CN118416197A - A drug for resisting tuberculosis infection and its application - Google Patents

Abstract

The present invention discloses the application of SLC44A1 in the preparation of anti-tuberculosis drugs, belonging to the field of biotechnology. For the first time, the solute carrier family 44 member 1 protein SLC44A1 was identified, which significantly inhibited the proliferation of Mycobacterium tuberculosis in macrophages. SLC44A1 can be used as a new potential drug for anti-tuberculosis infection. In addition, it can form a combination drug with other active substances that promote the body's anti-tuberculosis and/or drugs that improve tuberculosis symptoms, providing a new treatment method and drug for anti-tuberculosis infection.

Description

A drug for resisting tuberculosis infection and its application

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to discovering drugs for resisting Mycobacterium tuberculosis infection by screening important target sites of anti-tuberculosis drugs.

Background technique

Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (M.tb). According to a report released by the World Health Organization, approximately 10.6 million people were infected with tuberculosis worldwide in 2021. China is the third country in the world in terms of the number of tuberculosis cases, with an annual number of patients of approximately 780,000. The incidence of tuberculosis is not optimistic (WHO. The Global Tuberculosis Report [J]. 2022.). Although the current mainstream strategy for tuberculosis treatment relies on anti-tuberculosis drugs with bactericidal or antibacterial functions, the success rate of traditional anti-tuberculosis treatment has been greatly affected by the emergence of drug-resistant tuberculosis patients, the existence of serious adverse reactions to anti-tuberculosis drugs, and poor compliance caused by long treatment cycles. At present, studies have shown that host-directed anti-tuberculosis immunotherapy can target highly conserved host signaling pathways, improve the treatment success rate on the basis of conventional anti-tuberculosis treatment, and even shorten the treatment time, especially in the treatment of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. It can bring better therapeutic effects (Guo Xueying et al. (2023) Chinese Journal of Zoonoses, 2023, 39(11): 1124-1129.). Using molecular biology techniques to screen and identify genes or proteins involved in resistance to M.tb infection and pathogenesis can provide important drug targets for the design and screening of new tuberculosis agents, which has very important theoretical significance and application value for the comprehensive prevention and treatment of tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a typical intracellular bacterium. After M.tb infects the body, it mainly parasitizes macrophages. The innate immune response mediated by macrophages is the first line of defense for the host against tuberculosis infection, and the game between macrophages and M.tb determines the outcome of the infection. M.tb has evolved efficient and complex methods to evade or interfere with the host immune signaling pathways, including preventing the maturation of phagocytic-lysosomes in macrophages, inducing the expression of anti-inflammatory factors, destroying macrophage pattern recognition receptors and hindering macrophage aggregation. Therefore, in-depth exploration of the molecular mechanism of intracellular survival of Mycobacterium tuberculosis can provide new targets for the development of anti-tuberculosis drugs. Finding genes or proteins involved in resisting M.tb infection and pathogenesis, and targeting these genes or proteins to develop corresponding therapeutic drugs will be an important way to achieve host-directed anti-tuberculosis immunotherapy.

Solute carriers (SLCs) are a group of membrane transporters that include more than 300 members, most of which are located on the cell membrane. Their main function is to facilitate the transport of various substrates across biological membranes, including small molecule absorption by cells. Transporters of the SLC44 family have become promising new targets in the treatment and diagnosis of immune and degenerative diseases. SLC44A1, as a choline carrier, is involved in choline transport and transmembrane transport. It is widely expressed in neurons and dendritic glial cells throughout the nervous system and can be detected in both the plasma membrane and mitochondrial membrane. Therefore, it is involved in the regulation of some diseases such as the proliferation of lung cancer cell lines (Traiffort E et al. (2013) MolAspects Med.10.1016/j.mam.2012.10.011.). Driven by the new host-directed anti-tuberculosis treatment strategy, new drugs with anti-tuberculosis effects are discovered through the study of target sites.

Summary of the invention

In order to solve the deficiencies in the prior art, the present invention has discovered a solute carrier family 44 member 1 (SLC44A1) that can significantly regulate and inhibit the proliferation of Mycobacterium tuberculosis in host macrophages and lung tissues, and the present invention is completed based on this.

The technical solution of the present invention is as follows:

In a first aspect of the present invention, there is provided a use of SLC44A1 in the preparation of a drug for preventing Mycobacterium tuberculosis infection, wherein the amino acid sequence of SLC44A1 is shown in SEQ ID NO.1, wherein SLC44A1 is a solute carrier family 44 member 1 functional protein present in the cytoplasm and mitochondria, and has the effect of significantly regulating and inhibiting the proliferation of Mycobacterium tuberculosis in host macrophages.

SEQ ID NO.1:

Furthermore, the amino acid sequence of the SLC44A1 protein also includes 80%-99% homologous sequences, preferably 80%-85%; preferably 85%-90%; preferably 90%-95% homologous sequences.

Furthermore, the amino acid sequence of the SLC44A1 protein has amino acid deletion, mutation or addition at one or several sites.

Furthermore, the SLC44A1 protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecule.

Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, and extrapulmonary infection.

Furthermore, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

Furthermore, the hosts infected by Mycobacterium tuberculosis include humans, cattle, mice, badgers, deer, primates, etc.

Furthermore, the drug has at least one of the following effects:

(1) Inhibit the activity of Mycobacterium tuberculosis;

(2) Anti-Mycobacterium tuberculosis infection;

(3) prevention and/or treatment of diseases caused by Mycobacterium tuberculosis;

Furthermore, the drug includes at least one of a pharmaceutical carrier, an excipient, and an auxiliary material.

Furthermore, the excipients include at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, antioxidants, adsorbents, filter aids, and release retardants.

Furthermore, the pharmaceutical preparation includes a biological preparation or a pharmaceutical preparation.

Furthermore, the dosage forms of the anti-tuberculosis drug preparations include: sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, powders, wine preparations, preparations, drops, injections, powder injections, creams, sustained-release preparations, targeted agents, etc.

Furthermore, the administration methods of the anti-tuberculosis drug preparation include oral administration, injection, implantation, external use, spraying, and inhalation.

Furthermore, the drug can be delivered into the host body through a delivery system such as a gene transfer device, AVV, liposome, etc. to achieve in vivo treatment.

In a second aspect, the present invention provides an anti-Mycobacterium tuberculosis pharmaceutical composition, which comprises SLC44A1 and another anti-Mycobacterium tuberculosis drug, wherein the amino acid sequence of SLC44A1 is shown in SEQ ID NO.1, and SLC44A1 is a solute carrier family 44 member 1 functional protein present in the cytoplasm and mitochondria, and has the effect of significantly regulating and inhibiting the proliferation of Mycobacterium tuberculosis in host macrophages.

SEQ ID NO.1:

Furthermore, another anti-Mycobacterium tuberculosis drug is a drug that can promote the body's anti-tuberculosis active substances and/or improve the symptoms of tuberculosis.

Furthermore, another active ingredient against Mycobacterium tuberculosis includes, but is not limited to, in the process of anti-tuberculosis infection: active ingredients and drugs that act on the mycobacterial cell wall, active ingredients and drugs that inhibit protein synthesis, active ingredients and drugs that inhibit the activity of energy metabolism ATP synthase, and active ingredients and drugs that inhibit DNA synthesis.

Beneficial effects of the present invention:

The present invention provides an important target gene or target protein for developing host-oriented anti-tuberculosis treatment strategies. It is of great significance to reveal the mechanism of macrophage-mediated innate immune response in host anti-tuberculosis infection. Drugs targeting this gene or protein can inhibit the proliferation of Mycobacterium tuberculosis in host macrophages and control the pathogenicity of tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: SLC44A1 expression in peripheral blood mononuclear cells (HC: n=8; LTBI: n=10; TB: n=13).

Figure 2: A. Expression changes of SLC44A1 in THP-1 cells after H37Rv infection (n=3); B. Relative expression of SLC44A1 protein in THP-1 cells after H37Rv infection; C. Expression changes of SLC44A1 in primary human macrophages after H37Rv infection (n=17).

Figure 3: A-B. Comparison of the expression changes of SLC44A1 in primary macrophages of infected and uninfected mice (n=3); C-D. The expression changes of SLC44A1 in mouse lung tissues before and after H37Rv infection (n=3).

Figure 4: A. Silencing efficiency of SLC44A1 in THP-1 cells; B. CFU detection at 4h, 24h and 48h after H37Rv infection (n=3).

Figure 5: siRNA inhibition of SLC44A1 leads to a decrease in the apoptosis rate of THP-1 cells; A. Schematic representation of cell apoptosis 24 hours after H37Rv infection; B. The apoptosis rate of cells infected and not infected with H37Rv (n=3).

Figure 6: A. Detection of caspase-3 protein in SLC44A1 silenced cells and control empty vector cells after H37Rv infection; B. Relative expression level of cleaved caspase-3 protein (n=5).

Figure 7: A. Detection of caspase-8 and caspase-9 proteins in SLC44A1-silenced cells and control cells after H37Rv infection; B-C. Relative expression levels of cleaved caspase-8 and cleaved caspase-9 proteins.

Figure 8: A. Co-localization detection of SLC44A1 and mitochondrial internal reference protein VDAC1; B. Co-localization detection of SLC44A1 and endoplasmic reticulum stress-related protein calnexin (blue fluorescence: DAPI; red fluorescence: SLC44A1; green fluorescence: VDAC1 and Calnexin).

FIG. 9 : A. Expression levels of SLC44A1 in the nucleus and cytoplasm; expression levels of SLC44A1 in mitochondria and cytoplasm excluding mitochondria.

Figure 10: Protein levels of Bax and cytochrome c in mitochondria and cytosol deprived of mitochondria.

FIG. 11 : CFU in the lungs of wild-type mice (WT) and SLC44A1 ^−/− mice (n=3).

Detailed ways

The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

the term:

As used herein, the term "specimen" or "sample" refers to material that is specifically associated with a subject from which specific information about the subject can be determined, calculated or inferred. A sample may consist entirely or in part of biological material from the subject.

PBMC (peripheral blood mononuclear cell), peripheral blood mononuclear cells, its main cell type is the cells with a single nucleus in the blood, mainly including lymphocytes (T/B), monocytes, macrophages, dendritic cells and a small number of other cell types. Among them, lymphocytes account for a large part. The main purpose of isolating PBMC is to remove multinuclear cells and red blood cells, so as to easily simulate the blood immune environment in vitro.

Ficoll separation: Ficoll is a polymer of sucrose, which is neutral and has an average molecular weight of 400,000. When the density is 1.2g/ml, it still does not exceed the normal physiological osmotic pressure and does not pass through biological membranes. Red blood cells and granulocytes have a high specific gravity and sink to the bottom of the tube after centrifugation; the specific gravity of lymphocytes and monocytes is less than or equal to the specific gravity of the layering liquid. After centrifugation, they float on the surface of the layering liquid, and a small number of cells may also be suspended in the layering liquid. By aspirating the cells on the surface of the layering liquid, mononuclear cells can be separated from peripheral blood. Ficoll is a separation liquid used to separate cells of specific density (three types: 1.077, 1.084 and 1.073g/mL), which is suitable for the separation of cells of known density.

Target gene: also known as the target gene, a structural gene that encodes a protein. Common methods for obtaining target genes include obtaining from gene libraries, amplifying using PCR technology, and artificial synthesis.

Macrophages: Macrophages are thought to develop from hematopoietic stem cells in the bone marrow → monocytes. After undergoing differentiation steps, they come to the peripheral blood and become circulating monocytes. Two types have been identified: called "inflammatory" and "resident" monocytes, mainly based on how long they stay in the blood before migrating to tissues. After migrating to tissues, they differentiate into tissue-specific macrophages: including the skeletal system (osteoclasts), central nervous system (microglia), lungs (alveolar macrophages), liver (Kupffer cells) and connective tissue (histiocytes), as well as spleen, gastrointestinal tract and peritoneum.

H37Rv: The first Mycobacterium tuberculosis (MTB) standard strain in 1998. The genome of the H37Rv standard strain is about 4 million bases long, containing 3906 protein-coding genes that can encode various enzymes involved in lipid metabolism, as well as two glycine-rich protein families PE and PPE with repetitive structures, the latter two of which are the difference between MTB and other bacteria.

Example 1 Quantitative RT-qPCR verification of the expression of SLC44A1 in monocytes of tuberculosis patients and healthy donors and in THP-1 macrophages infected with H37Rv and human primary macrophages (A) Experimental method: Peripheral blood mononuclear cells (PBMCs) of active tuberculosis patients, latent infections and healthy people were separated by Ficoll lymphocyte separation fluid density gradient centrifugation, and CD14 ⁺ monocytes were further obtained by magnetic bead sorting. The expression of solute carrier family 44 member 1 (Solute carrier family 44 member 1, SLC44A1) was detected by qPCR. Similarly, the expression of SLC44A1 was also detected in H37Rv-infected THP-1 macrophages and human primary macrophages. The specific experimental steps are as follows:

1. Ficoll separation of PBMCs

Magnetic beads were used to negatively sort human peripheral blood mononuclear cells (PBMCs) to obtain CD14+ monocytes. Peripheral blood was collected from the subjects in heparin anticoagulation tubes, and an equal volume of RPMI 1640 medium was added to dilute the blood. A certain volume of TBD human whole blood mononuclear cell separation solution was added to a 15 ml centrifuge tube in advance, and the diluted peripheral blood was slowly added to the separation solution along the tube wall (separation solution: diluted whole blood = 3:4). It was then moved to a horizontal centrifuge with an acceleration of 9 and a deceleration of 0, and centrifuged at a centrifugal force of 1000g for 16 minutes. After the centrifugation, the middle buffy coat cells (mononuclear cells) were drawn into a new 15 ml centrifuge tube, RPMI 1640 was added to resuspend the cells, and the cells were washed twice at 400g for 5 minutes. The cells were then resuspended in a complete medium containing RPMI 1640 plus 10% fetal bovine serum, and the cells were counted.

2. Stimulate the activation of monocytes in PBMCs into macrophages

Before the experiment, colony stimulating factor (GM-CSF) was dissolved. Before opening the reagent, it was centrifuged to make its contents reach the bottom. After Ficoll separation of PBMCs, the cells were counted and plated at a density of 2×10 ⁶ /ml/well in a 12-well plate. The cells were cultured with complete medium added with GM-CSF (GM-CSF: complete medium = 1:1000, with a final concentration of 25ng/ml). The cells were cultured continuously for 7 days in a cell culture incubator at 37°C and 5% CO2. During this period, the cells were replaced with medium every two days. When replacing the medium, the medium in the 12-well plate was aspirated and centrifuged at 400g for 5min, the supernatant was discarded, and the medium was resuspended with complete medium containing GM-CSF and re-added to the original 12-well plate. The monocytes in the PBMCs were activated into macrophages by continuous stimulation and attached to the 12-well plate. After washing the suspended cells with RPMI 1640 medium, the cells were infected.

3. THP-1 cell recovery, culture and induction

The cell lines THP-1 and 293T cells used in the study were purchased from the cell bank of the Chinese Academy of Medical Sciences. Before the experiment, phorbol 12-myristate 13-acetate (PMA) working solution was prepared, PMA dry powder was centrifuged at high speed, and then 1 ml DMSO was used to dissolve it (concentration 1 mg/ml) and aliquoted. THP-1 cells frozen in liquid nitrogen were quickly taken to a 37°C water bath until the liquid in the cell cryopreservation tube thawed, washed with 5 ml RPMI medium, centrifuged at 200g for 2 minutes, the supernatant was discarded, and 10 ml complete medium was added to the cell culture bottle. The cell medium was changed every two days. When the cells grew to the logarithmic growth phase, the cells were plated at a culture density of 5×10 ⁶ /well in 24-well plates, 1×10 ⁶ /well in 12-well plates, 2×10 ⁶ /well in 6-well plates, and 1×10 ⁶ /cell culture dishes.

4. Induce THP-1 cells with PMA

Prepare complete medium containing PMA (PMA: complete medium = 1:10000, final concentration is 100ng/ml), add the above-prepared complete medium containing 1/10000 PMA according to the liquid volume of 0.5ml for 24-well plate, 1ml for 12-well plate, 2ml for 6-well plate and 10ml for cell culture dish, induce for 36h, and when THP-1 cells adhere to more than 90%, stop inducing differentiation of cells, replace with complete medium without PMA and continue to recover culture for 12h. Then, carry out cell infection.

5. H37Rv resuscitation and expansion culture

The M.tb standard strain H37Rv comes from the Beijing Major Disease Clinical Data and Sample Resource Library - Tuberculosis Library. Take the frozen bacteria from the -80℃ refrigerator and streak them in Middlebrook 7H10 medium. Incubate at 37℃ for about 3 weeks to revive the H37Rv strain. Pick a single colony from the Middlebrook 7H10 medium and inoculate it into the Middlebrook 7H9 medium. Also incubate it in a 37℃ incubator for about 2 weeks. When the optical density value (A) _A600 reaches 0.6-0.8, it means that H37Rv has grown to the logarithmic growth phase and is used for infection. The whole process is sterile.

6. Construction of cell infection model

After inducing cell adhesion, the cells were infected with the H37Rv strain. Take 1ml of bacteria in the logarithmic growth phase and 1ml of sterile culture medium (7H9+10% OADC) and put them into a cuvette for OD measurement (1OD=3×10 ⁸ bacteria), and infect the cells at a multiplicity of infection (MOI) of 10. Calculate the required amount of bacteria, pipette it into a 15ml centrifuge tube, add an appropriate amount of RPMI1640 culture medium, 4500rpm, and centrifuge at room temperature for 7min for washing. After centrifugation, discard the supernatant, add 1ml of complete culture medium for resuspending, and then add it to a dispersion tube for ultrasonic dispersion. Finally, add the ultrasonically dispersed bacterial solution to a fixed volume of complete culture medium, blow and mix, add it to each well plate respectively, and place it in a 37°C, 5% CO ₂ incubator for continued cultivation. After 4h, remove the culture medium in the well plate, and wash the cells three times with RPMI 1640 culture medium to wash off the extracellular M.tb. Cells, RNA, proteins or intracellular bacteria are collected at different time periods as needed to perform corresponding cell phenotype and CFU tests.

7. RNA extraction

The total RNA of the sample was extracted using miRNeasy Mini Kit (217004, Qiagen). 30 ml of anhydrous ethanol (analytical grade) was added to wash solution 1 (rinsing solution RWT) and mixed by inverting. 44 ml of anhydrous ethanol (analytical grade) was added to wash solution 2 (rinsing solution RPE) and mixed. The sample lysed with 700 μl QIAzol was taken out of the -80°C refrigerator and placed for 5 minutes to melt; 140 μl of chloroform was added, and the mixture was fully shaken and mixed, placed for 2-3 minutes, and then centrifuged at 4°C and 12000g for 15 minutes. After the centrifugation, the upper transparent aqueous phase was transferred to a new enzyme-free centrifuge tube, 1.5 times the volume of anhydrous ethanol was added, and the mixture was transferred to the collection column after being mixed by pipetting. The mixture was centrifuged at 10000g for 15 seconds, the filtrate was discarded, and 700 μl of wash solution 1 was added, and the mixture was centrifuged at 10000g for 15 seconds. Discard the filtrate, add 80μl DNA enzyme (52μl enzyme-free water, 20μl DNA enzyme, 8μl reaction buffer), and place at room temperature for 15min; add 700μl wash solution 1 and 500μl wash solution 2 in batches, centrifuge at 10000g for 15s at room temperature, and discard the filtrate; add 500μl wash solution 2, centrifuge at 10000g for 1min, put the collection column into a new centrifuge tube, open the lid, and centrifuge at 20000g for 1min at room temperature. Put the collection column into a centrifuge tube, add 30μl RNase-free ddH ₂ O, place for 2min, centrifuge at 10000g for 1min, recover the liquid in the centrifuge tube into the collection column, repeat the centrifugation once to increase the total RNA yield, and measure the RNA concentration and purity with an ultra-micro UV spectrophotometer (NanoDrop2000).

8. mRNA reverse transcription and qPCR, the specific methods are as follows:

1) The kit used for mRNA reverse transcription was ReverTraAce qPCRRT Kit (TOYOBO, Japan). The mRNA reverse transcription system is shown in Table 1.

Table 1 mRNA reverse transcription system (dye method)

2) The reverse transcription reaction conditions were: 37°C for 15 min; 98°C for 5 min; 4°C infinity.

3) GAPDH was the internal reference gene in mRNA detection; the kit used for qPCR detection was PowerUp SYBR Green Master Mix (Applied Biosystems, USA), and the PCR reaction system was shown in Table 2.

Table 2 qPCR reaction system (dye method)

4) The PCR reaction program is set as follows:

5) After the PCR reaction is completed, the experimental data are exported and the expression level of mRNA is analyzed using the relative expression method (2 ^-ΔΔCt ).

9. The primers used in this study are shown in Table 3

Table 3 Primers and their sequences used in this study

(B) Results: The expression of SLC44A1 in monocytes of patients with active tuberculosis was significantly higher than that in the control group. At the THP-1 cell level, the expression level of SLC44A1 was significantly increased after 24h and 48h of H37Rv infection compared with the uninfected group (P<0.01); at the human primary macrophage level, the expression of SLC44A1 in cells was significantly increased after H37Rv infection compared with the control group (P<0.001). The expression of SLC44A1 in the H37Rv infected group was significantly increased compared with the control group (P<0.01); similarly, the expression of SLC44A1 in the lung tissue of mice was significantly increased compared with the control group (P<0.05). The results showed that H37Rv infection led to upregulation of SLC44A1 expression.

Example 2 SLC44A1 protein against mycobacterial infection (A) Experimental method: The three SLC44A1-specific silencing RNAs (siRNAs) and their control siRNA-NC provided by Guangzhou Ruibo Biotechnology Co., Ltd. were pre-tested for transfection efficiency, and siRNA-SLC44A1-02 was finally selected for subsequent functional studies of SLC44A1. SLC44A1 low-expressing THP-1 cells and normal THP-1 cells were infected with H37Rv, and the CFU in the cells at different time periods were measured to prove the functional role of SLC44A1 in the process of resisting pathogens. Taking the transfection of 12-well plate cells as an example, the transfection concentration of siRNA was 50nM, and the amount of transfection reagent used was LipofectamineTM RNAiMAX:siRNA=5:1.

The specific transfection steps are as follows:

(1) Add 150 μl to tube A culture medium, then add 12.5 μl LipofectamineTM RNAiMAX and gently pipette to mix.

(2) Add 150 μl to tube B culture medium, add 2.5 μl siRNA, and mix well by pipetting.

(3) Add the liquid in tube B to tube A, mix well, and let stand for 15 minutes.

(4) Take out the plated THP-1 cells and wash off the PMA from the cell culture incubator, discard the original culture medium, and quantitatively add 700 μl of complete culture medium.

(5) Add the mixed liquid in tubes A and B to the 700 μl complete culture medium added in advance and shake well. Return the 12-well plate to the incubator and continue culturing.

(B) Results: Comparison of the CFU differences between the normal THP-1 cell group (NC siRNA) and the SLC44A1 low-expression THP-1 cell group (SLC44A1 siRNA group) showed that the survival of H37Rv in the SLC44A1 siRNA group was significantly higher than that in the NC siRNA group 24h and 48h after infection. The results showed that the mycobacterium was cleared faster in THP-1 cells with normal SLC44A1 expression, while the mycobacterium survival ability was enhanced in THP-1 cells with low SLC44A1 expression.

Example 3 SLC44A1 protein regulates cell apoptosis and inhibits the survival of H37Rv in cells (A) Experimental method: SLC44A1-specific siRNA and control siRNA were used to transfect THP-1 cells to construct SLC44A1-silenced cells and control cells. SLC44A1-silenced THP-1 cells and normal THP-1 cells were infected with H37Rv, respectively, and analyzed by flow cytometry after AnnexinV/7-AAD staining. Protein samples of SLC44A1-silenced cells and control cells infected with and not infected with H37Rv were collected, and after BCA concentration determination, the expression of caspase-3 and cleaved caspase-3 was detected by western blot.

The steps for Western blot detection of protein expression are as follows:

1. Cell protein collection and extraction

(1) THP-1 cells were induced in 6-well plates at a cell density of 2×10 ⁶ /well. After PMA induction for 36 h, the complete medium without PMA was replaced and the culture was restored for 12 h. The cells were infected with H37Rv at an MOI of 10, and cell samples were collected at uninfected, 24 h infected, and 48 h infected: the plate was washed once with 1×PBS, 1 ml of 1×PBS was added, the cells were gently scraped off, pipetted and mixed, and then the liquid was transferred to a 1.5 ml centrifuge tube, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cell pellet at the bottom of the tube was frozen and stored at -80°C for later use.

(2) Prepare protein lysis buffer before extracting protein samples: add 100 μl phosphatase inhibitor and 10 μl protease inhibitor to 890 μl RIPA lysis buffer. Take out protein samples from -80℃ freezer and place on ice. Add 60 μl protein lysis buffer to each sample, mix by pipetting, lyse on ice for 20 min, then centrifuge at 4℃, 20000g for 20 min, aspirate supernatant (60 μl) into a new 1.5 ml centrifuge tube, mix and centrifuge for later use (keep samples at low temperature throughout the process).

2. Determination of protein concentration by BCA method

(1) Take 1.2 ml of protein standard solution and add it to a tube of protein standard (30 mg BSA). After fully dissolving, prepare a 25 mg/ml protein standard solution. Use RIPA lysis buffer to dilute the 25 mg/ml protein standard solution to a final concentration of 0.5 mg/ml. Then divide it into 70 μl per tube. At the same time, prepare BCA working solution (prepare it before use) before the experiment. According to the number of samples (200 μl per sample), prepare an appropriate amount of BCA working solution at a ratio of 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), mix it thoroughly and set aside.

(2) Add 0.5 mg/ml standard to the standard wells of a 96-well plate at the following levels: 0, 1, 2, 4, 8, 12, 16, 20 μl. Add standard diluent (RIPA lysis buffer) to make up to 20 μl, which is equivalent to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml, respectively. Pipette 4 μl of sample into 16 μl RIPA lysis buffer (sample diluted 5 times), mix and centrifuge, and transfer to the sample wells of a 96-well plate. Then, use a spray gun to add 200 μl of BCA working solution to the standard wells and sample wells at the same time. Place at 37°C for 20-30 min. It can be seen that the color in the standard wells deepens with the increase of standard concentration. Use an ELISA reader to measure the absorbance between 540-595 nm (550 nm), and then calculate the protein concentration of the sample based on the standard curve and the sample volume used. After the protein concentration determination, add 11.2 μl of 6× loading buffer to each sample tube and boil in a 95°C metal bath for 5 min to denature the protein and prevent its degradation.

3. SDS-PAGE polyacrylamide gel electrophoresis

(1) Fix the glass plate horizontally, prepare the separation gel (see Table 4 for the formula), inject 8 ml of separation gel between the glass plates, add anhydrous ethanol to press the line, and after the separation gel agglutinates, pour out the anhydrous ethanol and let the remaining anhydrous ethanol evaporate naturally; then prepare the concentrated gel (see Table 4 for the formula), fill the glass plates with concentrated gel and insert the corresponding comb, and let it stand at room temperature for 30 minutes. After the gel agglutinates, carefully pull out the comb between the glass plates, put the glass plate into the electrophoresis tank, fill the inside with electrophoresis liquid, and rinse the sample well with electrophoresis liquid to avoid bubbles in the well, and 1/2 of the outside with electrophoresis liquid; load 10 μg of protein and 5 μl of marker, use 80V voltage for the upper gel, adjust the voltage to 120V when the sample migrates to the separation gel, and continue electrophoresis until the sample migrates to the bottom of the separation gel.

Table 4 Separation gel and stacking gel formula for protein electrophoresis

(2) After electrophoresis, peel the gel from the glass plate and cut off the excess gel. Soak a stack of filter papers (7 sheets) in the transfer solution completely. Activate the nitrocellulose membrane (PVDF membrane) in methanol for about 15 seconds, balance it in the transfer solution for 1 minute, and then put it on the soaked filter paper. Then put the gel on the PVDF membrane and cover it with another stack of filter paper. Use a press plate to empty the bubbles between the gaps, put it in the transfer tank, and cover it with a safety cover. Use 1.3A current and 25V voltage to transfer one gel for 20-30 minutes; use 2.5A current and 25V voltage to transfer two gels together for 20-30 minutes. After the transfer is completed, take out the PVDF membrane, cut the PVDF according to the target protein, and keep the cut blank membrane (for subsequent full membrane photography). Place the PVDF membrane in blocking solution (select 5% BSA or 5% skim milk according to the antibody instructions), block at room temperature for 1 hour, pour out the blocking solution after blocking, add the primary antibody diluted in the blocking solution, and incubate overnight at 4°C on a shaker (14-16 hours). After the primary antibody incubation, recover the antibody and add PBST (1×PBS plus 10% Tween 20) to wash 4 times, 10 minutes each time. Add HRP-labeled secondary antibody diluted in blocking solution and incubate on a shaker for 1 hour. After the secondary antibody incubation, pour out the antibody and add PBST to wash 4 times, 7 minutes each time. After washing, prepare the developer (A solution: B solution = 1:1), cover the developer on the PVDF membrane, and use a chemiluminescence instrument to expose the protein bands. Image J was used to measure the protein gray value to calculate the relative protein expression.

The steps for analyzing cell apoptosis by flow cytometry are as follows:

Annexin V combined with 7-AAD double staining was used to detect cell apoptosis. THP-1 cells were induced in 12-well plates and then infected with H37Rv. Cell samples were collected at 0h, 24h, and 48h for apoptosis detection. First, the cells in the well plate were washed with 1×PBS, 400μl 0.25% trypsin was added, and the cells were digested at 37℃ for 5min. Then, an equal volume of complete medium was added to terminate the digestion reaction. The liquid in the well plate was aspirated into a centrifuge tube, centrifuged at 400g for 5min, the supernatant was discarded, 1×PBS was added to rinse the cells, centrifuged at 400g for 5min, and the supernatant was discarded; 100μl 1×bindingbuffer was used to resuspend the supernatant, and 5μl PE or APC-labeled Annexin V and 5μl 7-AAD were added, incubated at room temperature in the dark for 30min, the cells were rinsed twice with 1×PBS, and finally 200μl 1×PBS was used to resuspend the cells, and apoptosis was detected using flow cytometry (FACS).

(B) Results: After 24h and 48h of infection, the survival of H37Rv in the SLC44A1 siRNA group was significantly higher than that in the NCsiRNA group, indicating that SLC44A1 can inhibit the survival of H37Rv in macrophages. After 24h and 48h of H37Rv infection, the expression of cleaved caspase-3 protein in the SLC44A1 siRNA group was significantly lower than that in the control group. The results indicate that SLC44A1 has the function of promoting macrophage apoptosis.

Example 4 SLC44A1 protein inhibits the survival of H37Rv in cells by regulating endogenous cell apoptosis

(A) Experimental method: The SLC44A1 siRNA group and the control group cells were infected with H37Rv, and the expressions of caspase-8, cleaved caspase-8 and caspase-9, cleaved caspase-9 in the SLC44A1 siRNA group and the control group after H37Rv infection were detected.

(B) Results: The expression of cleaved caspase-9 in SLC44A1 silenced cells was significantly lower than that in the uninfected group at 24h and 48h after H37Rv infection, while the expression of cleaved caspase-8 after H37Rv infection did not change significantly compared with that in the uninfected group. These results suggest that SLC44A1 may regulate macrophage apoptosis through the caspase-9-mediated endogenous apoptosis pathway.

Example 5 SLC44A1 inhibits the survival of H37Rv in cells through mitochondria-mediated endogenous apoptosis (A) Experimental method: The co-localization of SLC44A1 with mitochondrial and endoplasmic reticulum proteins was detected by confocal microscopy. The subcellular components of THP-1 were separated, and the localization of SLC44A1 protein in cells was detected by western blot. SLC44A1 silenced cells and control group cells were infected with H37Rv, and the translocation of mitochondrial apoptosis-related proteins Bax and cytochrome c from mitochondria to the cytoplasm was tested.

The specific steps of immunofluorescence staining and confocal analysis of the intracellular localization of SLC44A1 are as follows:

(1) THP-1 cells were induced in a glass dish (containing a glass slide) at a cell density of 4×10 ⁶ /dish. After 36 hours of induction, the complete medium without PMA was replaced and the culture was resumed for 12 hours. The cells were washed with 1×PBS for 5 minutes, 1 ml of 4% paraformaldehyde was added and placed at room temperature for 10 minutes to fix the cells, and then the cells were washed with 1×PBS 3 times, each time for 5 minutes; next, the cells were permeabilized to detect the target protein in the cells, and the cells were incubated with 1×PBS containing 0.2% Triton X-100 for 5 minutes, and the cells were washed with 1×PBS 3 times, each time for 5 minutes; then the cells were incubated with 1×PBS containing 1% BSA, 1‰ Tween 20 and 22.52mg/ml glycine for 30 minutes to block the nonspecific binding of the antibody. Antibodies were diluted with 1×PBS containing 1% BSA and 1‰ Tween 20 (SLC44A1 antibody concentration 1:50; VDAC1 antibody concentration 1:100; Calnexin antibody concentration 1:100), added to the corresponding glass dishes (SLC44A1 was co-incubated with VDAC1 and Calnexin, respectively), incubated overnight at 4°C, and then the cells were washed 3 times with 1×PBS, each time for 5 minutes; then the fluorescent antibodies were diluted (secondary antibody 1:1000; goat anti-rabbit for red fluorescence, goat anti-mouse for green fluorescence), incubated in the dark for 1 hour, and the cells were washed 3 times with 1×PBS in the dark for 5 minutes; DAPI was used to label the cell nucleus (DAPI: 1×PBS = 1:99), incubated in the dark for 5 minutes, and then the cells were rinsed 3 times with 1×PBS in the dark for 5 minutes; finally, the cell slides were sealed with mounting medium (mounting medium: glycerol = 1:50) and stored in the dark at -20°C or 4°C. (2) Use a laser confocal scanning microscope to capture fluorescence, observe the cell field of view under a 60x objective lens, select cy3 (red fluorescence), FITC (green fluorescence), DAPI (blue fluorescence) and bright field in the dye list, and adjust the detector sensitivity (HV) and other parameters of each color fluorescence. Finally, scan the spectrum and take pictures to obtain the target image. Finally, image J was used to perform co-localization analysis on the image results.

The steps for cell subcomponent separation are as follows:

1. Separation of nucleus and cytoplasm to detect the localization of SLC44A1 in cells

The cells were inoculated in 6-well plates at a density of 2×10 ⁶ /well. H37Rv infection was performed 36 hours after PMA induction. Cell components were separated using a nuclear cytoplasm separation kit 24 hours after infection. 400 μl of trypsin cell digestion solution was added to the well plate and placed in a 37°C incubator for digestion. After 5 minutes, complete medium was added to terminate the trypsin digestion. The cells were collected by centrifugation at 200g for 5 minutes. The cells were washed with pre-cooled 1×PBS, centrifuged at 200g for 5 minutes, the supernatant was discarded, 300 μl of pre-cooled Fractionation buffer was added, gently mixed, and incubated on ice for 7 minutes. Subsequently, the cells were centrifuged at 4°C and 500g for 5 minutes. The precipitate was the total nuclear protein, and the supernatant was the total cytoplasmic protein. The nuclear protein was lysed using RIPA lysis buffer, and the protein concentration was determined by BCA for subsequent protein detection.

2. Isolate cell mitochondria to detect mitochondrial apoptosis-related proteins

(1) Thaw the various solutions in the kit (mitochondrial isolation reagent, mitochondrial lysis buffer, and protease inhibitor PMSF) at room temperature. Immediately place on ice and mix well after thawing. Aliquot into 15 ml and 1.5 ml centrifuge tubes.

(2) THP-1 cells were seeded in a 10 cm diameter cell culture dish at a cell density of 1×10 ⁷ /well, PMA was added for induction for 36 h, and then siRNA was used to inhibit the expression of SLC44A1. Cell mitochondria were isolated 24 h after H37Rv infection. First, cells were washed with 1×PBS, 3 ml of trypsin cell digestion solution was evenly added and placed in a 37°C cell culture incubator for digestion. After 5 min, an equal volume of complete culture medium was added to terminate the trypsin digestion. 400g, centrifuged at room temperature for 5 min to collect cells. Wash cells with pre-cooled 1×PBS and centrifuged at 4°C for 5 min to precipitate cells. Then 1 ml of mitochondrial isolation reagent was added to resuspend the cells and ice-bathed for 10-15 min. The cell suspension was transferred to a glass homogenizer, homogenized, centrifuged at 4°C, 600g for 10min, the supernatant was transferred to another centrifuge tube, centrifuged at 4°C, 11000g for 10min, the supernatant was transferred to a new centrifuge tube, the collected precipitate was the isolated cell mitochondria, and 100μl mitochondrial lysis solution was used for lysis. Then the supernatant was centrifuged at 4°C, 12000 for 10min, and the supernatant was the cytoplasmic protein without mitochondria. The BCA method was used to determine the protein concentration of mitochondrial protein and cytoplasmic protein without mitochondria. Finally, western blot was used to detect the expression changes of proteins.

(B) Results: Colocalization detection revealed that SLC44A1 overlapped with the fluorescent spots of the mitochondrial internal reference protein VDAC1, but did not overlap with the fluorescent spots of the endoplasmic reticulum stress-related protein calnexin. Western blot detection showed that SLC44A1 was mainly present in cytoplasmic mitochondria. In the process of mitochondrial-mediated intrinsic apoptosis, SLC44A1 inhibited the entry of Bax into mitochondria and the translocation of cytochrome c from mitochondria to cytoplasm, and the apoptosis-induced state of mitochondria was low. The above results indicate that SLC44A1 controls the survival of intracellular Mycobacterium tuberculosis by regulating the mitochondrial-mediated intrinsic cell apoptosis pathway.

Example 6: Verification of SLC44A1 in mice to promote host resistance to Mycobacterium tuberculosis infection

(A) Experimental methods: Specific pathogen-free (SPF) female C57BL/6J mice, aged 4-6 weeks, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. SLC44A1 ^-/- mice were provided by Guangzhou Saiye Biotechnology Co., Ltd. The mice were housed in a temperature-controlled negative pressure animal room, with 6 mice per cage, and the animals had easy access to food and water. The mouse experiments were conducted after approval by the Animal Ethics Committee of Beijing Chest Hospital. Wild-type mice and SLC44A1 ^-/- mice were infected with H37Rv, and lung tissues were taken from the mice for CFU detection and pathological detection 14 days later.

The steps for establishing a mouse tuberculosis infection model are as follows:

Dilute H37Rv (10 ⁷ CFU/ml) with sterile PBS + 0.05% Tween-80, sonicate for 1 minute, and finally adjust the volume of the bacterial solution to 5 ml. Put the mice to be infected into the basket, close the cabin, and inhale the bacterial suspension into the nebulizer. Perform nebulization infection and sterilization and disinfection according to the preheating for 15 minutes, nebulization for 30 minutes, smoke attenuation for 30 minutes, and purification for 15 minutes. After infection, the mice were caged and raised in a negative pressure infection animal room. On the first day of infection, 3 mice were dissected and CFU counted to determine whether the model was successful.

(B) Results: The degree of pathological inflammatory damage in wild mice was significantly lower than that in SLC44A1 ^-/- mice. The results showed that SLC44A1 could inhibit the survival of Mycobacterium tuberculosis in macrophages and mouse lung tissues, that is, SLC44A1 has the effect of inhibiting the proliferation of M.tb in vivo.

Claims (9)

1. Use of SLC44A1 in the preparation of a drug for preventing Mycobacterium tuberculosis infection, wherein the amino acid sequence of SLC44A1 is shown in SEQ ID NO.1, wherein SLC44A1 is a solute carrier family 44 member 1 functional protein present in the cytoplasm and mitochondria, and has the effect of significantly regulating and inhibiting the invasion of Mycobacterium tuberculosis into host macrophages. SEQ ID NO.1: 1MGCCSSASSAAQSSKREWKP LEDRSCTDIP WLLLFILFCI GMGFICGFSI ATGAAARLVS 61GYDSYGNICG QKNTKLEAIP NSGMDHTQRKYVFFLDPCNL DLINRKIKSVALCVAACPRQ121ELKTLSDVQKFAEINGSALC SYNLKPSEYT TSPKSSVLCP KLPVPASAPIPFFHRCAPVN 181ISCYAKFAEA LITFVSDNSVLHRLISGVMT SKEIILGLCL LSLVLSMILM VIIRYISRVL 241VWILTILVIL GSLGGTGVLWWLYAKQRRSP KETVTPEQLQ IAEDNLRALL IYAISATVFT 301VILFLIMLVM RKRVALTIAL FHVAGKVFIH LPLLVFQPFW TFFALVLFWV YWIMTLLFLG361TTGSPVQNEQ GFVEFKISGP LQYMWWYHVV GLIWISEFIL ACQQMTVAGAVVTYYFTRDK421RNLPFTPILA SVNRLIRYHL GTVAKGSFII TLVKIPRMIL MYIHSQLKGKENACARCVLK 481SCICCLWCLE KCLNYLNQNAYTATAINSTNFCTSAKDAFV ILVENALRVA TINTVGDFML541FLGKVLIVCS TGLAGIMLLN YQQDYTVWVL PLIIVCLFAF LVAHCFLSIYEMVVDVLFLC 601FAIDTKYNDG SPGREFYMDKVLMEFVENSRKAMKEAGKGGVADSRELKPM LKKR. 2. The use according to claim 1, wherein the amino acid sequence of the SLC44A1 protein further comprises 80%-99% homologous sequences. 3. The use according to claim 1 or 2, wherein the SLC44A1 protein further comprises its fusion protein, conjugate, nucleic acid encoding the fusion protein or conjugate, and a vector expressing the aforementioned nucleic acid molecule. 4. The use according to any one of claims 1 to 3, wherein the tuberculosis infection includes but is not limited to pulmonary tuberculosis infection, lymphatic tissue tuberculosis infection, bone and joint and spinal tuberculosis infection, and brain tissue tuberculosis infection. 5. An anti-Mycobacterium tuberculosis pharmaceutical composition, comprising SLC44A1 and another anti-Mycobacterium tuberculosis drug, wherein the amino acid sequence of SLC44A1 is the amino acid sequence of claim 1. 6 . The pharmaceutical composition according to claim 5 , wherein the amino acid sequence of the SLC44A1 protein further comprises 80%-99% homologous sequences. 7 . The pharmaceutical composition according to claim 5 , wherein the SLC44A1 protein further comprises a fusion protein, a conjugate thereof, a nucleic acid encoding the fusion protein or the conjugate thereof, and a vector expressing the aforementioned nucleic acid molecule. 8. The pharmaceutical composition according to claim 5, characterized in that the other anti-tuberculosis drug is an active substance that can promote the body's resistance to tuberculosis bacteria and/or a drug that improves tuberculosis symptoms. 9. The pharmaceutical composition according to claim 5 or 6, wherein the drug comprises at least one of a pharmaceutical carrier, an excipient, and an auxiliary material.