Target identification of anti-mycobacterium tuberculosis and application thereof

Abstract

The invention provides a pharmaceutical composition for preventing and / or treating diseases caused by mycobacterium tuberculosis infection. The pharmaceutical composition contains a substance for inhibiting the activity of Rv2521 protein or a substance for reducing the content of the Rv2521 protein. According to the invention, an Rv2521 large-fragment knockout strain delta Rv2521 is constructed, and a complement strain delta Rv2521:: 2521 is further constructed, compared with cells infected with delta Rv2521, the death rate of the cells infected with the WT or delta Rv2521: 2521 strain is obviously increased, and the intracellular LPO (marker of ferroptosis) level displayed by the cells infected with the WT or delta Rv2521: 2521 strain is obviously higher than that displayed by the cells infected with the delta Rv2521 strain, so that the death rate of the cells infected with the delta Rv2521 strain is obviously increased. The Mtb Rv2521 is a key ferroptosis promoting effector in the mycobacterium tuberculosis infection process, can promote the survival and dissemination of mycobacterium tuberculosis in macrophages, and can provide a new target spot for clinical tuberculosis treatment and antituberculosis drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the anti-tuberculosis target Rv2521 and its application. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) infection (The tuberculosis vaccine challenge[J]. Bull World Health Organ, 2023, 101(5):303-4.). It is one of the global public health problems (DANIAE, STIEGLERN, BOUCHARD JP. [Tuberculosis remains a public health issue in South Africa]. [J]. Rev Infirm, 2023, 72(290):39-40.), and also a major cause of death worldwide (NATARA JANA, BEENA PM, DEVNIKARAV, et al. A systemic review on tuberculosis[J]. Indian J Tuberc, 2020, 67(3):295-311.). Currently, the prevention and control of tuberculosis faces many challenges, mainly in the following aspects: First, the diagnostic tools for tuberculosis suffer from poor specificity, long processing time, and low sensitivity (SINGHV, KABRA S K. Advances in Tuberculosis: Diagnostics[J]. Indian JPediatr, 2019, 86(5):439-40.), which leads to many tuberculosis patients not receiving timely and accurate diagnosis, thus delaying treatment. Second, the current mainstream strategy for tuberculosis treatment relies on anti-tuberculosis drugs with bactericidal or bacteriostatic functions. However, due to the rising proportion of drug resistance, the existence of serious adverse reactions to anti-tuberculosis drugs, and poor compliance caused by long treatment cycles, traditional anti-tuberculosis treatment is facing great challenges, and the success rate of treating drug-resistant tuberculosis remains at around 60% (WHO, Global Tuberculosis Report, 2024).

[0003] The development and progression of tuberculosis depend on the interaction between Mycobacterium tuberculosis and the host. Existing theories suggest that Mycobacterium tuberculosis can utilize key virulence factors to resist host immune defenses and achieve immune escape (Qiyao Chai, Lin Wang, et al. New insights into the evasion of host innate immunity by Mycobacterium tuberculosis. Cell Mol Immunol, 2020, 7(9):901-913.). However, the mechanisms of interaction between Mycobacterium tuberculosis virulence factors and the host involved in immune escape are very complex, characterized by a large number of virulence factors and diverse interaction mechanisms. Therefore, in-depth analysis of the virulence factors and elucidation of their molecular mechanisms of interaction with the host are of great significance for the development of effective new anti-tuberculosis drugs.

[0004] The proteins encoded by genes Rv2521 and Rv1608c are identified by sequence homology as Prxs (also known as bacterial ferritin combinatorial proteins, Bcp) of the PrxQ subfamily. Among them, the protein encoded by Rv2521 is a eukaryotic-like secretory protein.

[0005] In recent years, many scholars have discovered an additional regulatory necrosis pathway called ferroptosis. Ferroptosis is an iron-dependent, regulated form of cell death, characterized by apoptosis and necrosis (Sun Y, Chen P, Zhai B, et al. The emerging role of ferroptosis ininflammation. Biomed Pharmacother, 2020, 127:110108.). Ferroptosis is a form of cell death characterized by iron-dependent lipid peroxidation. Imbalances in iron and lipid metabolism, mitochondrial dysfunction, and other factors can induce ferroptosis, such as Fenton's reaction-related lipid peroxidation and ferritin autophagy. The main mechanism of ferroptosis is the catalysis of lipid peroxidation of unsaturated fatty acids on the cell membrane by ester oxygenases or ferrous iron, thereby inducing cell death; in addition, it is also manifested by a decrease in the expression of the core enzyme GPX4 regulated by the glutathione system. Studies have found that downregulation of GPX4 expression in cells increases ferroptosis sensitivity, and knocking down GPX4 induces ferroptosis; conversely, upregulation of GPX4 expression leads to tolerance to ferroptosis (Xu C, Sun S, Johnson T, et al. The glutathione peroxidase Gpx4 prevents lipid peroxidation and ferroptosis to sustain Treg cell activation and suppression of antitumor immunity. Cell Rep, 2021, 35(11):109235.). When the cellular antioxidant system is metabolically abnormal, Fe... 2+ Accumulation and mediating the Fenton reaction to produce excessive ROS, which undergo peroxidation with polyunsaturated fatty acids (PUFA) on the cell membrane, leading to the disruption of lipid bilayer stability, cell membrane disintegration, and ultimately cell death (Tang D, Chen X, Kang R, et al. Ferroptosis: molecular mechanisms and health implications. Cell Res, 2021, 31(2): 107-125.). Ferroptosis is highly dependent on iron and Mtb. It has been reported that elevated iron levels can increase the risk of active pulmonary tuberculosis in patients (Li Wei, Zhang Wanjiang. Research progress on the role of trace iron in the pathogenesis of tuberculosis. Journal of Cellular and Molecular Immunology, 2012, 28(05): 554-556). Summary of the Invention

[0006] This invention discovered that the Rv2521 protein of Mtb is closely related to ferroptosis and can promote the survival and dissemination of Mycobacterium tuberculosis in macrophages, thus serving as a novel target for the treatment or adjuvant therapy of tuberculosis. Based on this, this invention was completed.

[0007] In a first aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of diseases caused by Mycobacterium tuberculosis infection, said pharmaceutical composition containing a substance that inhibits the activity of Rv2521 protein or a substance that reduces the content of Rv2521 protein.

[0008] Furthermore, the substance that inhibits the activity of Rv2521 protein may be a protein, polypeptide, or small molecule compound that inhibits the function of Rv2521 protein.

[0009] Furthermore, the substance that reduces the Rv2521 protein content may be a substance that inhibits Rv2521 protein synthesis, promotes Rv2521 protein degradation, or knocks down or eliminates the Rv2521 gene.

[0010] Furthermore, the substance that knocks down the Rv2521 gene can be any substance that can prevent the gene encoding the Rv2521 protein from being expressed, such as substances that silence the Rv2521 gene (e.g., miRNA, siRNA, dsRNA, shRNA, etc.).

[0011] Furthermore, the substance that knocks out the Rv2521 gene can be any substance that prevents Mycobacterium tuberculosis cells from producing the functional protein product of the Rv2521 gene. Specific methods include removing all or part of the coding gene sequence, introducing frameshift mutations to prevent the production of functional proteins, removing or altering regulatory components (e.g., promoter editing) to prevent the coding gene sequence from being transcribed, or preventing translation by binding to mRNA.

[0012] Furthermore, the substance that knocks out the Rv2521 gene can be any substance that can cause the Rv2521 gene to mutate (the mutation can be a deletion mutation and / or an insertion mutation and / or a base substitution) and thus lose its activity.

[0013] Furthermore, the aforementioned Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by infected individuals.

[0014] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the pharmaceutical composition.

[0015] Furthermore, the carrier material includes, but is not limited to, one or more of water-soluble carrier materials, poorly soluble carrier materials, and / or enteric carrier materials.

[0016] Furthermore, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acids.

[0017] Furthermore, the poorly soluble carrier material includes, but is not limited to, one or more of ethyl cellulose and / or cholesterol stearate.

[0018] Furthermore, the enteric carrier material includes, but is not limited to, one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0019] Furthermore, the pharmaceutical composition can be formulated into various dosage forms, including but not limited to one or more of the following: tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and / or lyophilized powder injections.

[0020] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, a controlled-release formulation, and / or a variety of microparticle delivery systems.

[0021] Furthermore, the tablets can widely utilize a variety of carriers known in the art, including one or more of diluents and absorbents, humectants and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0022] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0023] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinylpyrrolidone.

[0024] Furthermore, the disintegrant includes, but is not limited to, one or more of the following: dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose and / or ethylcellulose.

[0025] Furthermore, the disintegration inhibitors include, but are not limited to, sucrose, tristearate, cocoa butter, and / or hydrogenated oils.

[0026] Furthermore, the absorption enhancer includes, but is not limited to, one or more of quaternary ammonium salts and / or sodium dodecyl sulfate.

[0027] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0028] Furthermore, the tablets can be further formulated into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, bilayer tablets, and multilayer tablets.

[0029] Furthermore, the pills can utilize a wide range of carriers known in the art, including diluents and absorbents, binders and / or disintegrants.

[0030] Furthermore, the diluent and absorbent include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin and / or talc.

[0031] Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, and / or flour paste.

[0032] Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose and / or ethylcellulose.

[0033] Furthermore, the suppository can widely use various carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and / or semi-synthetic glycerides.

[0034] Furthermore, the injectable formulation includes, but is not limited to, one or more of the following: solutions, emulsions, lyophilized powder for injection, and / or suspensions.

[0035] Furthermore, the injectable formulation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and / or polyoxyethylene sorbitan fatty acid esters.

[0036] Furthermore, in order to prepare an isotonic injection solution, appropriate amounts of one or more of sodium chloride, glucose, glycerol, conventional solubilizers, buffers, and / or pH adjusters may be added to the injectable formulation.

[0037] Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary.

[0038] In a second aspect, the present invention provides the use of substances that inhibit the activity of Rv2521 protein or substances that reduce the content of Rv2521 protein in the preparation of medicaments for the prevention and / or treatment of diseases caused by Mycobacterium tuberculosis infection, wherein the medicament contains substances that inhibit the activity of Rv2521 protein or substances that reduce the content of Rv2521 protein.

[0039] Furthermore, the substance that inhibits the activity of Rv2521 protein may be a protein, polypeptide, or small molecule compound that inhibits the function of Rv2521 protein.

[0040] Furthermore, the substance that reduces the Rv2521 protein content may be a substance that inhibits Rv2521 protein synthesis, promotes Rv2521 protein degradation, or knocks down or eliminates the Rv2521 gene.

[0041] Furthermore, the substance that knocks down the Rv2521 gene can be any substance that can prevent the gene encoding the Rv2521 protein from being expressed, such as substances that silence the Rv2521 gene (e.g., miRNA, siRNA, dsRNA, shRNA, etc.).

[0042] Furthermore, the substance that knocks out the Rv2521 gene can be any substance that prevents Mycobacterium tuberculosis cells from producing the functional protein product of the Rv2521 gene. Specific methods include removing all or part of the coding gene sequence, introducing frameshift mutations to prevent the production of functional proteins, removing or altering regulatory components (e.g., promoter editing) to prevent the coding gene sequence from being transcribed, or preventing translation by binding to mRNA.

[0043] Furthermore, the substance that knocks out the Rv2521 gene can be any substance that can cause the Rv2521 gene to mutate (the mutation can be a deletion mutation and / or an insertion mutation and / or a base substitution) and thus lose its activity.

[0044] Furthermore, the aforementioned Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by infected individuals.

[0045] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the medicament for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

[0046] Furthermore, the carrier material includes, but is not limited to, one or more of water-soluble carrier materials, poorly soluble carrier materials, and / or enteric carrier materials.

[0047] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be formulated into various dosage forms, including but not limited to one or more of the following: tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and / or lyophilized powder injections.

[0048] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, a controlled-release formulation, and / or a variety of microparticle delivery systems.

[0049] Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary.

[0050] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be administered by injection, cavity, respiratory tract, or mucosal administration.

[0051] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; the cavity administration includes rectal or vaginal administration; and the respiratory administration includes nasal administration.

[0052] Beneficial effects

[0053] This invention constructed a large fragment knockout strain of Rv2521, ΔRv2521, and further constructed a complement strain, ΔRv2521::2521. Compared with cells infected with ΔRv2521, the cell death rate of cells infected with either WT or ΔRv2521::2521 strains was significantly increased. Cells infected with either WT or ΔRv2521::2521 strains showed significantly higher intracellular LPO (a marker of ferroptosis) levels than cells infected with ΔRv2521 strains. Mtb Rv2521 is a key ferroptosis-promoting effector in Mycobacterium tuberculosis infection. Rv2521 can promote the survival and dissemination of Mycobacterium tuberculosis in macrophages, providing a new target for clinical tuberculosis treatment and anti-tuberculosis drug development. Attached Figure Description

[0054] Figure 1 Screening for ferroptosis-related genes using the ferroptosis inducer RSL3 (GPX4 inhibitor).

[0055] Figure 2Mtb Rv2521 is involved in inducing ferroptosis in host cells: a) Cells uninfected (con) or infected with Mycobacterium tuberculosis WT, ΔRv2521, or ΔRv2521::Rv2521 strains were treated with (Vehicle) or without Fer-1 (10 μM) for 24 hours. Dead cells (red) were stained with PI, and cell nuclei (blue) were stained with Hoechst. Scale bar, 25 μm; b) Quantitative analysis of PI-positive THP-1 cells treated with (a); c) Detection of lipid peroxides in THP-1 cells treated with the same method as (a) using a 1 μM LIPO probe; d) Quantitative analysis of LIPO-positive THP-1 cells treated with (a). Figure 3 Mtb Rv2521 promotes Mtb survival and spread in THP-1 cells: a) In vitro growth kinetics of Mycobacterium tuberculosis WT, ΔRv2521, and ΔRv2521::Rv2521 in 7H9 medium; b) Survival of Mycobacterium tuberculosis WT, ΔRv2521, and ΔRv2521::Rv2521 strains in THP-1 cells at 4 h post-infection; cd) Survival of Mycobacterium tuberculosis WT, ΔRv2521, and ΔRv2521::Rv2521 strains in THP-1 cells at 24 h and 48 h post-infection; ef) Effect of ferroptosis inhibitors on the survival of Mycobacterium tuberculosis WT, ΔRv2521, and ΔRv2521::Rv2521 strains in THP-1 cells.

[0056] Figure 4 Mtb Rv2521’s involvement in ferroptosis may depend on GPX4: a) GPX4 protein expression levels in THP-1 cells, whether uninfected (Con) or infected with WT, ΔRv2521, or ΔRv2521::Rv2521 strains, with or without Ferrostatin-1 (Fer-1) (10 μM); b) Quantification of GPX4 protein expression in (a)-treated cells; ce) Expression levels of GPX4, FTH1, and ACSL4 mRNA in THP-1 cells treated in the same manner as (a).

[0057] Figure 5Effects of Rv2521 on Mtb-triggered NF-κB and MAPK signaling pathway activation: a, c, e, THP-1 cells were either uninfected or infected with WT, ΔRv2521, or ΔRv2521::Rv2521, with or without JSH-23 (10 μM) treatment for 30 or 60 min. The expression of phosphorylated P65, total P65, phosphorylated ERK, total ERK, phosphorylated P38, total P38, phosphorylated JNK, total JNK, and β-actin or GAPDH was analyzed by Western blotting; b, d, f, g, density analysis was performed using ImageJ software grayscale intensity analysis, and the results were normalized to their respective non-phosphorylated total protein levels; i, qPCR was used to detect the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α.

[0058] Figure 6 Western blot analysis was used to detect the culture filtrate and bacterial precipitation of the Rv2521 protein labeled by this strain. GroEL1 was used as a marker for bacterial precipitation, and ESAT-6 was used as a marker for the culture filtrate.

[0059] Figure 7 Rv2521 co-localizes with NF-κB protein, and Rv2521 protein enhances ferroptosis in THP-1 cells via NF-κB p65-mediated GPX4: a) GPX4 protein expression levels in THP-1 cells, whether uninfected (Con) or infected with WT, ΔRv2521, or ΔRv2521::Rv2521 strains, MOI 10, with or without JSH-23 (10 μM); b) Quantification of GPX4 and GAPDH in THP-1 cells as described in (a); c) GPX4 mRNA expression levels in THP-1 cells; de) Co-immunoprecipitation of Rv2521 and NF-κB proteins; f) Co-localization of Rv2521 protein with NF-κB; g) ChIP-qPCR analysis of the GPX4 promoter in THP-1 cells using NF-κB p65 antibody. DETAILED DESCRIPTION

[0060] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0061] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0062] Example 1: Screening of the gene Rv2521 that promotes host ferroptosis

[0063] 1. Construction of a CRISPR library of eukaryotic secretory proteins from Mycobacterium tuberculosis

[0064] Based on the Mycobrowser database (https: / / mycobrowser.epfl.ch / genes / ) and the literature "Abacterial phospholipid phosphatase inhibits host pyroptosis by hijackingubiquitin. Science. 2022 Oct14; 378(6616):eabq0132," 201 eukaryotic-like secretory proteins were divided into essential genes and non-essential genes. Among the 201 eukaryotic-like secretory proteins, there were 29 essential genes and 172 non-essential genes, including the Mce family, PE / PPE family, as well as eukaryotic-like kinases / phosphatases, methyltransferases, redox-related enzymes, lipid metabolism-related enzymes, etc., and some proteins that have not yet been characterized or whose functions are unknown. Among the 172 non-essential genes, 13 genes lacked PAM sequences, so sgRNAs could not be designed; therefore, a total of 159 sgRNAs were designed. 159 sgRNA expression plasmids were constructed and amplified, and then transformed into plasmids containing helper plasmids. Knockout strains were verified by PCR and sequencing, and mutant strains of 137 genes were obtained. Mutant strains of 137 genes were obtained at two different sites. Most of the mutant strains were knocked out by less than 10 bp.

[0065] 2. Screening for the gene Rv2521 that promotes host ferroptosis.

[0066] 137 Mtb eukaryotic-like secretory protein knockout strains were infected with THP-1 cells. After screening with the ferroptosis inducer RSL3 (GPX4 inhibitor), Rv2521 was found to be involved in ferroptosis.

[0067] Example 2 confirmed that Mtb Rv2521 can participate in inducing ferroptosis in host cells.

[0068] To investigate the regulatory role of Rv2521 in ferroptosis during Mtb infection, a large Rv2521 knockout strain, ΔRv2521, was constructed and validated. The Rv2521 knockout strain (NCBI gene ID: 887694: ΔRv2521) was created using the pJV53 system. The complement strain MtbΔRv2521::Rv2521 of strain MtbΔRv2521 was constructed using the pYC874 plasmid (constructed by Beijing Chest Hospital, Capital Medical University). The Rv2521 gene was amplified from H37Rv genomic DNA by PCR and inserted into the eukaryotic expression vector pFlag-CMV to generate pFlag-Rv2521. These plasmids were subsequently used for transfection experiments in 293T cells. The full-length cDNA of Rv2521 was amplified from the genomic DNA of Mtb H37Rv, and the gene was inserted into the vector pMV261 to construct a prokaryotic expression plasmid. The plasmid was then electroporated into Mtb to generate the pMV261-Rv2521 strain.

[0069] The results showed that this invention successfully knocked out 309 bp of Rv2521 from Mtb, and subsequently constructed the complement strain MtbΔRv2521::2521. After infecting THP-1 cells with WT, ΔRv2521, and ΔRv2521::2521, the cell death rate of Mtb WT or MtbΔRv2521::2521-infected cells was significantly higher than that of cells infected with MtbΔRv2521. This phenomenon was eliminated by the ferroptosis inhibitor Ferrostatin-1 (Fer-1) (a potent lipid peroxidation inhibitor). Figure 2 ab). Cells infected with Mtb WT or MtbΔRv2521::2521 showed significantly higher levels of intracellular LPO (a marker of ferroptosis) than cells infected with MtbΔRv2521. Figure 2 These results demonstrate that Mtb Rv2521 is a key pro-ferroptosis effector during Mycobacterium tuberculosis infection.

[0070] Example 3: Rv2521 promotes the survival and dissemination of M. tb in macrophages by inhibiting ferroptosis.

[0071] To verify whether knocking out Rv2521 promotes intracellular survival of Mycobacterium tuberculosis by affecting ferroptosis, this invention first demonstrated in vitro that knocking out the Rv2521 gene does not affect the growth of Mycobacterium tuberculosis. Figure 3a) It is speculated that Rv2521 may promote the survival of Mycobacterium tuberculosis in macrophages by influencing ferroptosis. Bacterial load in intracellular and cell culture supernatants was measured at 4, 24, and 48 hours after infection with WT, ΔRv2521, or ΔRv2521::Rv2521 strains, respectively. The results showed no significant difference in bacterial load among the three groups at 4 hours of infection, indicating that THP-1 cells had the same phagocytic capacity for these three strains. Figure 3 b). However, 24 hours after infection, the number of ΔRv2521 strains in THP-1 cells decreased significantly and was lower than that of WT and ΔRv2521::Rv2521. At 48 hours, the number of WT and ΔRv2521::Rv2521 strains in THP-1 cells further increased, while the increase in ΔRv2521 strain load was not significant. This indicates that Rv2521 can promote the survival of M. tb in macrophages, while knocking out Rv2521 significantly inhibits the survival of M. tb. Figure 3 c). Meanwhile, the bacterial load results in the cell culture supernatant showed that the extracellular load of THP-1 cells infected with ΔRv2521 was significantly lower than that of cells infected with WT and ΔRv2521::Rv2521, indicating that knocking out Rv2521 can reduce the dissemination of M. tb to the cell culture supernatant. Figure 3 d). Furthermore, after adding the ferroptosis inhibitor Fer-1, the bacterial load in the intracellular and culture media of the ΔRv2521-infected group was not significantly different from that without Fer-1, while there were significant differences between the WT-infected group and the ΔRv2521::Rv2521-infected group, indicating that Rv2521 may enhance the intracellular survival and dissemination of *M. tb.* through promoting macrophage ferroptosis. Figure 3 ef).

[0072] Example 4: Mtb Rv2521's involvement in ferroptosis may be GPX4-dependent.

[0073] Compared with the MtbΔRv2521 strain, the expression levels of GPX4 protein and mRNA were significantly reduced in the infected WT or ΔRv2521::Rv2521 strains. Figure 4 Simultaneously, the mRNA expression levels of FTH1 and ACSL4, which are associated with ferroptosis, were also examined, and no significant difference in the mRNA expression of FTH1 and ACSL4 was found between those infected with Mycobacterium tuberculosis strain ΔRv2521::Rv2521. Figure 4 (de). In summary, these results indicate that Mtb Rv2521 promotes ferroptosis during Mtb infection, and this process is likely GPX4-dependent.

[0074] Example 5: Mtb Rv2521 evades the host's immune response by inhibiting the activation of NF-κB and MAPK.

[0075] Pro-inflammatory cytokines are key immune responses of macrophages to external stimuli, controlled by the NF-κB and MAPK signaling pathways. The NF-κB and MAPK signaling pathways play crucial roles in the induction of inflammatory cytokines. Virulence factors in Mtb have the ability to induce inflammation. Macrophages are the primary host of Mtb, and the NF-κB pathway is known to induce inflammation during infection.

[0076] To better understand the signaling pathway initiated by ΔRv2521, Western blot was used to detect the phosphorylation status of key inflammatory factors NF-κB subunits P65, P38, ERK, and JNK. Phosphorylation profiles of major transcription factors P65, ERK, P38, and JNK were measured after infection of THP-1 cells with WT, ΔRv2521, and ΔRv2521::2521. Western blot analysis showed that compared with cells infected with WT and ΔRv2521::2521, ΔRv2521 significantly increased the phosphorylation level of p65 in infected cells at 30 and 60 min, and increased the phosphorylation level of Erk1 / 2 in infected cells at 60 min; however, the phosphorylation levels of p38 and JNK did not change significantly. Figure 5 These results indicate that Rv2521 inhibits mycobacterium-induced macrophage activation, and this is at least in part mediated by the NF-κB and MAPK signaling pathways, which are key to Rv2521's role in evading the host's immune response during infection.

[0077] Example 6: Rv2521 inhibits the inflammatory response of Mtb-infected macrophages.

[0078] Studies have shown that inflammatory responses may play an important role in tuberculosis infection. This invention investigated the effects of Rv2521 on mycobacterial-induced inflammation. The levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were measured at different time points in THP-1 cells infected with WT, ΔRv2521, and ΔRv2521::2521 strains. THP-1 cells infected with ΔRv2521 produced more potent inflammatory cytokines, with significantly higher levels of IL-6, TNF-α, and IL-1β compared to cells infected with WT and ΔRv2521::2521 strains. Figure 5 These data indicate that Rv2521 negatively regulates mycobacterium-induced inflammatory responses in macrophages. In summary, these results suggest that Rv2521 can suppress host inflammatory responses during Mycobacterium tuberculosis infection, inhibit the induction of pro-inflammatory cytokines, and promote mycobacterial survival in macrophages.

[0079] Example 7: Rv2521 is a secretory protein.

[0080] To further verify the effect of Rv2521 on mycobacterial infection, a flag-tagged Rv2521 strain was constructed and identified. Western blot analysis showed that flag-Rv2521 was detectable in both whole-cell lysates and culture filtrates, while it was not detected in the control flag-Vector strain, indicating that Rv2521 protein is indeed a secreted protein. Figure 6 ).

[0081] Example 8: Rv2521 protein enhances host cell ferroptosis via NF-κB / p65-mediated GPX4.

[0082] To further investigate the regulatory mechanism of Rv2521's inhibition of the NF-κB signaling pathway on ferroptosis, cells were treated with the NF-κB inhibitor JSH-23. The results showed that infection with ΔRv2521 failed to rescue GPX4 expression, indicating that Rv2521 blocked the activation of the NF-κB signaling pathway, further leading to the downregulation of GPX4. The downregulated GPX4 promoted ferroptosis in host cells. Figure 7 These results indicate that activation of the NF-κB signaling pathway plays an important role in Rv2521-mediated ferroptosis.

[0083] This invention overexpressed pLvx-Flag-Rv2521 (Flag-Rv2521) in 293T cells. Immunoprecipitation experiments revealed that the overexpressed Flag-Rv2521 protein directly interacts with NF-κB. Figure 7 d). Further immunoprecipitation using anti-NF-κB in 293T cells overexpressing Flag-Rv2521 demonstrated, inversely, that Flag-Rv2521 protein interacts with NF-κB. Figure 7 e). Simultaneously, immunofluorescence analysis revealed significant co-localization between the overexpressed Flag-Rv2521 protein and NF-κB, suggesting a direct interaction between the two. Figure 7 f).

[0084] Furthermore, this invention discovered through bioinformatics analysis that the GPX4 promoter region may contain NF-κB binding sites. Therefore, ChIP-qPCR was used to detect the enrichment of NF-κB / P65 in the GPX4 promoter region. The results showed that, compared with macrophages infected by WT and ΔRv2521::Rv2521 strains, the binding level of NF-κB / P65 in the GPX4 gene promoter region was significantly increased in THP-1 cells infected by ΔRv2521 strain. Figure 7 g) indicates that Rv2521 may inhibit GPX4 transcription and thus downregulate tuberculosis infection-induced GPX4 expression by inhibiting the binding of NF-κB / P65 to the GPX4 gene promoter region.

[0085] In summary, the data from this invention indicate that Rv2521 may inhibit the activation and transport of NF-κB / P65 phosphorylation modification into the nucleus by targeting and binding to NF-κB, thereby inhibiting its enrichment in the GPX4 promoter region, thus downregulating GPX4 transcription levels and causing ferroptosis.

Claims

1. A pharmaceutical composition for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection, wherein the pharmaceutical composition contains a substance that inhibits the activity of Rv2521 protein or a substance that reduces the content of Rv2521 protein.

2. The pharmaceutical composition according to claim 1, wherein the substance that inhibits the activity of the Rv2521 protein can be a protein, polypeptide or small molecule compound that inhibits the function of the Rv2521 protein.

3. The pharmaceutical composition as claimed in claim 1, wherein the substance that reduces the content of Rv2521 protein can be a substance that inhibits Rv2521 protein synthesis or promotes Rv2521 protein degradation or knocks down or knocks out the Rv2521 gene.

4. The pharmaceutical composition according to claim 1, further comprising one or more pharmaceutically acceptable carriers or excipients.

5. The pharmaceutical composition according to claim 1, which can be prepared into various dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

6. Use of a substance that inhibits the activity of Rv2521 protein or a substance that reduces the content of Rv2521 protein in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection, wherein the drug contains a substance that inhibits the activity of Rv2521 protein or a substance that reduces the content of Rv2521 protein.

7. The use according to claim 6, wherein the substance that inhibits the activity of the Rv2521 protein can be a protein, polypeptide or small molecule compound that inhibits the function of the Rv2521 protein.

8. The use as claimed in claim 6, wherein the substance that reduces the content of Rv2521 protein can be a substance that inhibits Rv2521 protein synthesis or promotes Rv2521 protein degradation or knocks down or knocks out the Rv2521 gene.

9. The use according to claim 6, wherein one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

10. The use according to claim 6, wherein the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

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