safe and effective treatments for enterohemorrhagic Escherichia coli (EHEC) infection and the difficulty in precisely inhibiting virulence and the lack of in vivo protective efficacy verification of existing antiviral candidates. This invention provides the application of taurocholate and taurodeoxycholate in the preparation of products for inhibiting the virulence of EHEC. This application can significantly inhibit the expression of key genes of the LEE virulence island (such as ler, espB, eae, etc.) at sub-inhibitory concentrations, reduce the synthesis and secretion of the type III secretion system effector protein EspB, weaken the adhesion of bacteria to host epithelial cells, and effectively reduce intestinal colonization, alleviate small intestinal tissue pathological damage, and improve the survival rate of infected individuals in animal models. Therefore, it provides a safe, efficient, and novel antiviral approach with a complete chain of evidence for the prevention and control of EHEC infection.

[0007] A first aspect of the invention provides the use of taurocholate and / or taurodeoxycholate in the preparation of products for inhibiting the virulence of enterohemorrhagic Escherichia coli.

[0008] Furthermore, the application involves using taurocholate and / or taurodeoxycholate at a sub-inhibitory concentration; and / or, the sub-inhibitory concentration is 0.1 µM-1 µM.

[0009] Furthermore, the inhibition of enterohemorrhagic Escherichia coli (EHEC) virulence includes any one or more of the following:

[0010] (a) Inhibits the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC);

[0011] (b) Inhibit the expression and / or secretion of proteins associated with the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (T3SS);

[0012] (c) Inhibits the adhesion ability of enterohemorrhagic Escherichia coli (EHEC) to host cells;

[0013] (d) Inhibits the colonization of enterohemorrhagic Escherichia coli (EHEC) in the host gut;

[0014] (e) Reduce host intestinal tissue damage caused by enterohemorrhagic Escherichia coli (EHEC) infection.

[0015] Furthermore, the genes on the LEE virulence island include at least one of ler, espB, eae, escV, and sepZ.

[0016] Furthermore, the type III secretion system (T3SS) related proteins include the EspB protein.

[0017] A second aspect of the invention provides the use of taurocholate and / or taurodeoxycholate in the preparation of medicaments for the prevention and / or treatment of enterohemorrhagic Escherichia coli infection.

[0018] Furthermore, the prevention and / or treatment of enterohemorrhagic Escherichia coli infection includes:

[0019] (a) Reduce the mortality rate of infected individuals;

[0020] (b) Reduce intestinal pathological damage in infected individuals;

[0021] (c) Reduce the amount of bacteria colonizing the gut of infected individuals;

[0022] (d) Inhibit the expression of bacterial virulence genes in the gut of infected individuals.

[0023] A third aspect of the present invention provides a composition for inhibiting the virulence of enterohemorrhagic Escherichia coli, characterized in that the active ingredient of the composition comprises taurocholate and / or taurodeoxycholate.

[0024] Furthermore, the concentration of taurocholate and / or taurodeoxycholate in the composition is a sub-inhibitory concentration; and / or, the sub-inhibitory concentration is 0.1 µM-1 µM.

[0025] Furthermore, the composition is a pharmaceutical composition, a feed additive, or a food preservative; and / or, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, and the dosage form of the pharmaceutical composition is an oral formulation.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention provides the application of taurocholate and / or taurodeoxycholate in the preparation of products for inhibiting the virulence of enterohemorrhagic Escherichia coli. Through multi-level validation, this invention confirms that taurocholate and / or taurodeoxycholate can significantly downregulate the expression of key genes in the LEE virulence island, inhibit the synthesis and secretion of the virulence factor EspB, and reduce the adhesion ability of bacteria to host cells; in animals, it can effectively inhibit virulence gene expression, reduce intestinal colonization, improve the survival rate of infected mice, and alleviate intestinal pathological damage.

[0028] 2. This invention provides the application of taurocholate and / or taurodeoxycholate in the preparation of drugs for the prevention and / or treatment of enterohemorrhagic Escherichia coli (EHEC) infection. The drug exerts its efficacy at a sub-inhibitory concentration (0.1 µM-1 µM), without inhibiting bacterial growth. This is expected to fundamentally avoid the SOS response and Shiga toxin burst release risks that may be induced by antibiotic treatment, while also potentially reducing the selective pressure of bacterial resistance, suggesting its potential to improve clinical drug safety. In animal infection models, the drug significantly inhibits the expression of key genes (ler, espB, eae, etc.) of the LEE virulence island in the small intestine, effectively reducing the number of EHEC colonizations in the intestine, significantly improving the survival rate of infected mice, and significantly alleviating pathological damage to small intestinal tissue. Based on the high biocompatibility of taurocholate and taurodeoxycholate as endogenous bile acids in the human body, and experimental studies confirming their non-toxicity to the body, the drug described in this invention has clear prospects for clinical translation, providing a safe, efficient, and mechanistically sound novel treatment option for the clinical prevention and control of EHEC infection.

[0029] 3. The composition provided by this invention uses taurocholate and / or taurodeoxycholate as active ingredients. This composition utilizes the high biocompatibility of endogenous bile acids in the human body to achieve precise inhibition of EHEC virulence at sub-inhibitory concentrations. Experiments have confirmed that this composition can inhibit LEE virulence island activity at the transcriptional level, block the secretion of T3SS effector proteins, weaken bacterial adhesion ability, and significantly reduce bacterial colonization, alleviate tissue damage, and improve survival rate in infected animal models. Based on its good safety and clear antiviral effect, this composition can be flexibly prepared into various product forms such as pharmaceutical formulations, feed additives, or food preservatives, providing a safe, efficient, and promising new product for the prevention and control of EHEC infection. Attached Figure Description

[0030] Figure 1 Figure showing the results of the analysis of the expression of the virulence gene of EHEC O157:H7 after treatment with different concentrations of taurine and taurine deoxycholate.

[0031] Figure 2The growth curves of EHEC O157:H7 under different concentrations of taurine and taurine deoxycholate treatment are shown.

[0032] Figure 3 Comparative diagram of the effects of different bile acid components on the expression of the EHEC O157:H7 virulence gene (cholate, deoxycholate, chenodeoxycholate, taurocholate, taurodeoxycholate).

[0033] Figure 4 The figure shows the effect of taurocholate and taurodeoxycholate treatments on the activity of the ler promoter of EHEC O157:H7.

[0034] Figure 5 Figure 1 shows the effect of Western Blot analysis on the amount of EspB protein, a virulence factor secreted by EHEC O157:H7 into the culture supernatant.

[0035] Figure 6 Figure showing the effect of taurocholate and taurodeoxycholate treatments on the ability of EHEC O157:H7 cells to adhere to HeLa cells.

[0036] Figure 7 Figure showing the effect of taurocholate and taurodeoxycholate treatments on the expression level of virulence genes of EHEC O157:H7 in the small intestine of mice.

[0037] Figure 8 Figure showing the effect of taurocholate and taurodeoxycholate treatments on the colonization of EHEC O157:H7 in the small intestine of mice.

[0038] Figure 9 Figure 1 shows the effect of taurocholate and taurodeoxycholate treatments on the survival rate of mice infected with EHEC O157:H7.

[0039] Figure 10 The images show pathological sections of small intestinal tissue from mice infected with the control group and the taurocholate treatment group, illustrating the differences in histological damage. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0041] The pathogenicity of EHEC is highly dependent on its encoded Type III Secretion System (T3SS) and its related effector proteins. The core components of this system are encoded by virulence islands at the Locus of Enterocyte Effacement (LEE), including the regulatory factor Ler, the secretory device protein EscV, the secretory effector protein EspB, and the adhesion factor Intimin (encoded by the eae gene). These virulence factors work synergistically to mediate bacterial adhesion to host intestinal epithelial cells and the formation of attachment and efffacing (A / E) damage, which is a key step in EHEC pathogenicity. Therefore, specifically inhibiting LEE virulence island expression or T3SS function, without affecting bacterial survival—i.e., antiviral methods—is considered an ideal direction for controlling EHEC infection, avoiding the selective pressure and toxin release risks associated with traditional antibiotics.

[0042] In recent years, researchers have attempted to screen active substances that can inhibit the toxicity of EHEC from natural products and small molecule compound libraries. However, the existing antiviral candidates have the following shortcomings: (1) some compounds still have antibacterial activity at high concentrations, making it difficult to achieve precise regulation that only inhibits toxicity without affecting growth; (2) most studies only focus on in vitro morphological observations and lack a complete chain of evidence from molecular mechanisms to in vivo protective effects; (3) the safety or in vivo efficacy of some compounds has not been verified, and their prospects for clinical translation are unclear.

[0043] The first aspect of this embodiment provides the use of taurocholate and / or taurodeoxycholate in the preparation of products for inhibiting the virulence of enterohemorrhagic Escherichia coli.

[0044] This invention has demonstrated through multi-level verification that taurocholate and / or taurodeoxycholate can significantly downregulate the expression of key genes in the LEE virulence island, inhibit the synthesis and secretion of the virulence factor EspB, and reduce the adhesion ability of bacteria to host cells. In animals, it can effectively inhibit the expression of virulence genes, reduce intestinal colonization, improve the survival rate of infected mice, and alleviate intestinal pathological damage.

[0045] In some embodiments, the application involves using taurocholate and / or taurodeoxycholate at a sub-inhibitory concentration; and / or, the sub-inhibitory concentration is 0.1 µM-1 µM. In some embodiments, for example, sub-inhibitory concentrations of 0.1 µM, 0.2 µM, 0.3 µM, 0.4 µM, 0.5 µM, 0.6 µM, 0.7 µM, 0.8 µM, 0.9 µM, and 1 µM can all achieve the effect of inhibiting toxicity without killing bacteria.

[0046] In some embodiments, the inhibition of enterohemorrhagic Escherichia coli (EHEC) virulence includes any one or more of the following:

[0047] (a) Inhibits the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC);

[0048] (b) Inhibit the expression and / or secretion of proteins associated with the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (T3SS);

[0049] (c) Inhibits the adhesion ability of enterohemorrhagic Escherichia coli (EHEC) to host cells;

[0050] (d) Inhibits the colonization of enterohemorrhagic Escherichia coli (EHEC) in the host gut;

[0051] (e) Reduce host intestinal tissue damage caused by enterohemorrhagic Escherichia coli (EHEC) infection.

[0052] In some embodiments, the genes on the LEE virulence island include at least one of ler, espB, eae, escV, and sepZ.

[0053] In some embodiments, the type III secretion system (T3SS)-related proteins include EspB protein.

[0054] The second aspect of this embodiment provides the use of taurocholate and / or taurodeoxycholate in the preparation of medicaments for the prevention and / or treatment of enterohemorrhagic Escherichia coli infection.

[0055] This drug exerts its efficacy at a sub-inhibitory concentration (0.1 µM-1 µM), without inhibiting bacterial growth. Therefore, it is expected to fundamentally avoid the SOS response and the risk of explosive release of Shiga toxin that may be induced by antibiotic treatment. Simultaneously, it is expected to reduce the selective pressure of bacterial resistance, suggesting its potential to improve the safety of clinical use. In animal infection models, this drug significantly inhibited the expression of key genes (ler, espB, eae, etc.) of the LEE virulence island in the small intestine, effectively reducing the number of EHEC colonizations in the intestine, significantly improving the survival rate of infected mice, and significantly alleviating pathological damage to small intestinal tissue. Based on the high biocompatibility of taurocholate and taurodeoxycholate as endogenous bile acids in the human body, and experimental studies confirming their non-toxicity to the body, the drug described in this invention has clear prospects for clinical translation, providing a safe, efficient, and mechanistically sound novel treatment option for the clinical prevention and control of enterohemorrhagic Escherichia coli infection.

[0056] In some embodiments, the prevention and / or treatment of enterohemorrhagic Escherichia coli infection includes:

[0057] (a) Reduce the mortality rate of infected individuals;

[0058] (b) Reduce intestinal pathological damage in infected individuals;

[0059] (c) Reduce the amount of bacteria colonizing the gut of infected individuals;

[0060] (d) Inhibit the expression of bacterial virulence genes in the gut of infected individuals.

[0061] A third aspect of this embodiment provides a composition for inhibiting the virulence of enterohemorrhagic Escherichia coli, characterized in that the active ingredient of the composition comprises taurocholate and / or taurodeoxycholate.

[0062] In some embodiments, the concentration of taurocholate and / or taurodeoxycholate in the composition is a sub-inhibitory concentration; and / or, the sub-inhibitory concentration is 0.1 µM-1 µM. In some embodiments, for example, sub-inhibitory concentrations of 0.1 µM, 0.2 µM, 0.3 µM, 0.4 µM, 0.5 µM, 0.6 µM, 0.7 µM, 0.8 µM, 0.9 µM, and 1 µM can all achieve the effect of inhibiting toxicity without killing bacteria.

[0063] In some embodiments, the composition is a pharmaceutical composition, a feed additive, or a food preservative; and / or, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, and the dosage form of the pharmaceutical composition is an oral formulation.

[0064] To better understand the technical solutions of the above embodiments, the following more detailed implementation examples will be provided for further explanation.

[0065] In the following examples, the taurocholate (CAS No.: 345909-26-4, purity ≥97%) used was purchased from Sigma-Aldrich (catalog number: 86339).

[0066] Taurodeoxycholate (CAS No.: 207737-97-1, purity ≥97%) was purchased from Sigma-Aldrich (item number: T0557).

[0067] EHEC O157:H7 standard strain EDL933 source: China Microbial Culture Collection Center, platform code bio-106964 (ATCC 700927).

[0068] All other reagents were commercially available analytical grade.

[0069] In this invention, the terms taurocholate and taurodeoxycholate respectively include any salt form formed by taurocholic acid and taurodeoxycholic acid with pharmaceutically acceptable cations, such as, but not limited to, sodium, potassium, calcium, magnesium, and ammonium salts. The embodiments of this invention specifically use sodium taurocholate and sodium taurodeoxycholate, which is merely illustrative and does not constitute a limitation on the types of salts. Those skilled in the art will understand that other pharmaceutically acceptable salts (such as potassium and calcium salts) can also dissociate into active forms of taurocholate or taurodeoxycholate in vivo or in vitro, thereby exerting the same inhibitory effect on the virulence of enterohemorrhagic Escherichia coli. Therefore, unless specifically specified, the taurocholate and taurodeoxycholate referred to in this invention are preferably sodium salts, but other salt forms with the same pharmacological activity are also included.

[0070] Example 1

[0071] The EHEC O157:H7 standard strain EDL933 was inoculated into LB medium and allowed to grow to the OD level. 600 When the concentration was 0.8, 0 µM (control), 0.1 µM, and 1 µM taurocholate or taurodeoxycholate were added, respectively. After culturing for another hour, the bacterial cells were collected, and total RNA was extracted according to the RNA extraction kit instructions. The extracted total RNA was reverse transcribed into cDNA. The transcriptional levels of key genes (ler, espB, eae, escV, sepZ) of the LEE virulence island were detected using real-time quantitative PCR (qPCR). The 16S rRNA of EDL933 was used as an internal control, and the relative gene expression levels were calculated after normalization.

[0072] The results are as follows Figure 1 As shown, the results indicate that, compared with the control group, the treatment groups of 0.1 µM taurocholate, 0.1 µM taurodeoxycholate, 1 µM taurocholate and 1 µM taurodeoxycholate all downregulated the transcriptional level of LEE virulence island-related genes to varying degrees; among them, the treatment of 1 µM taurocholate or 1 µM taurodeoxycholate had the most significant downregulation effect on key genes of LEE virulence island, and the expression level of virulence-related genes of the strain decreased most significantly.

[0073] Example 2

[0074] EHEC O157:H7 standard strain EDL933 was inoculated into LB medium, with 0 µM (control), 0.1 µM, and 1 µM taurocholate or taurodeoxycholate added respectively, and incubated in a 37°C shaker. OD was monitored every 2 hours using a microplate reader. 600 The optical density value was recorded for 24 hours, and a growth curve was plotted.

[0075] The results are as follows Figure 2As shown, the results indicated that, compared with the blank control group, there were no significant differences in growth rate, growth trend, and final bacterial density among the EDL933 strains treated with 0.1 µM taurocholate, 1 µM taurocholate, 0.1 µM taurodeoxycholate, and 1 µM taurodeoxycholate, suggesting that the above concentrations of taurocholate and taurodeoxycholate had no significant effect on the in vitro growth and proliferation of the EDL933 strain.

[0076] Example 3

[0077] EDL933 was inoculated into LB medium and allowed to grow to OD. 600 When the concentration was 0.8, 0 µM (control) and 1 µM of cholate, deoxycholate, chenodeoxycholate, taurocholate, or taurodeoxycholate were added, respectively. After culturing for another hour, the transcriptional levels of key genes (ler, espB, eae) of the LEE virulence island were detected. Using EDL933 16S rRNA as an internal control, the relative gene expression levels were calculated after normalization.

[0078] The results are as follows Figure 3 As shown, at the same concentration, taurocholate, deoxycholate, and chenodeoxycholate did not significantly inhibit the toxicity of EDL933, indicating that the inhibitory effect of taurocholate and taurodeoxycholate on toxicity is not a universal property of bile acid compounds.

[0079] Example 4

[0080] A lacZ fusion reporter strain containing the LEE1 promoter region was constructed. The activity of bacterial β-galactosidase was determined using the following method: 50 µL of bacterial culture was placed in a 1.5 mL EP tube, and 420 µL of Z buffer, 20 µL of chloroform, and 10 µL of 0.1% SDS were added. The mixture was thoroughly mixed and incubated at 30 °C for 1 h. Then, 100 µL of pre-prepared substrate (4 mg / mL ONPG, 40 mM NaH2PO4, 60 mM Na2HPO4, 2.7 µL / mL β-mercaptoethanol) activated on a shaker at 30 °C was added, and the mixture was thoroughly mixed by inversion. The mixture was incubated at 30 °C for an extended period, and the color change was observed. When the reaction color turns yellow, add 250 µL of Na2CO3 solution (concentration: 1 M) to terminate the reaction (the reaction time is from the start of substrate addition to the end of the reaction (min)). After the reaction is terminated, let it stand for 15 min, then take 200 µL of the supernatant in a fume hood and measure the absorbance values ​​of OD420 and OD550, and record the data. The enzyme activity calculation formula is: Miller enzyme activity unit = 1000 × (OD420 - 1.75 × OD550) / [bacterial solution OD600 × reaction time (min) × bacterial solution volume (L)].

[0081] The results are as follows Figure 4 As shown in the results, compared with the control group (0 µM) without bile salts, treatment with 1 µM taurocholate and 1 µM taurodeoxycholate significantly reduced the β-galactosidase activity of the lacZ fusion reporter strain. Furthermore, the Miller enzyme activity units in each bile salt treatment group were significantly lower than those in the control group, indicating that bile salt treatment can significantly inhibit the transcriptional activity of the LEE1 promoter, thereby reducing the expression level of the lacZ gene it drives.

[0082] Example 5

[0083] An espB gene deletion mutant was constructed using the CRISPR-Cas9 system, and then the His-tagged espB gene was reintroduced in situ using the CRISPR-Cas9 system. The His-espB-tagged EHEC O157:H7 standard strain EDL933 was inoculated into LB medium and allowed to grow to OD. 600 When the concentration was 0.8, 0 µM (control), 1 µM taurocholate, and 1 µM taurodeoxycholate were added, respectively. After culturing for another hour, the supernatant was collected by centrifugation. Bacteria were first removed using a sterile membrane, then proteins were adsorbed using a protein adsorption membrane, and finally the proteins were denatured using 1×SDS. SDS-PAGE and Western blotting were performed, and the amount of EspB protein secreted extracellularly was detected using anti-His antibody.

[0084] The results are as follows Figure 5 As shown, Western blotting results indicated that both 1 µM taurocholate and 1 µM taurodeoxycholate significantly inhibited the synthesis of intracellular EspB and the secretion of extracellular EspB in strain EDL933, further validating the inhibitory effect of bile salts on the virulence of strain EDL933 from the perspective of protein secretion.

[0085] Example 6

[0086] HeLa cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. After cell adhesion, cells were sputtered at 1 × 10⁶ cells per well. 5 HeLa cells were reseeded in 24-well DMEM containing 10% fetal bovine serum and no antibiotics at a density of 10% cell line, and cultured at 37°C for 48 hours in a 5% CO2 cell culture incubator before bacterial infection. EDL933 cells were cultured in LB medium until OD2000. 600 The concentration was set at 0.8, and 0 µM (control), 1 µM taurocholate, and 1 µM taurodeoxycholate were added, respectively. After culturing for another hour, bacterial cells were collected, washed, and resuspended in PBS. The final cell count was diluted to 5 × 10⁸. 7CFU mL−1. HeLa cells monolayer adherently cultured in 24-well plates were infected with EDL933 strain at 100-fold infection rate (MOI) and incubated in a cell culture incubator at 37°C and 5% CO2 for 1 h. Infected cells were lysed with 1% Triton X-100, and the precipitate was collected by centrifugation, diluted, and plated to count the number of CFU of adhering bacteria.

[0087] The results are as follows Figure 6 As shown, compared with the control group, the adhesion rates of the strains treated with 1 µM taurocholate and 1 µM taurodeoxycholate were significantly reduced. These results indicate that taurocholate and taurodeoxycholate can significantly reduce the adhesion ability of the EDL933 strain to host epithelial cells.

[0088] Example 7

[0089] Female BALB / c mice aged 4-5 weeks were selected and acclimatized for one week in an SPF-grade environment. Three days prior to infection, streptomycin (50 μg / ml) was administered orally to clear the normal intestinal flora. EDL933 cells were resuspended and diluted with PBS to adjust the concentration to 1 × 10⁻⁶. 10 CFU / mL. After fasting for one day, the animals were randomly divided into groups of 13 each. Experimental groups:

[0090] Group 1: Control. Gavage with 0.2 mL of PBS to prevent bacterial infection.

[0091] Group 2: Infection control group: 0.2 mL of EDL933 bacterial solution was administered by gavage.

[0092] Group 3: Taurocholate treatment group: oral administration of sodium taurocholate (200 μg) + 0.2 mL EDL933 bacterial culture.

[0093] Group 4: Taurine deoxycholate treatment group: oral administration of sodium taurine deoxycholate (200 μg) + 0.2 mLEDL933 bacterial suspension.

[0094] Group 5: Control: Taurocholate (200 μg) administered orally, without bacterial infection.

[0095] Group 6: Control: Taurine deoxycholate (200 μg) administered orally, without bacterial infection.

[0096] After fasting for 12 hours, mice in the infection group were administered 0.2 mL of bacterial solution (i.e., an infection dose of approximately 2 × 10⁻⁶) via gavage. 9CFU / mouse). The control group was administered equal volumes of PBS, taurocholate, and taurodeoxycholate by gavage, respectively. Two hours after inoculation, the infection treatment group received the first gavage administration of taurocholate and taurodeoxycholate, respectively, followed by three consecutive days of administration. Three days post-infection, mice were sacrificed, and small intestinal tissue was harvested. Total RNA (containing RNA from colonizing bacteria) was extracted, and the expression of bacterial virulence genes was specifically detected by qPCR.

[0097] The results are as follows Figure 7 As shown, the relative expression levels of virulence genes (ler, espB, eae) in the EDL933 strain infection group remained at a high level. The expression levels of virulence genes in the taurocholate and taurodeoxycholic acid treatment groups were significantly lower than those in the EDL933-only infection group. These results indicate that taurocholate and taurodeoxycholate can effectively inhibit the expression of virulence genes in the EDL933 strain in mice.

[0098] Example 8

[0099] Following Example 7, after 3 days of treatment, mice were killed, and small intestinal tissue was collected, ground, and spread onto MacConkey agar plates containing kanamycin using a serially diluted sterile PBS solution. The plates were incubated overnight at 37°C, and the colony count (CFU / g) of strain EDL933 in the intestine was calculated.

[0100] The results are as follows Figure 8 As shown, the results indicated that no EHEC colonies appeared on kanamycin-containing sorbitol MacConkey agar plates after homogenization of small intestinal tissue from the uninoculated groups (Group 1, Group 5, and Group 6). The group infected with EDL933 alone had the highest colony count in the mouse intestine, and the colonies after treatment with taurocholate and taurodeoxycholate were significantly lower than those after EDL933 infection. These results indicate that taurocholate and taurodeoxycholate significantly inhibited the colonization ability of the EDL933 strain in the mouse small intestine.

[0101] Example 9

[0102] Following Example 7, the survival status of the mice was recorded daily, and survival curves were plotted.

[0103] The results are as follows Figure 9 As shown, all mice in Group 1 (blank control group 1), Group 5 (blank control group 2), and Group 6 (blank control group 3) survived without any symptoms (such as lethargy, loss of appetite, or diarrhea). The survival curves maintained a 100% survival rate, indicating that 200 μg of taurocholate and taurodeoxycholate are non-toxic to mice and do not affect their normal physiological state. Compared with EDL933 infection, the survival rates of mice treated with taurocholate and taurodeoxycholate were significantly improved.

[0104] Example 10

[0105] Following the small intestinal tissue of mice in Example 7, sections were stained and the pathological morphology of the small intestinal tissue was observed under a microscope to assess the degree of small intestinal damage in each group of mice.

[0106] The results are as follows Figure 10 As shown, small intestinal tissue sections from Group 1 (blank control group 1), Group 5 (blank control group 2), and Group 6 (blank control group 3) of mice revealed intact intestinal mucosal structure, neatly arranged and regularly shaped intestinal villi, exhibiting normal finger-like projections, uniform villi length, and tightly packed, intact villi epithelial cells without shedding or breakage. The lamina propria of the intestinal mucosa was clearly defined, with a very small number of white granular cells (mainly inflammatory cells, including neutrophils and macrophages) that were evenly distributed, showing no obvious inflammatory infiltration. This indicates that under normal physiological conditions and after simple bile salt treatment, the mouse small intestinal mucosa showed no damage and only mild inflammatory response. Infection with the EDL933 strain induced characteristic damage to the mouse small intestinal mucosa (shortening and breakage of intestinal villi and increased inflammatory cell infiltration), while treatment with taurocholate and taurodeoxycholate significantly alleviated the above-mentioned small intestinal tissue damage and reduced inflammatory cell infiltration.

[0107] 1. This invention provides the first systematic demonstration that taurocholate and taurodeoxycholate exert a potent "antiviral" effect at sub-inhibitory concentrations. This mechanism is expected to avoid the explosive release of Shiga toxin and drug resistance selection that may be induced by direct bactericidal action, suggesting that its safety may be superior to that of traditional antibiotics.

[0108] 2. Clear and comprehensive efficacy: From gene transcription, protein secretion, cell adhesion to colonization and pathogenicity in animals, it provides a multi-level and complete chain of evidence for efficacy, proving that it can fundamentally weaken the pathogenicity of bacteria.

[0109] 3. Promising prospects for transformation: Taurocholate and taurodeoxycholate are endogenous substances with high safety. They can be directly used to develop oral preventive drugs, feed additives or food preservatives for the prevention and control of EHEC infection, with clear application scenarios.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of taurocholate and / or taurodeoxycholate in the preparation of products for inhibiting the virulence of enterohemorrhagic Escherichia coli.

2. The application according to claim 1, characterized in that, The application involves using taurocholate and / or taurodeoxycholate at sub-inhibitory concentrations; and / or, the sub-inhibitory concentration is 0.1 µM – 1 µM.

3. The application according to claim 1 or 2, characterized in that, The inhibition of enterohemorrhagic Escherichia coli (EHEC) virulence includes any one or more of the following: (a) Inhibits the expression of virulence genes in enterohemorrhagic Escherichia coli; (b) Inhibit the expression and / or secretion of proteins associated with the secretion system of enterohemorrhagic Escherichia coli type III; (c) Inhibits the adhesion ability of enterohemorrhagic Escherichia coli to host cells; (d) Inhibits the colonization of enterohemorrhagic Escherichia coli in the host intestine; (e) Reduce host intestinal tissue damage caused by enterohemorrhagic Escherichia coli infection.

4. The application according to claim 3, characterized in that, The genes on the LEE virulence island include at least one of ler, espB, eae, escV, and sepZ.

5. The application according to claim 3, characterized in that, The type III secretory system-related proteins include EspB protein.

6. Use of taurocholate and / or taurodeoxycholate in the preparation of medicaments for the prevention and / or treatment of enterohemorrhagic Escherichia coli infection.

7. The application according to claim 6, characterized in that, The prevention and / or treatment of enterohemorrhagic Escherichia coli infection includes: (a) Reduce the mortality rate of infected individuals; (b) Reduce intestinal pathological damage in infected individuals; (c) Reduce the amount of bacteria colonizing the gut of infected individuals; (d) Inhibit the expression of bacterial virulence genes in the gut of infected individuals.

8. A composition for inhibiting the virulence of enterohemorrhagic Escherichia coli, characterized in that, The active ingredients of the composition include taurocholate and / or taurodeoxycholate.

9. The composition according to claim 8, characterized in that, The concentration of taurocholate and / or taurodeoxycholate in the composition is a sub-inhibitory concentration; and / or, the sub-inhibitory concentration is 0.1 µM-1 µM.

10. The composition according to claim 8 or 9, characterized in that, The composition is a pharmaceutical composition, a feed additive, or a food preservative; and / or, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, and the dosage form of the pharmaceutical composition is an oral formulation.

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