Application of gallic acid in preparation of salmonella typhimurium CDSH inhibitor

Abstract

The invention discloses an application of gallic acid in preparation of a salmonella typhimurium CDSH inhibitor, and belongs to the technical field of biological medicine. CDSH enzyme activity inhibition experiment, H2S detection experiment, antibiotic stimulation experiment and greater wax moth infection model experiment analysis finds that the gallic acid can reduce the stress tolerance of S.Typhimurium by inhibiting the activity of CDSH enzyme, reduce the stress tolerance of S.Typhimurium, reduce the stress tolerance of S.Typhimurium, reduce the stress tolerance of S.Typhimurium, and reduce the stress tolerance of S.Typhimurium by inhibiting the activity of CDSH enzyme. The cysteine thiotransferase has the advantages that the cysteine thiotransferase can be used as a transferase inhibitor, the antibiotic sensitivity and the in-vivo removal effect of the cysteine thiotransferase are enhanced, the gallic acid is used for inhibiting the enzyme activity of CDSH and reducing the salmonella tolerance, and the cysteine thiotransferase inhibitor and the medicine for treating the caused infectious diseases are prepared and are used for preventing or treating salmonella typhimurium infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and discloses the application of gallic acid in the preparation of CDSH inhibitors of Salmonella typhimurium, which relates to a new medical use of gallic acid. Background Technology

[0002] Salmonella typhi ( S Salmonella Typhimurium is a foodborne pathogen whose symptoms include diarrhea, fever, and abdominal cramps. Commonly used drugs such as ampicillin, tetracycline, and fluoroquinolones can produce drug-resistant strains of Salmonella, and the emergence of multidrug-resistant strains makes clinical management more complicated.

[0003] Hydrogen sulfide (H2S) is a known survival strategy for bacteria under antibiotic and oxidative stress; therefore, inhibiting bacterial H2S production is a promising antibacterial strategy. H2S possesses antioxidant properties, can enhance biofilm formation, and increase bacterial tolerance and survival during antibiotic treatment. S In Typhimurium, L-cysteine ​​degradation by cysteine-degrading enzyme CDSH is the main source of H2S. However, the importance of CDSH for stress tolerance and infection is not fully understood, and no inhibitors targeting CDSH have been reported to date.

[0004] Gallic acid (GA), with the molecular formula C7H6O5 and a molecular weight of 170.12, is a natural polyphenol compound widely found in plants. It mainly functions as a potent natural antioxidant, reducing oxidative stress and creating a more favorable microenvironment for the survival of probiotics. It also reduces oxidative damage to the cell membranes and DNA of probiotics, indirectly enhancing the overall stability and activity of encapsulated probiotics during processing, storage, and passage through the gastrointestinal tract. Summary of the Invention

[0005] The purpose of this invention is to disclose the application of gallic acid in the preparation of CDSH inhibitors for Salmonella Typhimurium, thereby solving the problem of antibiotic resistance in Salmonella Typhimurium.

[0006] The application of gallic acid in the preparation of CDSH inhibitors for Salmonella typhimurium described in this invention utilizes the characteristic of gallic acid to inhibit CDSH enzyme activity, thereby reducing the tolerance of Salmonella.

[0007] This invention further provides the application of gallic acid in the preparation of drugs for the prevention / treatment of Salmonella typhimurium infection, which reduces Salmonella tolerance by inhibiting CDSH enzyme activity, thereby preventing or treating Salmonella typhimurium infection.

[0008] The present invention also provides the use of gallic acid and gentamicin in combination in the preparation of drugs for the prevention / treatment of Salmonella typhimurium infection.

[0009] The application of gallic acid and gentamicin in combination as described in this invention in the preparation of drugs for the prevention / treatment of Salmonella typhimurium infection.

[0010] The Salmonella typhimurium used in the application described in this invention is specifically... S Typhimurium SL1344.

[0011] Gallic acid, as an active ingredient for inhibiting Salmonella typhimurium, is used in the preparation of pharmaceutical inhibitors. The carrier can be any pharmaceutically acceptable solvent, excipient, or formulation.

[0012] The positive effects of this invention are: it discloses a new medical use of gallic acid, which inhibits the enzyme activity of CDSH to reduce Salmonella tolerance, prepares inhibitors of cysteine ​​thiotransferase and drugs for infectious diseases caused by it, and prevents or treats Salmonella typhimurium infection.

[0013] This invention, through CDSH enzyme activity inhibition experiments, H2S detection experiments, antibiotic stimulation experiments, and experiments using a large wax moth infection model, discovered that gallic acid can reduce [the risk of disease / damage] by inhibiting CDSH enzyme activity. S Gallic acid can enhance the stress tolerance of Typhimurium and improve its sensitivity to antibiotics and its clearance in vivo, thereby enabling the preparation of CDSH inhibitors and subsequently, drugs for treating infectious diseases caused by Salmonella typhimurium. Attached Figure Description

[0014] Figure 1 This is a graph showing the inhibition of CDSH enzyme activity by gallic acid, provided in Example 1 of the present invention. Figure 2 The WT and Δ values ​​before and after gallic acid treatment provided in Example 2 of this invention cdsH Graph showing the determination of H2S content in the body; Figure 3 The WT and Δ values ​​before and after gallic acid treatment provided in Example 3 of this invention are... cdsH Quantitative graph of the number of bacteria surviving gentamicin antibiotic stimulation; Figure 4 The survival rate of the large wax moth infection model before and after treatment with gallic acid alone or in combination with gentamicin antibiotics, as provided in Example 4 of this invention; In the attached figures, statistical analysis was performed using GraphPad Prism 9.0. All experiments were independently repeated three times. Data are expressed as mean ± standard deviation (SD). The significance of differences between groups was assessed using one-way ANOVA, and the significance level was indicated by an asterisk (*) in the results. p < 0.05, ** p < 0.01). Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] The Salmonella typhimurium used in the embodiments of this invention is specifically... S Typhimurium SL1344 was a gift from Professor Qiu Jiazhang's research group at Jilin University.

[0017] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0018] Example 1: Assay on the inhibition of CDSH enzyme activity by gallic acid: by S Using Typhimurium SL1344 genomic DNA as a template, the sample was amplified by PCR. cdsH The full-length gene was inserted into the pCold-SUMO vector, successfully constructing a CDSH recombinant plasmid with a His tag at the N-terminus. This plasmid was then transformed into Origami 2(DE3) competent cells, and expression was induced at 20 °C and 180 rpm for 24 h using 0.1 mM IPTG. After induction, the cells were collected and resuspended in lysis buffer (containing 20 mM Tris-HCl, pH 8.5, 300 mM NaCl, and 5 mM β-mercaptoethanol). The cells were then disrupted by sonication, and the lysate was centrifuged at 12000 g to obtain the supernatant. This supernatant was then purified using a Ni-NTA affinity chromatography column and eluted with 250 mM imidazole. The eluent was collected, and the protein concentration was determined using Nanodrop. Purity was assessed by SDS-PAGE, and the eluent was aliquoted and stored at -80 °C for later use. Enzyme activity was measured in 96-well plates, with PbS production reflecting enzyme activity. A precise 200 μL reaction mixture was prepared, containing 50 μM PLP and 0.25 mg / mL purified CDSH protein. Different concentrations of gallic acid (0 μM, 50 μM, 100 μM, 200 μM, and 400 μM) were pre-incubated with CDSH enzyme and PLP buffer in PBS for 15 minutes. Subsequently, 10 mM L-cysteine ​​and 0.4 mM lead acetate were added to initiate the reaction. The reaction was carried out at 37 °C for 30 minutes. Enzyme activity was measured by PbS production. The absorbance was measured at 390 nm using a microplate reader, with the enzyme activity of the 0 μM gallic acid treatment group serving as a 100% control. The inhibition rate of each concentration group was calculated. The results are shown below. Figure 1 As shown in the figure, it can be seen that the CDSH enzyme activity is significantly inhibited with the increase of gallic acid concentration, which confirms that gallic acid can be used as an effective small molecule inhibitor of CDSH.

[0019] Example 2: Wild-type strain WT (S. Typhimurium SL1344) and Δ strain before and after gallic acid treatment cdsH Experiment to determine H2S content in vivo: Constructing CDSH knockout strains (Δ cdsH (Refer to the existing Red recombination system for knocking out Salmonella typhimurium) cdsH Genes, first designed and then PCR amplified, are characterized by having ends with [specific components]. cdsH A linear DNA fragment containing a kanamycin resistance marker, derived from upstream and downstream homologous sequences, was electroporated into competent bacteria induced to express Red recombinase. The recombinase mediated homologous recombination, causing the resistance marker to replace the one on the chromosome. cdsH The gene coding region was then identified. Transformants were screened using antibiotic resistance plates, and the correctness of the gene knockout was verified using PCR and sequencing. Finally, the gene was obtained... cdsH Mutant strains with missing genes.

[0020] The lead acetate test paper method was used to qualitatively detect H2S generated above the culture medium. The WT and Δ values ​​of overnight culture were then analyzed. cdsHThe cells were transferred at a low ratio of 1:100 to fresh M9 medium containing 0.5 mM L-cysteine, which provides sufficient substrate for H2S production. The inoculated bacterial culture was then cultured at 37 ℃ and 180 rpm with shaking. Experimental groups were prepared with 50 μM, 100 μM, 200 μM, and 400 μM gallic acid, while the control group received no gallic acid. The culture was continued for 3 h. To accurately quantify intracellular H2S levels, the WSP-5 fluorescent probe (Cayman, China) was used. Logarithmic growth phase cells were collected, and a final concentration of 10 μM WSP-5 fluorescent probe was added to the culture medium. The cells were incubated at 37 ℃ in the dark for 40 min to ensure sufficient probe entry into the cells and reaction with intracellular H2S. After incubation, the cells were washed twice with PBS to remove unreacted probe and background fluorescence from the culture medium. The cells were then resuspended in PBS and analyzed on a microplate reader at an excitation wavelength of 488 nm and a wavelength of 524 nm. Fluorescence quantification was performed at an emission wavelength of nm. The fluorescence intensity directly reflects the relative level of intracellular H2S, and the results are as follows: Figure 2 As shown, the intensity of intracellular WSP-5 fluorescence signal decreases with increasing drug concentration, demonstrating that gallic acid effectively inhibits CDSH-mediated hydrogen sulfide generation at the cellular level.

[0021] Example 3: The effect of combined treatment with gallic acid and gentamicin (GM) on... S Quantitative determination of viable bacterial count in Typhimurium SL1344: WT and Δ cdsH The overnight culture of the strain was diluted 1:20 in M9 medium containing 0.5 mM L-cysteine ​​and pre-cultured at 180 rpm and 37 °C with shaking for 3 h to induce the bacteria into the logarithmic growth phase. Experimental groups included: WT antibiotic alone (5×MIC GM), GM (5×MIC) combined with gallic acid treatment groups (50 μM, 100 μM, 200 μM, and 400 μM), and Δ... cdsH The antibiotic-treated strain served as a control. Gallic acid-pretreated bacteria in the logarithmic growth phase were added to a predetermined concentration of antibiotic and incubated at 37 °C for 3 h. After incubation, the bacterial culture was centrifuged at 12000 rpm, washed twice with PBS to remove residual drug, and then serially diluted (e.g., 10⁻⁶). -1 Up to 10 -6 ), and 100 μL was seeded onto an LB agar plate. After incubating the plate overnight at 37 °C, the number of surviving colonies (CFU / mL) was accurately counted, and the results are as follows. Figure 3As shown in the figure, in the antibiotic stimulation experiment, for 5×MIC GM treatment, the number of surviving colonies in the combined gallic acid and antibiotic treatment group was significantly lower than that in the antibiotic-only treatment group after 3 h, and its killing effect was related to Δ cdsH The similarity between the missing strains strongly suggests that gallic acid can enhance the killing ability of GM against Salmonella typhimurium.

[0022] Example 4: Experimental analysis of the effect of gallic acid on GM treatment in a Salmonella typhimurium infection model of the large wax moth: A *Gnaphalium affine* infection model (purchased from Tianjin Huiyude Biotechnology Co., Ltd.) was established according to standard procedures for in vivo survival experiments. Bacterial cells in the logarithmic growth phase were collected and then washed twice with sterile PBS. The infection dose was precisely controlled at 1×10⁻⁶. 5 An infection model was established by injecting the bacterial suspension into the second abdominal leg of each larva using a microsyringe at a dose of CFU / larva. After infection, larvae were randomly assigned to four groups for treatment: GM monotherapy (4 mg / kg GM), solvent control (PBS), gallic acid monotherapy (8 mg / kg gallic acid), and gallic acid combined with GM (8 mg / kg gallic acid + 4 mg / kg GM). Survival rates under different treatments were compared. All treatment groups were incubated at 37 ℃, and larval survival was precisely recorded every 6 h. Kaplan-Meier survival curves were plotted. The results are shown below. Figure 4 As shown, the combined treatment group of gallic acid and GM exhibited the best efficacy, with a significant increase in larval survival rate, demonstrating that gallic acid combined with GM has significant synergistic therapeutic effects in the in vivo model.

[0023] In summary, the embodiments of the present invention demonstrate that gallic acid can reduce Salmonella tolerance by inhibiting CDSH enzyme activity, providing a new approach and potential lead compound for the prevention and treatment of Salmonella infection, and verifying that gallic acid can have a synergistic therapeutic effect when used in combination with gentamicin. Conclusion: Gallic acid can be used as a CDSH inhibitor in any pharmaceutically acceptable carrier. Gallic acid can be used as a CDSH inhibitor in the preparation of drugs for treating infectious diseases caused by Salmonella typhimurium. Gallic acid can be used in combination with gentamicin to prepare drugs for the prevention / treatment of infectious diseases caused by Salmonella typhimurium.

[0024] 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 gallic acid for the preparation of a Salmonella typhimurium CDSH inhibitor, characterized in that: The application discloses a method for preparing cysteine sulfotransferase inhibitor by inhibiting the enzyme activity of CDSH with gallic acid to reduce the tolerance of salmonella.

2. Use according to claim 1, characterized in that: Application of gallic acid and gentamicin in combination in preparation of a medicine for preventing / treating salmonella typhimurium infection.

3. Use according to claim 1, characterized in that: Salmonella typhimurium is in particular S . Typhimurium SL1344.

4. The use according to claim 2, characterized in that: Gallic acid and gentamicin in combination are used as active ingredients for inhibiting salmonella typhimurium, and are used for preparing a medicine inhibitor, and the carrier can be any solvent, excipient or preparation form acceptable in pharmacy.

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