Identification and application of mycobacterium tuberculosis bacteriophage susceptibility gene

Abstract

The invention discloses identification and application of a mycobacterium tuberculosis bacteriophage susceptibility gene, and a gene target which affects mycobacterium bacteriophage infection efficiency is obtained through combined application of a mycobacterium tuberculosis CRISPR-KO library and a CRISPRi library and bacteriophage D29 infection screening. Reagents or drugs targeting phage susceptibility genes or encoded proteins thereof are screened based on the gene targets, and the reagents or drugs can knock out the phage susceptibility genes or inhibit expression of the phage susceptibility genes, or inhibit activity of the encoded proteins of the phage susceptibility genes, serve as phage synergists, or are combined with antibiotics to be used with phages, and can be used for preparing drugs for treating the phage susceptibility genes or the encoded proteins of the phage susceptibility genes. The bacteriophage-antibiotic composition has the advantages that a synergistic sterilization effect is achieved, a new strategy is provided for bacteriophage-antibiotic combined treatment of drug-resistant mycobacterium infection, rapid sterilization is expected to be achieved, the treatment course is shortened, and finally the target of rapid treatment of tuberculosis and other pathogenic mycobacterium infection is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to key cell wall synthesis pathway gene targets of Mycobacterium tuberculosis that determine the sensitivity of Mycobacterium phage D29 obtained by CRISPR library screening, and the synergistic bactericidal effect of drugs targeting corresponding genes and phages, providing a new strategy for phage-antibiotic combination therapy for drug-resistant Mycobacterium infections. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), and is one of the main infectious diseases causing death globally. Existing anti-tuberculosis drugs face two core problems: (1) the exacerbation of drug resistance; (2) the long treatment course (the standard regimen requires 6 - 24 months), resulting in poor patient compliance and high recurrence rates. Therefore, it is urgent to develop anti-tuberculosis strategies based on new mechanisms.

[0003] Mycobacteriophage is a type of virus that can specifically infect and lyse bacteria of the genus Mycobacterium, and has played an important role in the research of Mycobacterium molecular biology and genetic engineering. In 1947, Gardner first discovered and named Mycobacteriophage from Mycobacterium smegmatis. So far, more than 13,000 Mycobacteriophages have been isolated using Mycobacterium smegmatis MC 2 155, and the sequencing and annotation of 2,315 of them have been completed. Mycobacteriophages show significant genetic diversity. According to the similarity of nucleotide sequences, they are divided into 32 clusters, and another 7 are listed as monomers due to genomic specificity. Among them, only phages in clusters K and subclusters A2 and A3 can effectively infect Mycobacterium tuberculosis.

[0004] Mycobacteriophages have made breakthrough progress in the clinical treatment of drug-resistant non-tuberculous mycobacterial infections. Using the mycobacteriophage cocktail therapy, multiple patients with extensively drug-resistant Mycobacterium abscessus infections have been successfully cured, demonstrating its clinical safety, feasibility, and effectiveness. Mycobacteriophage D29 is a member of the A2 subgroup and has not only been applied to the clinical treatment of drug-resistant Mycobacterium abscessus infections but also shown significant potential in the treatment of Mycobacterium tuberculosis. In vitro experiments have shown that by preparing a mixture of five mycobacteriophages including phage D29, the occurrence of phage drug resistance and cross-resistance can be effectively reduced, and Mycobacterium tuberculosis can be killed. Moreover, by aerosol inhalation of phage D29, the infection of Mycobacterium tuberculosis in a clinical mouse model can be significantly reduced. Therefore, mycobacteriophage therapy not only provides a new treatment strategy for drug-resistant non-tuberculous mycobacterial infections but also can be used as a potential alternative therapy for tuberculosis. Among them, phage D29 has become a key candidate phage with both basic research and clinical translation value due to its relatively efficient infectivity characteristics against Mycobacterium tuberculosis.

[0005] Despite the broad application prospects, mycobacteriophage therapy still faces challenges such as narrow host range and the evolution of bacterial resistance. Exploring the susceptibility and resistance mechanisms of bacteria to phages is crucial for understanding the interaction between bacteria and phages. Relevant research not only helps to clarify the evolutionary trade-off between the two but also provides theoretical support for the clinical application of phages. For example, optimizing the preparation of phage cocktails to reduce the occurrence probability of resistant mutations; guiding the combined use of phages and antibiotics to exert a synergistic effect and achieve a better bactericidal effect, etc. Currently, there is still a lack of strategies to enhance phage efficacy through host gene-targeted regulation, and the molecular mechanism of mycobacteriophage-drug synergy is also unclear. Identifying host-determining genes that affect mycobacteriophage infection using the high-throughput screening platforms of the Mycobacterium tuberculosis CRISPR-KO knockout library and the CRISPRi inhibition library can provide a theoretical basis for developing targeted small molecules against phage-infectible susceptible genes to enhance the efficacy of mycobacteriophage treatment regimens and shorten the treatment cycle. Summary of the Invention

[0006] In view of the problems existing in the existing phage therapy for the treatment of mycobacterial infections, such as limited host range, evolution of bacterial resistance, unclear synergistic effect with drugs and molecular mechanism, the present invention proposes a phage-based synergistic antibacterial strategy based on host gene targeted regulation. The core content of the present invention includes the following two aspects: 1) By jointly applying the Mycobacterium tuberculosis CRISPR-KO and CRISPRi libraries and screening through the infection of Mycobacterium phage D29, 36 host susceptibility genes that significantly regulate the infection efficiency (|log2FC|>1, p-value<0.05) were systematically identified. Functional enrichment analysis showed that these genes were mainly enriched in the cell wall synthesis pathway (17, 47.2%); knocking out or inhibiting these genes (such as rv1411c, rv3794, rv2673) significantly improved the infection efficiency of phage D29 against Mycobacterium tuberculosis; 2) The first-line anti-tuberculosis drug ethambutol (EMB) targeting embA (rv3794) and embB (rv3795) enhanced the bactericidal effect of phage D29 against Mycobacterium tuberculosis and Mycobacterium abscessus by improving the adsorption ability of phage D29.

[0007] In the first aspect, the present invention provides a method for enhancing the efficiency of Mycobacterium phage based on host gene targeted regulation. The present invention first uses the screening strategy of the Mycobacterium tuberculosis CRISPR-KO and CRISPRi dual libraries, overcomes the off-target effect and coverage bias of a single library, systematically identifies the key host genes affecting the infection efficiency of Mycobacterium phage D29, and reveals that the cell wall synthesis pathway plays a core role in the phage infection process. The present invention has identified a total of 36 phage susceptibility genes, of which 17 genes belong to the cell wall synthesis pathway, accounting for the highest proportion among all the pathways enriched by the susceptibility genes. These susceptibility genes can be used as molecular targets for developing phage potentiators or phage-antibiotic combination therapies, providing new treatment options for drug-resistant tuberculosis and non-tuberculous mycobacterial infections.

[0008] The phage susceptibility genes include, but are not limited to: arabinan synthesis pathway genes (rv3794, rv3795, rv2673), lipoprotein-related genes (rv1411c, rv1166), transporter-related genes (rv3679, rv3783), conserved membrane protein genes (rv3277, rv1476, rv0007, rv0364, rv2174, rv1824, rv2133) and other related genes (rv0431, rv3682, rv2347c).

[0009] Using the phage-susceptible gene or its encoded protein as a screening target, screening for targeting reagents or drugs that can knockout, inhibit, or regulate the expression thereof, for enhancing the adsorption efficiency and bactericidal effect of mycobacteriophages. The reagent or drug targeting the phage-susceptible gene or its encoded protein can knockout the phage-susceptible gene or inhibit its expression, or inhibit the activity of the protein encoded by the phage-susceptible gene, thereby serving as a phage synergist or a phage-antibiotic combination therapy drug to achieve the therapeutic effect of anti-mycobacterial infection.

[0010] The phage-susceptible gene encodes an amino acid sequence shown in any one of SEQ ID NO: 1-17 in the sequence listing, or a polypeptide sequence having ≥80% sequence homology with any one of SEQ ID NO: 1-17 and retaining the same biological function.

[0011] Homologous genes of the phage-susceptible gene are present in other pathogenic mycobacteria, including Mycobacterium abscessus, Mycobacterium avium complex, and strains having a genomic homology of ≥85% therewith.

[0012] The reagent or drug targeting the phage-susceptible gene or the encoded protein includes, but is not limited to: small molecule inhibitors, siRNA, antisense oligonucleotides, CRISPR gene editing systems, or monoclonal antibodies.

[0013] Among them, the small molecule inhibitor, siRNA, antisense oligonucleotide, or monoclonal antibody serves as a targeting agent for the susceptible gene and can be combined with antibiotics and phages to form a synergistic treatment regimen.

[0014] The reagent or drug targeting the phage-susceptible gene or its encoded protein and mycobacteriophage form a composite preparation, and the phage is preferably engineered to adapt to cell wall-deficient strains.

[0015] On the other hand, the present invention provides a combination therapy method for enhancing the bactericidal efficacy of mycobacteriophages, by co-administering an inhibitor or drug targeting the cell wall synthesis pathway or key enzymes of lipid metabolism of Mycobacterium tuberculosis with mycobacteriophages. Among them, the mycobacteriophages include, but are not limited to, D29 or its engineered variants. D29 is genetically engineered to enhance the adsorption efficiency, and the engineering methods include: optimization of the tail fiber protein gene, host range expansion mutation, or insertion of a promoter regulatory element.

[0016] Research of the present invention shows that ethambutol (EMB), a drug targeting cell wall synthesis, can synergistically enhance the antibacterial efficacy of mycobacteriophage D29. By specifically inhibiting the activities of arabinosyltransferases EmbA (Rv3794) and EmbB (Rv3795), EMB blocks the synthesis of arabinogalactan, disrupts the structural integrity of the cell wall, significantly improves the adsorption efficiency of phage D29, and enhances its lytic ability against Mycobacterium tuberculosis and Mycobacterium abscessus. Pretreatment with delamanid (DLM), an anti-tuberculosis drug targeting mycolic acid biosynthesis, has no effect on the infection efficiency of phage D29.

[0017] The combined use form of the inhibitor or drug and the phage is sequential administration or synchronous administration, and it is applicable to Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium avium complex, and pathogenic mycobacteria with ≥80% genomic homology thereto.

[0018] The treatment further includes the combined use with other anti-tuberculosis drugs, and the drugs include rifampicin, isoniazid, pyrazinamide, and bedaquiline.

[0019] Advantages of the present invention: The present invention identifies multiple new targets that can be used as potential mycobacteriophage enhancers, and provides a combined application strategy of anti-tuberculosis drugs and phages. Inhibitors or drugs studied with these proteins or genes as targets can be used in combination with mycobacteriophages, which is expected to achieve rapid bactericidal effect, shorten the treatment course, and ultimately achieve the goal of rapidly treating tuberculosis and other pathogenic mycobacterial infections. Description of the Drawings

[0020] Figure 1 . Schematic diagram of the phage D29 infection screening process based on the Mycobacterium tuberculosis CRISPR-KO / CRISPRi library in Example 1. Phage D29 with an MOI≈0.1 was inoculated into CRISPR-KO and CRISPRi library strains respectively, and samples were collected after 48 h and 120 h of infection; genomic DNA was extracted, the sgRNA region was amplified and high-throughput sequencing was performed; finally, by comparing the differences in the abundances of each sgRNA between the phage-treated group and the control group without phage, the resistance genes and susceptible genes affecting the infection efficiency of D29 were identified.

[0021] Figure 2 . Screening results of the Mycobacterium tuberculosis CRISPR library of phage D29 in Example 2. A. Volcano plot of differential genes of the CRISPRi library at 48 h (left) and 120 h (right) time points; B. Volcano plot of differential genes of the CRISPR-KO library at 48 h (left) and 120 h (right) time points.

[0022] Figure 3 . For Figure 2Statistical analysis of the functional classification of differentially expressed genes between the CRISPR-KO library (upper panel) and the CRISPRi library (lower panel) in the 120-hour screening as shown.

[0023] Figure 4 . Results of the drop plate of Mycobacterium tuberculosis susceptible gene knockout or inhibition strain D29 in Example 3. Among them, rv1411c is a non-essential gene, and the knockout strain was used for the experiment; rv2673 and rv3794 are essential genes, and CRISPRi inhibition strains were constructed for the experiment.

[0024] Figure 5 . In Example 4, EMB enhances the adsorption ability of phage D29 to improve its infection efficiency against Mycobacterium tuberculosis. A. 0.5×MIC 50 Results of the dilution drop plate of phage D29 after pretreatment of Mycobacterium tuberculosis with EMB and DLM; B. 0.5×MIC 50 Statistical analysis of the adsorption rate of phage D29 to Mycobacterium tuberculosis after pretreatment with EMB; C. Statistical analysis of the number of particles of phage D29 adsorbed to the cell surface under transmission electron microscopy, with 15 cells randomly counted in the EMB pretreatment group and the control group; D. Evaluation of the inhibitory effect of the combination of phage D29 and EMB on the growth of Mycobacterium tuberculosis by the checkerboard method.

[0025] Figure 6 . Study on the combined inhibition of Mycobacterium abscessus by EMB and phage D29 in Example 5. A. 0.5×MIC 50 Statistical analysis of the adsorption rate of phage D29 to Mycobacterium abscessus after pretreatment with EMB; B. Evaluation of the inhibitory effect of the combination of phage D29 and EMB on the growth of Mycobacterium abscessus by the plate checkerboard method. Detailed implementation manners

[0026] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0027] Example 1. Screening of genes related to the infection of Mycobacterium phage D29 by combining CRISPR-KO and CRISPRi libraries

[0028] As Figure 1 shown, the cryopreserved solutions of the Mycobacterium tuberculosis H37Ra CRISPR-KO and CRISPRi libraries were placed in a culture flask for resuscitation culture. When the resuscitated bacterial solution grew to an OD 600 of 1.0, it was transferred to fresh 7H9 + OADC medium at a ratio of 1:50, and the initial OD 600 was 0.02. Among them, 100 ng / mL anhydrotetracycline (ATc) was added to the CRISPRi library to induce the expression of dCas9 to inhibit the expression of sgRNA-targeted genes. When the bacterial solution grew to an OD600 When it was 0.8, after washing the bacteria twice with 7H9 + OADC + 2 mM CaCl2 without Tween 80, resuspend it to the original volume and add phage D29 with MOI≈0.1. Mix the mixture with the same volume of upper agar and quickly spread it on the surface of a 7H10 + OADC solid plate. At the same time, set up a control group without phage. For the CRISPRi library, add 100 ng / mL ATc to the solid plate. After infecting at 37 °C for 48 h and 120 h, scrape the upper agar, add solubilization solution, solubilize it at 50 °C, and then centrifuge at 5000 g for 10 min to collect the bacterial precipitate. In addition, take the supernatant of the solubilization solution at 12 h and 24 h to evaluate the release of easily lysed mutant DNA. Finally, after extracting genomic DNA from the bacterial precipitate, amplify the sgRNA sequence region and perform amplicon sequencing to compare the abundance differences of each sgRNA between the phage treatment group and the control group. Use MAGeCK or similar software for enrichment analysis, with p-value < 0.05 and |log2FC| > 1 as the screening threshold to identify host genes affecting phage D29 infection.

[0029] In this example, a set of efficient CRISPR-KO / CRISPRi parallel screening platforms was established, which can systematically reveal the key genes affecting phage D29 infection in Mycobacterium tuberculosis, providing important candidates for subsequent targeted modification or drug combination strategies.

[0030] Example 2: Differential enrichment and functional clustering analysis of phage D29 infection-related resistant and susceptible genes in Mycobacterium

[0031] According to the method described in Example 1, collect the bacterial solutions of the treatment groups and the phage-free control groups of the CRISPR-KO and CRISPRi libraries after phage D29 infection for 48 h and 120 h respectively, and extract genomic DNA. After amplifying sgRNA, construct a sequencing library and perform high-throughput sequencing using the Illumina platform. Use MAGeCK software to compare the abundances of each sgRNA between the treatment group and the control group, and set the thresholds p-value < 0.05 and |log2FC| > 1 to identify significantly enriched (resistant) or depleted (susceptible) genes. Align the screened differential genes to the Mycobacterium database for functional classification statistics, and focus on analyzing the enrichment of susceptible genes in each functional category.

[0032] As Figure 2As shown, at the two time points of 48 h and 120 h, the target genes of the classical anti-tuberculosis drug ethambutol, rv3794 (embA), rv3795 (embB), and the lipid transport regulator rv1411c (lprG), all showed a significant depletion (downregulation) trend; while genes such as rv3092c that may be involved in membrane transport were significantly enriched (upregulated). The amplitude of enrichment or depletion of differential genes increased with the prolongation of the infection time, suggesting their dynamic regulatory role during the infection process of phage D29 ( Figure 2 ). In the 120 h sampling, 37 differential genes were identified by the CRISPR-KO library, and 54 differential genes were identified by the CRISPRi library. Functional clustering analysis showed that these genes were significantly enriched in two functional categories: cell wall synthesis and dynamic regulation, and intermediate metabolism and energy respiration ([[ID=③]] Figure 3 )). Further combined analysis of 36 CRISPR-KO and CRISPRi susceptible genes showed that 17 of them were enriched in the cell wall synthesis pathway (Table 1).

[0033] In this example, through high-throughput sgRNA abundance analysis and functional clustering, a series of resistant and susceptible genes were systematically identified, revealing the key role of Mycobacterium tuberculosis cell wall structure remodeling and metabolic reprogramming during the infection process of phage D29, laying a foundation for subsequent targeted gene verification and combined intervention strategies.

[0034] Table 1. List of susceptible genes in the cell wall synthesis pathway screened by the CRISPR library

[0035] ORF_ID Gene Name Log2Foldchange Description of Encoded Protein Function Rv3277 — -1.1078 Putative Conserved Transmembrane Protein Rv3679 — -1.1309 Putative Anion Transport ATPase Rv1411c lprG -1.7153 Conserved Lipoprotein LprG Rv3795 embB -2.5083 Integral Membrane Indoleacetylinositol Arabinosyltransferase EmbB Rv3794 embA -2.3996 Integral Membrane Indoleacetylinositol Arabinosyltransferase EmbA Rv2673 aftC -2.1647 Putative Arabinosyltransferase AftC Rv1476 — -1.9555 Putative Membrane Protein Rv3783 rfbD -1.5135 Putative O-Antigen / Lipopolysaccharide Transport Integral Membrane Protein ABC Transporter RfbD Rv0431 — -1.327 Putative Tuberculin-Related Peptide Rv3682 ponA2 -1.3148 Putative Bifunctional Membrane-Associated Penicillin-Binding Protein 1A / 1B PonA2 Rv0007 — -1.1355 Putative Conserved Membrane Protein Rv0364 — -1.1332 Putative Conserved Transmembrane Protein Rv2347c esxP -1.1325 Putative ESAT-6-Like Protein EsxP Rv2174 — -1.1273 α(1→6) Mannosyltransferase Rv1824 — -1.106 Conserved Hypothetical Membrane Protein Rv2113 — -1.06 Putative Integral Membrane Protein Rv1166 lpqW -1.0018 Putative Conserved Lipoprotein LpqW

[0036] Example 3. Inhibiting or knocking out susceptible genes in the cell wall synthesis pathway enhances the infectivity of phage D29

[0037] In this example, the enhancing effect of knocking out genes and inhibiting susceptible genes related to cell wall synthesis by CRISPRi on the infectivity efficiency of phage D29 was evaluated. The knockout strain of rv1411c was obtained using the CRISPR-Cas9-assisted NHEJ method. Using the dCas9-sgRNA system, sgRNAs were designed for rv2673 and rv3794 respectively, and stably expressed through an integration vector to construct CRISPRi inhibitory strains of the corresponding genes. Bacterial liquid in the logarithmic growth phase (OD 600 ≈0.6) was mixed with upper-layer agar and plated onto 7H10 + OADC solid plates (100 ng / mL Atc was added for CRISPRi inhibitory strains), and phage D29 was diluted and plated, and the infectivity efficiency was evaluated by the number of plaques (PFU). As Figure 4 shown, the knockout of rv1411c and the CRISPRi inhibition of rv2673 and rv3794 significantly improved the infectivity efficiency of phage D29.

[0038] It can be seen that by knocking out or inhibiting CRISPRi against the key susceptible genes (rv1411c, rv2673, rv3794) in the Mycobacterium tuberculosis cell wall synthesis pathway, the infection efficiency of phage D29 against the host bacteria can be significantly enhanced, providing a new technical route for the combined strategy of phage and genetic engineering or drugs.

[0039] Example 4. Study on the synergistic effect of the Mycobacterium tuberculosis cell wall synthesis inhibitor EMB and phage D29

[0040] In this example, the enhancing effect of sub-inhibitory concentration of ethambutol (EMB) on the infectivity and bactericidal effect of phage D29 was evaluated.

[0041] Use 0.5×MIC 50 of the EMB solution (MIC 50 predetermined by the microdilution method) to pre-incubate the H37Ra bacterial solution for about 5 days. The double-layer agar phage drop plate method was used to evaluate the difference in the infection efficiency of phage D29 between the EMB treatment group and the control group through PFU. Under the same conditions, 0.5×MIC 50 of DLM was used to replace EMB to evaluate the effect of DLM on the infection efficiency of phage D29. Phage D29 was used to infect H37Ra at MOI = 0.001, and samples were taken at different time points (0.5 h, 1 h, 1.5 h, 2 h). The unadsorbed phages were removed by centrifugation, and the phage titer in the supernatant was determined by the double-layer agar method to calculate the adsorption rate. The adsorbed samples at the 0.5 h time point were negatively stained and then photographed under a transmission electron microscope to observe the phage adsorption on the surface of the bacteria. To evaluate the bacterial survival rate and synergistic effect, the checkerboard dilution method was used to calculate the bacterial growth rate to determine the synergistic effect.

[0042] As Figure 5 shown, pretreatment of Mycobacterium tuberculosis with sub-inhibitory concentration (0.5×MIC 50 ) of EMB can significantly improve the infectivity of mycobacteriophage D29 ( Figure 5 A in). Through adsorption experiments and electron microscopy observations, we showed that pretreatment of Mycobacterium tuberculosis with EMB significantly enhanced the adsorption ability of phage D29 ( Figure 5 B, C in). Further, the combined bactericidal effect of phage D29 and EMB was evaluated by the checkerboard method, and the results showed that phage D29 and EMB had a significant synergistic effect ( Figure 5 D in).

[0043] It can be seen that by pre-treating with sub-inhibitory concentration drugs against the key targets of Mycobacterium tuberculosis cell wall synthesis, not only the adsorption efficiency of phage D29 was significantly enhanced, but also a synergistic bactericidal effect was achieved in vitro, providing a new strategy for phage therapy of drug-resistant mycobacterial infections.

[0044] Research on the Combined Killing of Mycobacterium abscessus by the Cell Wall Synthesis-Targeting Drug EMB and Bacteriophage D29 in Example 5

[0045] Select the clinical strain Mab_94 of Mycobacterium abscessus, which is characterized by being rough. Mix gradient PFU bacteriophage D29 and gradient CFU Mab_94 in a 96-well plate according to a checkerboard layout, and plate them separately onto 7H10 + OADC and 7H10 + OADC + 0.5×MIC 50 EMB plates, and determine the synergistic effect by observing the differences in colony growth. For the adsorption experiment, determine the MIC according to the microdilution method 50 , and select 0.5×MIC 50 Ethambutol (EMB) solution, pre-incubate the Mab_94 bacterial solution for about 3 days, infect Mab_94 with bacteriophage at MOI = 0.001, and sample at different time points (0.5 h, 1 h, 1.5 h). Centrifuge to remove unadsorbed bacteriophage, and determine the supernatant bacteriophage titer by the double-layer agar method, and calculate the adsorption rate.

[0046] As Figure 6 shown, the results of the adsorption experiment show that the adsorption rate of the EMB pretreatment group is significantly higher than that of the non-pretreatment group, indicating that EMB can enhance the adsorption ability of D29 to Mycobacterium abscessus ( Figure 6 A in). The results of the plate checkerboard method show that the combination of EMB and bacteriophage makes the growth state of Mab_94 significantly worse than that of using EMB or D29 alone, indicating that the two have a synergistic bactericidal effect ( Figure 6 B in).

[0047] This example shows that by pre-treating Mycobacterium abscessus with sub-inhibitory concentration of EMB, not only the binding efficiency of bacteriophage D29 is significantly improved at the adsorption stage, but also the synergistic effect of the two is achieved in vitro, providing a new combination strategy for phage therapy of non-tuberculous mycobacterial infections.

[0048] The present invention has been described in detail with the above embodiments, but it should be understood that the above embodiments are only several preferred modes of the present invention, and the present invention is not limited to the described embodiments. Without departing from the general concept and technical solution of the present invention, those skilled in the art can make various equivalent deformations, improvements or replacements to the above embodiments. All changes, modifications or alternative solutions made by them should be regarded as equivalent technical solutions of the present invention and fall within the protection scope of the present invention.

Claims

1. Use of a phage-susceptible gene of Mycobacterium or its encoded protein as a screening target in screening reagents or drugs for anti-Mycobacterium infection, characterized in that, The phage-susceptible genes are selected from at least one of the following genes: Mycobacterium tuberculosis genes rv3794, rv3795, rv2673, rv1411c, rv1166, rv3679, rv3783, rv3277, rv1476, rv0007, rv0364, rv2174, rv1824, rv2133, rv0431, rv3682, rv2347c, and homologous genes of these Mycobacterium tuberculosis genes in other pathogenic mycobacteria; the reagent or drug for anti-mycobacterial infection targets the phage-susceptible gene or its encoded protein, and is capable of knocking out the phage-susceptible gene or inhibiting its expression, or inhibiting the activity of the protein encoded by the phage-susceptible gene.

2. The application according to claim 1, characterized in that The phage-susceptible gene encodes an amino acid sequence shown in any one of SEQ ID NO: 1-17 in the sequence listing, or a polypeptide sequence having ≥80% sequence homology with any one of SEQ ID NO: 1-17 and retaining the same biological function.

3. The application according to claim 1, characterized in that, The mycobacteria include Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium avium complex, and strains having ≥85% genomic homology therewith.

4. The application according to claim 1, characterized in that, The reagent or drug targeting the phage-susceptible gene or its encoded protein includes: small molecule inhibitors, siRNA, antisense oligonucleotides, CRISPR gene editing systems, and / or monoclonal antibodies.

5. Use of a reagent or drug targeting a Mycobacteriophage-susceptible gene or its encoded protein in the preparation of a Mycobacteriophage synergist or a phage-antibiotic combination therapy drug, characterized in that, The phage-susceptible genes are selected from at least one of the following genes: Mycobacterium tuberculosis genes rv3794, rv3795, rv2673, rv1411c, rv1166, rv3679, rv3783, rv3277, rv1476, rv0007, rv0364, rv2174, rv1824, rv2133, rv0431, rv3682, rv2347c, and homologous genes of these Mycobacterium tuberculosis genes in other pathogenic mycobacteria; the reagent or drug targeting the mycobacterial phage-susceptible gene or its encoded protein is capable of knocking out the phage-susceptible gene or inhibiting its expression, or inhibiting the activity of the protein encoded by the phage-susceptible gene.

6. The application according to claim 5, characterized in that The phage-susceptible gene encodes an amino acid sequence shown in any one of SEQ ID NO: 1-17 in the sequence listing, or a polypeptide sequence having ≥80% sequence homology with any one of SEQ ID NO: 1-17 and retaining the same biological function; the mycobacteria include Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium avium complex, and strains having ≥85% genomic homology therewith.

7. The application according to claim 5, characterized in that, The reagent or drug targeting the mycobacterial phage-susceptible gene or its encoded protein includes: small molecule inhibitors, siRNA, antisense oligonucleotides, CRISPR gene editing systems, and / or monoclonal antibodies.

8. A compound preparation for treating tuberculosis or other pathogenic mycobacterial infections, comprising a reagent or drug targeting a mycobacteriophage-susceptible gene or its encoded protein and a mycobacteriophage, wherein, The phage-susceptible genes are selected from at least one of the following genes: Mycobacterium tuberculosis genes rv3794, rv3795, rv2673, rv1411c, rv1166, rv3679, rv3783, rv3277, rv1476, rv0007, rv0364, rv2174, rv1824, rv2133, rv0431, rv3682, rv2347c, and homologous genes of these Mycobacterium tuberculosis genes in other pathogenic mycobacteria; the reagent or drug targeting the phage-susceptible gene of mycobacteria or its encoded protein can knockout the phage-susceptible gene or inhibit its expression, or inhibit the activity of the protein encoded by the phage-susceptible gene.

9. The composite preparation according to claim 8, wherein, The reagent or drug targeting the phage-susceptible gene of mycobacteria or its encoded protein includes: small molecule inhibitors, siRNA, antisense oligonucleotides, and / or monoclonal antibodies.

10. The composite preparation according to claim 9, characterized in that, The composite preparation contains ethambutol and mycobacteriophage D29 or a phage engineered to be adapted to mycobacterial cell wall-deficient strains.

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