Application of indole in preparation of helicobacter pylori inhibitor

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of indole in preparation of a helicobacter pylori inhibitor. Experiments show that indole can effectively inhibit the growth of helicobacter pylori, can cause the negative chemotactic response of helicobacter pylori through a chemotactic receptor TlpA, can inhibit the formation of a helicobacter pylori biofilm, and is expected to be used as a precursor to be developed into a new drug for resisting the helicobacter pylori.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of indole in the preparation of Helicobacter pylori inhibitors. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Helicobacter pylori infection is one of the most common infectious diseases of the human digestive system worldwide. Curing Helicobacter pylori infection is very difficult, and the currently used treatment method is triple or quadruple antibiotic therapy, which has led to the emergence of a large number of multidrug-resistant strains. Therefore, studying the pathogenic mechanism of Helicobacter pylori and developing novel, specific antibiotic alternatives to reduce antibiotic use and curb bacterial resistance are urgent and important issues that need to be addressed. Summary of the Invention

[0004] Through experiments, this invention has shown that indole can effectively inhibit the growth of Helicobacter pylori, induce a negative chemotactic response in Helicobacter pylori through the chemotactic receptor TlpA, and inhibit the formation of biofilms of Helicobacter pylori. It is expected to be developed as a precursor into a new anti-antibiotic drug for this bacterium.

[0005] Based on the above research findings, this invention proposes the application of indole in the preparation of Helicobacter pylori inhibitors.

[0006] Specifically, the present invention provides the following technical solution:

[0007] This invention provides an application of indole in the preparation of Helicobacter pylori inhibitors.

[0008] The inhibitors described in this invention can be drugs intended to treat diseases, or experimental reagents for use in basic research.

[0009] This invention also provides the application of combinations of indole and any active ingredient in the preparation of Helicobacter pylori inhibitors. This invention does not limit the active ingredient or its ratio in the combination; the application of any combination of active ingredient and indole in the preparation of Helicobacter pylori inhibitors is within the scope of protection of this invention. Examples of active ingredients include amoxicillin, clarithromycin, furazolidone, metronidazole, tinidazole, and levofloxacin.

[0010] This invention also provides the application of indole, or a combination of indole and any active ingredient, with pharmaceutical excipients in the preparation of Helicobacter pylori inhibitors. This invention does not limit the proportions of the active ingredient or pharmaceutical excipients in the combination; the application of any combination of active ingredient, pharmaceutical excipients, and indole in the preparation of Helicobacter pylori inhibitors is within the scope of protection of this invention.

[0011] In some embodiments, the pharmaceutical excipient includes a pharmaceutical carrier. Examples of such pharmaceutical carriers include alumina, aluminum stearate, glycerin, polyethylene glycol, beeswax, lanolin, etc.

[0012] In some embodiments, the pharmaceutical excipient includes a formulation agent. Examples of formulation agents include binders, fillers, preservatives, lubricants, disintegrants, etc.

[0013] In some implementations, indole reduces the formation of Helicobacter pylori biofilms by directly binding to TlpA.

[0014] In some implementations, indole reduces the initial attachment of Helicobacter pylori to inhibit the formation of mature biofilms.

[0015] In some embodiments, the indole concentration is 300 μM to 1 mM. In this invention, M represents mol / L.

[0016] In some implementations, the Helicobacter pylori inhibitor is administered orally, by spray inhalation, rectally, or by intraperitoneal instillation.

[0017] In some embodiments, the Helicobacter pylori inhibitor is in the form of a liquid or a solid dosage form. The liquid dosage form may be a suspension, emulsion, solution, etc. The solid dosage form may be a capsule, tablet, pellet, or suppository.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention demonstrates that indole is a ligand capable of directly binding to TlpA with a strong binding affinity. TlpA mediates the negative chemotactic response of Helicobacter pylori to indole, causing wild-type Helicobacter pylori to migrate downwards along the indole concentration gradient. Further research shows that indole begins to inhibit the growth of Helicobacter pylori at 300 μM and completely inhibits its growth at 1 mM. Simultaneously, TlpA-mediated indole negative chemotaxis reduces the initial attachment of Helicobacter pylori to surfaces, thereby inhibiting biofilm formation. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The following is an identification diagram of indole as a ligand for TlpA-LBD in the embodiments of the present invention; (A) ITC analysis of indole binding to TlpA-LBD; Top: raw titration data, "a" represents the indole titration buffer, and "b" represents the indole titration of TlpA-LBD; Bottom: raw data after dilution heat correction and concentration normalization and fitting of the single ligand binding to macromolecule model; the concentration of TlpA-LBD is 203 μM, and the concentration of indole is 4 mM; the curve was obtained by fitting the "One binding site model" of Malvern MicroCal PEAQ ITC analysis software; (B) Molecular docking analysis of the interaction between TlpA-LBD and indole. The left image shows the three-dimensional structure of the TlpA-LBD monomer, which is a dcache domain. The top pocket is the distal membrane cache subdomain, and the bottom pocket is the proximal membrane cache subdomain. The right image is a magnified schematic diagram of the binding of indole to the proximal membrane pocket of TlpA-LBD. The key residues involved in indole binding in the ligand-binding pocket (Phe203, Tyr228, Tyr252, Val238, Ile287) are shown as magenta rod-like structures. Hydrogen bonds are shown as yellow dashed lines; LBD is the ligand-binding domain.

[0022] Figure 2This is a microfluidic analysis diagram of the indole chemotactic response of Helicobacter pylori in an embodiment of the present invention; (A) Left side: Schematic diagram of the overall microfluidic device; Right side: Top and side views of the observation channel and agarose gel channel; Each chip contains 24 parallel units, each unit contains one source well (Sink) and one compound source well (Source); The source well and the compound source well are connected by 5 agarose channels (150 μm long, 20 μm wide, and 5 μm high) and 1 observation channel (600 μm long, 200 μm wide, and 18 μm high); The analytical area (150 × 200 μm) in the observation channel is represented by an orange rectangle; Before use, the agarose channels are blocked with agarose to prevent bacterial convection, but the compound can pass through the agarose and enter the observation channel to form a concentration gradient; (BE) Helicobacter pylori WT (B), ΔtlpA(C), CtlpA(D), and ΔcheA(E) chemotactic responses to different concentrations of indole; data show the relative chemotactic intensity in the analytical region of the channel 50 minutes after adding the indicated concentration of ligand (indole or L-lactic acid) or buffer (negative control without ligand); the corresponding chemotactic intensity in the analytical region of the ligand (indole or L-lactic acid) was normalized to the chemotactic intensity in the buffer to obtain the relative chemotactic intensity; error bars represent the standard error of three independent replicates, shown as mean ± SD; p-values ​​were calculated using paired t-tests: *p < 0.05, **p < 0.01, ***p < 0.001; (FN) before adding the compound to the well (t = 0 min) and after adding buffer, 15 mM, etc. Example of Helicobacter pylori cell distribution (WT, G, H), ΔtlpA (I, J, K), and CtlpA (L, M, N) after 50 min with L-lactic acid or 10 mM indole (scale bar: 50 μm); the x-axis (black arrow) indicates the direction of increasing indole concentration gradient. The chemotactic strength of the bacteria towards the compound (buffer, L-lactic acid, indole) is obtained by dividing the number of bacterial cells in the analysis area (150 × 200 μm) shown in (FN) by the number of bacterial cells before the addition of the ligand 50 min at the source well.

[0023] Figure 3 This is a graph showing the results of the Koch reagent detection of whether Helicobacter pylori produces indole in an embodiment of the present invention; Left tube: negative control group, Koch reagent was added to fresh BB10 medium; Middle tube: positive control group, overnight culture of Escherichia coli in LB medium, the color turned red after the addition of Koch reagent, consistent with the fact that E. coli produces indole; Right tube: Helicobacter pylori experimental group; there was no color change after the addition of Koch reagent to the Helicobacter pylori culture inoculated in BB10 medium, indicating that Helicobacter pylori does not metabolize tryptophan to produce indole; both LB medium and BB10 medium contain 1% peptone, which is rich in tryptophan.

[0024] Figure 4 The graph shows the effect of indole on the growth of Helicobacter pylori in this embodiment of the invention; the growth curve of Helicobacter pylori in the presence or absence of indole; indole begins to inhibit the growth of Helicobacter pylori at 300 μM and completely inhibits growth at 1 mM; the error bars represent the standard errors of three independent replicates, shown as mean ± standard deviation.

[0025] Figure 5 The diagram shows the effect of TlpA-mediated indole chemotaxis on Helicobacter pylori biofilm formation in this embodiment of the invention; (A) The percentage of adhering cells of Helicobacter pylori WT, ΔtlpA, and CtlpA after 72 hours of incubation with different concentrations of indole was determined by crystal violet staining; the percentage of adhering cells in the biofilm was calculated by dividing the absorbance of the adhering cell population by the sum of the absorbance of adhering cells and planktonic cells, and multiplying by 100%; the error bars represent the standard errors of 4 replicates, shown as mean ± standard deviation; (B) The difference in the percentage of adhering cells in the biofilm after 24 hours of adding different concentrations of indole compared with the control group without indole; the box plot shows the mean and standard deviation of at least 4 experimental replicates. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example

[0028] 1. Heterologous expression and purification of the periplasmic ligand-binding domain of TlpA (TlpA-LBD)

[0029] The pET28b plasmid was digested with NdeI and BamHI restriction endonucleases, and the DNA sequence encoding the chemokine receptor TlpA-LBD (amino acids 28-299) was introduced between the NdeI and BamHI sites of the pET28b plasmid via GB05 dir-mediated linear homologous recombination (LLHR). A stop codon was introduced after amino acid 299 in primer design to obtain a plasmid containing the DNA sequence encoding the TlpA-LBD recombinant protein with an N-His tag. The correctly sequenced plasmid was electroporated into BL21(DE3) cells, which were then cultured in 5L Erlenmeyer flasks containing 1L Luria Bertani (LB, 1% tryptone, 0.5% yeast extract, 0.5% NaCl) medium supplemented with 30 μg mL of lysate. -1 Kanamycin, temperature 37℃, rotation speed 200 rpm. When OD 600When the protein concentration reached 0.6-0.8, isopropyl β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM was added to the culture to induce protein expression, and the culture was carried out at 18°C ​​and 110 rpm for 12 hours. Cells were harvested by centrifugation at 6000 × g at 4°C. The collected cells were resuspended in PBS and centrifuged again at 6000 × g at 4°C. The cells were then resuspended in buffer A (5.8 g Na₂HPO₄, 0.59 g NaH₂PO₄, 0.5 M NaCl, pH 6.8), and then the cells were lysed using a high-pressure homogenizer. The lysed cells in buffer A were centrifuged at 35328 × g at 4°C for 1 h to remove undiluted cells and cell debris. After exporting the supernatant, the sample was filtered through a 0.4 μm aqueous filter and then loaded onto a 5 mL HisTrap column (GE Healthcare, USA) pre-equilibrated with buffer A using a peristaltic pump. At this point, the recombinant protein is bound to nickel ions in the HisTrap column via its N-terminal His tag. The loaded HisTrap column was washed with 10% elution buffer B (5.8 g Na₂HPO₄, 0.59 g NaH₂PO₄, 0.5 M NaCl, and 0.5 M imidazole, pH 6.8) to remove contaminating proteins. The target protein TlpA-LBD was eluted with 100% buffer B, and the protein was concentrated using a 10 kDa-cutoff centrifuge filter. The concentrated protein was then loaded onto a 5 mL desalting column (GE Healthcare, USA) pre-equilibrated with desalting buffer (25 mM Na₂HPO₄, 25 mM NaH₂PO₄, 150 mM NaCl, pH 6.8) to remove imidazole. To achieve the best imidazole removal effect for proteins, the maximum loading volume of a 5mL desalting column should not exceed 1.5mL per loading.

[0030] 2. ITC determination of the binding of TlpA-LBD to indole

[0031] The ITC experiment was performed on a PEAQ-ITC system (Malvern Instruments, UK) with a titration temperature of 25°C and a total of 19 drops. The first drop was 0.4 μL, and the remaining 18 drops were each 2 μL. Both the protein TlpA-LBD and the compound were dissolved in a desalting buffer, and the pH of the compound was adjusted to 6.8 to match the pH of the protein. In the experimental group, 203 μM TlpA-LBD was titrated with 4 mM indole. The 203 μM TlpA-LBD protein was placed in the sample cell, and the 4 mM indole solution was placed in the titration needle. In the control group, the desalting buffer was titrated with 4 mM indole. Data were fitted to a unit point binding model using MicroCal PEAQ-ITC analysis software (Malvern Instruments).

[0032] 3. Molecular docking prediction

[0033] To evaluate potential sites for indole binding to TlpA-LBD (PDB code: 6E09), molecular docking was performed using AutoDockTools-1.5.6. TlpA-LBD is a dCache domain. Since it was unclear which subdomain of TlpA-LBD was involved in indole recognition, indole was docked with both the distal and proximal subdomains of the TlpA-LBD membrane during molecular docking. The docking results showed that indole could bind well into the ligand-binding pocket of the proximal cache domain, but not into the distal cache domain. The binding free energy of each conformation was calculated to obtain the minimum binding energy and the optimal docking conformation. The optimal docking results were displayed using PyMOL 2.4.1 software (http: / / www.pymol.org).

[0034] 4. Helicobacter pylori chemotaxis assay

[0035] The culture media used in this step are: (1) Brain heart and heart extract agar plate solid culture medium: 5% brain heart and heart extract agar medium, sterilized after adjusting pH to 7.2, and then added with 7% sterile defibrinated sheep blood when cooled to about 50℃; (2) Liquid culture medium BB10: Brucella broth (BB) medium after heat extinguishing, and prepared by adding 10% fetal bovine serum before use. Each 100mL of BB medium contains 2.2g brain heart and heart extract liquid culture medium powder, 1g tryptone, 0.2g yeast powder, and is sterilized after adjusting pH to 7.2.

[0036] Helicobacter pylori 7.13 cells, stored at -80℃, were plated in 200 μL onto BHI blood agar plates and incubated at 37℃ in a microaerophilic environment (5% O2, 10% CO2, 85% N2) for 24 hours. After initial activation, the cells were transferred back onto blood agar plates and incubated under the same conditions for another 24 hours, at which point the Helicobacter pylori was fully activated. The fully activated Helicobacter pylori 7.13 cells were then seeded into 100 mL anaerobic flasks containing 20 mL of fresh BB10. OD 600 The concentration was 0.025. The anaerobic flask was filled with microaerophilic gas through gas replacement, and the culture was carried out at 37°C and 100 rpm until the OD value was reached. 600 The OD value was set to 0.15-0.20. Cells were harvested by centrifugation at 850×g for 3 minutes and washed twice with PBS. Finally, the cells were resuspended in PBS to allow the OD value to reach 0.15-0.20. 600 To achieve a concentration of 1.2-2, the microfluidic device requires pre-filling the agarose channels with 4% agarose to prevent bacterial convection. Cells are then loaded into the side orifices of the microfluidic device's water tank. Figure 2 A) Cells were equilibrated for one hour to allow them to migrate into the observation channels. The ligand solution was added to the source-side wells and allowed to diffuse into the observation channels within a specified time to establish a concentration gradient. Phase-contrast microscopy (Ti-E, Nikon, Japan) was used to record photographs of the observation channels before and 50 minutes after ligand addition in the source wells. Finally, chemotaxis was characterized by counting the number of cells in the analysis area (150 × 200 μM) of the observation channels before and after ligand addition. The chemotaxis intensity was obtained by dividing the number of cells in the analysis area 50 minutes after ligand addition by the number of cells before ligand addition. For each channel, 100–400 cells were counted to characterize the response. Relative chemotaxis intensity was obtained by normalizing the chemotaxis intensity in the ligand to the chemotaxis intensity in the buffer.

[0037] 5. Construction of Helicobacter pylori ΔtlpA, CtlpA and ΔcheA mutant strains

[0038] Preparation of competent Helicobacter pylori cells: Fully activated Helicobacter pylori cells were collected and placed in a pre-chilled sterile competent cell preparation buffer (15% glycerol and 9% sucrose). After incubating on ice for 10 min, the cells were centrifuged at 6000 rpm at 4°C to collect the cells. The cells were resuspended in the competent cell preparation buffer again. This process was repeated twice, and the cells were then collected and resuspended in the competent cell preparation buffer to allow OD... 600 The value is 2. The entire preparation of competent cells was carried out at 4 degrees Celsius or in an ice bath.

[0039] The Helicobacter pylori 7.13 mutant strains ΔtlpA and ΔcheA were generated by replacing the target gene with a fragment. This was achieved using the suicide plasmid pBJ114 (see DUAN J, ZHAO Q, WANG Y, et al. The dCache domain of the chemoreceptor Tlp1 in Campylobacter jejuni binds and triggers).

[0040] (chemotaxis toward formate[J].mBio,2023,e0356422.) was used to construct a vector to knock out tlpA or cheA. A linear fragment tlpAup-kan-tlpAdown containing an 800bp upper flanking homologous sequence of tlpA, the kanamycin resistance gene, and an 800bp lower flanking homologous sequence of tlpA was ligated using overlap PCR.

[0041] The linear fragment cheAup-kan-cheAdown includes an 800bp upper flanking homologous sequence of cheA, a kanamycin resistance gene, and an 800bp lower flanking homologous sequence of cheA. The pBJ114 plasmid was digested with BamHI and XbaI, and the fragment tlpAup-kan-tlpAdown or cheAup-kan-cheAdown was introduced into the BamHI and XbaI sites of pBJ114 via GB05-mediated LLHR, generating plasmids pBJ114-ΔtlpA or PBJ114-ΔcheA. PCR screening and sequencing were used to verify the inserted sequence in the transformants. Using PBJ114-ΔtlpA or PBJ114-ΔcheA as a template, the target fragment tlpAup-kan-tlpAdown or cheAup-kan-cheAdown was amplified by PCR and recovered via gel electrophoresis. 1 μL of the target fragment was mixed with 25 μL of competent cells and placed in a Bio-Rad 0.1 cm electroporation cuvette. The fragment was then electroporated using the EC1 program on the Bio-Rad electroporator to introduce the target fragment into competent Helicobacter pylori 7.13 cells. The mixture was then treated with 1% β-cyclodextrin and 15 μg / mL... -1 Mutants with the tlpA or cheA endogenous gene replaced by a kanamycin resistance gene were screened from Brucella agar medium containing kanamycin. Single colonies of Helicobacter pylori ΔtlpA and Helicobacter pylori ΔcheA were verified using PCR and DNA sequencing.

[0042] The *Helicobacter pylori* 7.13tlpA complement strain CtlpA was generated by replacing the *kan* gene of *Helicobacter pylori* ΔtlpA with a target fragment. The target fragment replacing *kan* was constructed using the suicide plasmid pK18mob as a vector. A linear fragment tlpAup-tlpA-cm-tlpAdown containing an 800 bp upper flanking homologous sequence of tlpA, tlpA, the chloramphenicol resistance gene, and an 800 bp lower flanking homologous sequence of tlpA was ligated using overlap polymerase chain reaction. The pK18mob plasmid was digested with HindIII and XbaI, and the fragment was then ligated using GB05 dir-mediated LLHR.

[0043] The tlpAup-tlpA-cm-tlpAdown plasmid is ligated into pK18mob to generate the plasmid pK18mob-CtlpA. Using the correctly sequenced pK18mob-CtlpA as a template, the target fragment is amplified by PCR.

[0044] tlpAup-tlpA-cm-tlpAdown was introduced into Helicobacter pylori 7.13 cells via electroporation. The solution was supplemented with 1% β-cyclodextrin and 15 μg / mL. -1 On Brucella agar plates containing chloramphenicol, colonies that match the tlpA and chloramphenicol resistance gene substitution of the kan gene in strain ΔtlpA were identified as complement strain CtlpA, and verified by DNA sequencing.

[0045] 6. Indole test for Helicobacter pylori

[0046] Fully activated Helicobacter pylori 7.13 cells were seeded into 100 mL anaerobic flasks containing 20 mL of fresh BB10. The initial inoculation OD 600 The concentration was 0.025. The anaerobic flask was filled with microaerophilic gas through gas replacement, and the culture was carried out at 37°C and 100 rpm until the OD value reached 0.025. 600 The concentration reached 0.5. Add three drops of Kovacs reagent (Kovacs Indole Reagent Kit) to 1 mL of culture and observe whether the liquid surface turns red. The ether in the reagent extracts indole to the surface, and the indole reacts with p-dimethylaminobenzaldehyde in the reagent to form rose indole, which turns the liquid surface red. Use BB10 medium as a negative control and overnight E. coli LB culture as a positive control. Both LB and BB10 contain 1% tryptone, which enriches the medium with tryptophan for indole production.

[0047] 7. Helicobacter pylori growth curve determination

[0048] Fully activated Helicobacter pylori 7.13 cells were seeded into 100 mL anaerobic flasks containing 25 mL of BB10 with different concentrations of indole, and inoculated with initial OD. 600The concentration was 0.05. The anaerobic flask was filled with microaerophilic gas through gas replacement, and the culture was carried out at 37°C and 100 rpm. The OD values ​​were measured at different time points. 600 Each indole concentration contains three replicates. A 10mM indole concentrate prepared with BB10 was filtered through a sterile 0.22μm aqueous filter to remove bacteria, and then mixed with BB10 to prepare culture media with different indole concentrations (10μM, 30μM, 100μM, 30μM, 1mM, 4mM).

[0049] 8. Helicobacter pylori biofilm assay

[0050] Biofilm formation typically involves several stages, including surface attachment, proliferation, maturation, and dispersion. The entire biofilm structure is the result of dynamic interactions between cells transitioning between adherent and planktonic states. Therefore, the percentage of adherent cells in the total cell population can be considered as the percentage of the biofilm, thus quantifying biofilm formation. Helicobacter pylori WT, ΔtlpA, and CtlpA cells were seeded in fresh BB10 or BB10 with different concentrations of indole, with initial OD... 600 The concentration was 0.1. 1 mL of culture, with or without indole, was added to a 12-well suspension plate containing 2 g of autoclaved glass fragments, which increases the surface area for cell attachment. The 12-well plates containing the culture were statically incubated at 37°C under microaerophilic conditions for 72 hours. To quantify bacteria in the biofilm and planktonic community, 1 mL of 0.2% crystal violet dye was added to each well, gently mixed, and incubated for 20 minutes. The liquid was then carefully aspirated and centrifuged at 35328×g for 1 minute to collect unattached planktonic cells. Adhesive bacteria attached to the wells and glass fragments were washed three times with 1 mL PBS to remove excess dye. Each wash volume was collected and stored as planktonic bacteria. After washing and drying, 1 mL of 90% ethanol was added to each well and the planktonic bacteria collection tube for decolorization to extract crystal violet from the cells of both the adhesive and planktonic populations. The absorbance of the supernatant was measured at 590 nm. The percentage of cells in a biofilm is calculated by dividing the absorbance of the adherent population by the absorbance of the combined absorbance of the adherent and planktonic populations, multiplied by 100.

[0051] result

[0052] 1. Indole is a ligand molecule that Helicobacter pylori chemotactic receptor TlpA directly binds to.

[0053] The periplasmic ligand-binding domain of receptor TlpA (TlpA-LBD) was heterologously expressed and purified in E. coli. Isothermal titration calorimetry (ITC) analysis showed that indole binds to TlpA-LBD in a 1:1 stoichiometric ratio (n = 0.84), with a dissociation constant Kd of 6 μM. Figure 1A). Therefore, it can be determined that indole is a ligand capable of directly binding to TlpA, exhibiting a strong binding ability. Molecular docking and binding experiments verified that indole binds to the pocket in the proximal subdomain of the TlpA-LBD membrane, and specifically binds to residues Phe203, Tyr228, and Tyr252 within the pocket. And Val238 and Ile287 (distance in) Hydrophobic interactions are formed between them, and their imino groups also form hydrogen bonds with Tyr252. Figure 1 B).

[0054] 2. TlpA mediates the negative chemotactic response of Helicobacter pylori to indole.

[0055] use Figure 2 The microfluidic device shown in Figure A measured the response of Helicobacter pylori strain 7.13 to indole. Helicobacter pylori cells were loaded into the source wells of the device, and the bacteria gradually migrated into the observation channel. After 40 minutes of free diffusion, a compound solution (indole or lactic acid) was added to the source wells of the microfluidic chip and gradually diffused into the observation channel, creating a concentration gradient. If the compound was an attractant, the bacterial cells sensed the attractant gradient, and more bacteria entered the observation channel from the source wells and accumulated towards the source of the compound wells. If the compound was a dispersant, the bacteria migrated from the observation channel to the source wells, thus reducing the number of cells in the observation channel. L-lactic acid, a previously reported Helicobacter pylori attractant, was used as a positive control for measuring the chemotactic response, and a buffer was used as a negative control. Wild-type (WT) Helicobacter pylori cells migrated in the observation channel along the direction of increasing L-lactic acid concentration, and the number of cells accumulated in the observation channel increased over time, indicating that the bacterium has a positive chemotactic response to L-lactic acid. Figure 2 G). Furthermore, the cell density in the buffer remains almost constant ( Figure 2 F). However, WT cells exhibited a strong repulsive response to indole, migrating down the indole concentration gradient and leaving the observation channel to enter the source well, resulting in a significant reduction in the number of cells in the observation channel (F). Figure 2 H). This negative chemotactic response is concentration-dependent (H). Figure 2 B) indicates that indole is a novel repellent for Helicobacter pylori.

[0056] To investigate whether the negative chemotactic response of Helicobacter pylori to indole is mediated by TlpA, the inventors measured the responses of Helicobacter pylori tlpA gene knockout strains (ΔtlpA) and tlpA gene complement strains (CtlpA). The ΔtlpA mutant still possesses general chemotactic ability and can produce a significant positive chemotactic response to the positive control L-lactic acid. Figure 2 C,J), but at the tested concentration, it did not show a chemotactic response to indole ( Figure 2C,K). Conversely, the complementation of the tlpA gene (CtlpA) restored the rejection response of the ΔtlpA mutant to indole to the WT level ( Figure 2 D,N), indicating that indole specificity is exerted through TlpA. As a control, the chemotactic defective mutant ΔcheA cannot produce a chemotactic response to L-lactic acid or indole. Figure 2 E) indicates that the response to indole is mediated by the chemotactic system. Therefore, the negative chemotactic response of Helicobacter pylori to indole depends on the chemotactic receptor TlpA of the chemotactic system.

[0057] 3. Indole inhibits the growth of Helicobacter pylori.

[0058] To investigate the physiological significance of Helicobacter pylori's negative chemotactic indole, the effect of indole on the growth of Helicobacter pylori was first studied. The Helicobacter pylori genome does not encode tryptophanase, therefore it cannot produce indole, a fact also confirmed by Koch's reagent analysis. Figure 3 The growth curves of Helicobacter pylori 7.13 were determined in culture media with different concentrations of indole. The results showed that indole began to inhibit the growth of Helicobacter pylori at 300 μM and completely inhibited cell growth at 1 mM. Figure 4 This matches the range of indole concentrations that trigger a negative chemotactic response. Therefore, TlpA-mediated indole chemotactic repulsion may also be related to Helicobacter pylori colonization. Indole is produced by the microbial community, and its concentration in the gastrointestinal tract varies widely, reaching up to 6.5 mM. As a non-indole-producing bacterium, Helicobacter pylori may be repelled and inhibited by indole-rich areas. Therefore, the negative chemotaxis of Helicobacter pylori towards indole may be related to its escape from indole attack. Thus, the inhibition of Helicobacter pylori growth by indole may be expected to be used to resist infection of the host.

[0059] 4. TlpA-mediated indole negative chemotaxis reduces the initial attachment of Helicobacter pylori to surfaces, thereby inhibiting biofilm formation.

[0060] Biofilm formation typically involves several stages, including surface attachment, proliferation, maturation, and dispersion. The overall biofilm structure is the result of dynamic interactions between the cells' adhering and planktonic states. Therefore, the percentage of adherent cells in the total cell population can be considered as the percentage of the biofilm, thus quantifying biofilm formation. Adding indole to WT Helicobacter pylori 7.13 medium significantly reduced the percentage of adherent cells in the total cell population after 72 hours, indicating that indole inhibits the formation of mature biofilms in Helicobacter pylori. Figure 5 A). This inhibitory effect begins with 10 μM indole and is dose-dependent. However, the inhibition of biofilm formation observed in WT cells was completely eliminated in the ΔtlpA mutant, but was completely restored in the tlpA gene complementation strain CtlpA. Figure 5A) indicates that indole reduces the formation of Helicobacter pylori biofilms by directly binding to TlpA.

[0061] The initial adhesion of *Helicobacter pylori* in WT 7.13 medium after static incubation with indole for 24 h was further measured. The results showed that the percentage of initially adherent cells in *Helicobacter pylori* WT was significantly reduced after the addition of indole. Figure 5 B). Compared with the control group without indole, the percentage of initial adherent cells decreased by 12% after the addition of 300 μM indole. Consistent with its inhibitory effect on mature biofilms, the inhibitory effect of indole on surface adhesion was almost eliminated in ΔtlpA, but recovered in CtlpA. Figure 5 B). These results suggest that the repulsion response of indole to TlpA may reduce initial attachment of Helicobacter pylori, thereby inhibiting the formation of mature biofilms.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of an indole for the preparation of an inhibitor of Helicobacter pylori.

2. Use of a combination of an indole and any active ingredient for the preparation of an inhibitor of Helicobacter pylori.

3. Use of a composition of an indole or a combination of an indole and any active ingredient, together with a pharmaceutically acceptable adjuvant, for the preparation of an inhibitor of Helicobacter pylori.

4. The use according to claim 3, wherein the compound is ###0002### The pharmaceutically acceptable adjuvant comprises a pharmaceutically acceptable carrier.

5. The use according to claim 3, wherein the compound is ###0002### The pharmaceutically acceptable adjuvant comprises an excipient.

6. The use according to any one of claims 1 to 3, characterized in that, The indole reduces the formation of Helicobacter pylori biofilm by directly binding to TlpA.

7. The use according to any one of claims 1 to 3, wherein the compound is ###0002### The indole reduces the initial attachment of Helicobacter pylori to inhibit the formation of mature biofilm.

8. The use according to any one of claims 1 to 3, characterized in that, The indole is administered at a concentration of 300 μM to 1 mM.

9. The application as described in any one of claims 1 to 3, characterized in that, The inhibitor of Helicobacter pylori is administered orally, by spray inhalation, rectally, or by intraperitoneal perfusion.

10. The use according to any one of claims 1 to 3, wherein the compound is ###00002### The inhibitor of Helicobacter pylori is in a liquid dosage form or a solid dosage form.

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