Rv2241 gene as an inhibitory target against Mycobacterium tuberculosis biofilm and its application

Abstract

The present invention discloses the Rv2241 gene as an inhibitory target for Mycobacterium tuberculosis biofilms and its application, relating to the field of biology. The inventors of this application discovered that the Rv2241 gene is a gene associated with Mycobacterium tuberculosis biofilm formation and that inhibiting the expression level or protein activity of the Rv2241 gene can inhibit the formation of Mycobacterium tuberculosis biofilms. This gene can serve as a target for the development of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm inhibitors. Small molecule inhibitors targeting this gene can effectively inhibit Mycobacterium tuberculosis biofilm formation, thereby increasing the sensitivity of Mycobacterium tuberculosis to antibiotics.

Description

Technical Field

[0001] The present invention relates to the biological field, in particular to an Rv2241 gene as an inhibitory target against Mycobacterium tuberculosis biofilm and an application thereof. Background Art

[0002] Mycobacterium tuberculosis is one of the oldest human pathogens, and tuberculosis (TB) caused by it remains the infectious disease with the highest mortality rate among single pathogenic infections. This is mainly due to the lack of sufficient understanding of its gene functions and pathogenic mechanisms.

[0003] One of the most intriguing discoveries in recent years in biofilm research is the discovery that Mycobacterium tuberculosis can form biofilms. Biofilms are a well-established mechanism of antibiotic resistance, and biofilm formation may also be linked to the pathogenicity of M. tuberculosis. The first clinical case of biomaterial-associated tuberculosis infection occurred following prosthetic joint replacement. Clinical observations revealed that even when the isolates were susceptible to the antibiotics used in vitro, removal of the prosthetic joint was essential for controlling these infections. This is presumably because M. tuberculosis forms biofilms on biomaterial surfaces, which are insensitive to anti-TB drugs and cannot be completely eliminated. Further studies have also demonstrated that M. tuberculosis can form biofilms in vitro, opening up a new research direction for the pathogenesis and anti-infective control of this disease. Furthermore, the importance of biofilms in tuberculosis is reflected in their possible involvement in the formation of caseous necrosis and cavitation in lung tissue. These findings have stimulated interest in the mechanisms of biofilm formation as potential targets for new tuberculosis treatments. In the future, it is necessary to consider tuberculosis as a biofilm-associated disease to improve cure rates and shorten treatment durations.

[0004] However, there is not much research information on the biofilm-related genes of this bacteria, its biofilm formation mechanism and its formation conditions.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The object of the present invention is to provide.

[0007] The present invention is achieved in that:

[0008] In a first aspect, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in the preparation of a product for inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm.

[0009] In a second aspect, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm.

[0010] In a third aspect, an embodiment of the present invention provides a product for inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm, wherein the product comprises an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein.

[0011] In a fourth aspect, an embodiment of the present invention provides a method for screening inhibitors of Mycobacterium tuberculosis and / or its biofilm, wherein an agent capable of inhibiting Mycobacterium tuberculosis and / or its biofilm is screened out based on the effect of the candidate agent on the expression level of the Rv2241 gene or the activity of the Rv2241 protein.

[0012] In a fifth aspect, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in the preparation of a drug for treating or assisting in the treatment of diseases or symptoms caused by Mycobacterium tuberculosis.

[0013] In a sixth aspect, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in the preparation of a product for increasing the sensitivity of Mycobacterium tuberculosis to antibiotics.

[0014] In a seventh aspect, embodiments of the present invention provide use of an agent that inhibits the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in improving the sensitivity of Mycobacterium tuberculosis to antibiotics.

[0015] In an eighth aspect, an embodiment of the present invention provides a composition, the active ingredients of which include: an antibiotic and an inhibitor, wherein the inhibitor includes an agent that inhibits the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein.

[0016] The present invention has the following beneficial effects:

[0017] The present invention discovered that the Rv2241 (aceE) gene is associated with biofilm formation in Mycobacterium tuberculosis. By inhibiting the expression level or protein activity of the Rv2241 gene, biofilm formation in Mycobacterium tuberculosis can be inhibited. This gene can serve as a target for the development of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm inhibitors. Small molecule inhibitors targeting this gene can effectively inhibit biofilm formation in Mycobacterium tuberculosis, thereby increasing the sensitivity of Mycobacterium tuberculosis to antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 PCR identification of Rv2241 gene knockout mutants; A is a schematic diagram of the construction of Rv2241 homologous recombination inactivation mutants; B is the electrophoresis results of PCR products of the left and right arms and the interior of the gene; M1: Trans2000 ladder; M2: Trans2000plus ladder;

[0020] Figure 2 Comparison of biofilm formation between the wild-type Mtb H37Rv wild-type strain and the MtbΔaceE mutant. Figure A shows the wild-type and mutant bacterial cultures inoculated in test tubes and cultured for 5 days. Biofilm formation was observed at the air-liquid interface of the wild-type culture tube, while the MtbΔaceE mutant failed to form a biofilm. Figure B shows biofilm formation in a 96-well plate. Figure C shows the results of crystal violet staining quantification. Figures A and B show the wild-type Mtb H37Rv wild-type strain: H37Rv; and the MtbΔaceE mutant: aceE-KO.

[0021] Figure 3 To investigate the effect of the aceE gene on the growth of Mycobacterium tuberculosis under high-concentration rifampicin pressure, the survival of the wild-type strain and the mutant strain after 7-day static culture at 37°C in liquid culture medium containing final concentrations of 0.5 μg / ml, 2.0 μg / ml, and 10.0 μg / ml rifampicin (A) and 1 μg / ml, 5.0 μg / ml, and 20.0 μg / ml levofloxacin (B) was compared.

[0022] Figure 4 The results are the analysis results of the cell wall lipid composition of the Rv2241 gene knockout Mycobacterium tuberculosis mutant;

[0023] Figure 5 Matrix diagram for amino acid sequence homology analysis of AceE proteins from different species. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0025] The inventors of the present application have found that by constructing Rv2241 (aceE) gene knockout mutants, there are significant differences in biofilm formation ability between mutants and wild strains. In biofilm formation culture medium without adding TW80, Mycobacterium tuberculosis H37Rv wild strain can form obvious biofilm at the gas-liquid interface, while Δ aceE mutants fail to form biofilm structure, confirming that aceE gene knockout has greatly reduced the biofilm formation ability of Mycobacterium tuberculosis. Meanwhile, in antibiotic tolerance experiments, it was found that the survival ability of Δ aceE mutants under the effect of high concentrations of antibiotics was significantly lower than that of H37Rv wild strains. When the amino acid sequence alignment analysis of homologous genes of different species was carried out on this gene, it was found that this gene is relatively conservative in different mycobacteria, and the sequence similarity in organisms such as Escherichia coli, mice and humans is lower, indicating that this gene may be used as a target for the development of mycobacterium tuberculosis or even mycobacterium biofilm inhibitors. Small molecule inhibitors using this gene as a target can effectively inhibit mycobacterium tuberculosis from forming biofilms, thereby improving the sensitivity of mycobacterium tuberculosis to antibiotics.

[0026] In one aspect, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in the preparation of a product for inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm.

[0027] Specifically, the Mycobacterium tuberculosis Rv2241 gene can also be called aceE gene, and its sequence ID number is 887246.

[0028] In some embodiments, the product includes any one of a reagent, a kit, and a drug.

[0029] In some embodiments, the inhibition can be understood as any one or more of: reducing, slowing down the growth rate, and inhibiting abnormal growth.

[0030] In some embodiments, the inhibition of Mycobacterium tuberculosis can be understood as anti-Mycobacterium tuberculosis, including: inhibiting the generation of Mycobacterium tuberculosis, slowing the growth rate of Mycobacterium tuberculosis, reducing the rate of abnormal growth of Mycobacterium tuberculosis, and reducing the survival rate of Mycobacterium tuberculosis.

[0031] In some embodiments, inhibiting Mycobacterium tuberculosis biofilm comprises: inhibiting the formation of Mycobacterium tuberculosis biofilm, reducing the formation rate of Mycobacterium tuberculosis biofilm, reducing the rate of abnormal growth of Mycobacterium tuberculosis biofilm, and reducing any one or more of the efficacy or activity of Mycobacterium tuberculosis biofilm.

[0032] On the other hand, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm.

[0033] In some embodiments, the environment that inhibits Mycobacterium tuberculosis biofilm includes: an in vivo environment and / or an in vitro environment.

[0034] In some embodiments, the specific definitions of inhibiting Mycobacterium tuberculosis and inhibiting the biofilm of Mycobacterium tuberculosis are the same as those described in the above corresponding embodiments and will not be repeated here.

[0035] On the other hand, an embodiment of the present invention provides a product for inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm, wherein the active ingredient includes: an agent that inhibits the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein.

[0036] In some embodiments, the active ingredient may further include other ingredients capable of inhibiting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis biofilm.

[0037] In some embodiments, the product includes any one of a reagent, a kit, and a drug.

[0038] On the other hand, an embodiment of the present invention provides a method for screening inhibitors of Mycobacterium tuberculosis and / or its biofilm, based on the effect of candidate agents on the expression level of the Rv2241 gene or the activity of the Rv2241 protein, to screen out agents capable of inhibiting Mycobacterium tuberculosis and / or its biofilm.

[0039] In some embodiments, the screening criteria may include: if the candidate agent can inhibit the expression level of the Rv2241 gene or the activity of the Rv2241 protein, then the candidate agent is judged to have the ability to inhibit Mycobacterium tuberculosis and / or its biofilm.

[0040] In some embodiments, the screening criteria may further include: if the candidate agent has a stronger ability to inhibit the expression level of the Rv2241 gene or the activity of the Rv2241 protein, then the candidate agent is judged to have a higher ability to inhibit Mycobacterium tuberculosis and / or its biofilm.

[0041] On the other hand, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in the preparation of a drug for treating or assisting in the treatment of diseases or symptoms caused by Mycobacterium tuberculosis.

[0042] In some embodiments, the diseases or symptoms caused by Mycobacterium tuberculosis include diseases or symptoms caused by biofilm formation by Mycobacterium tuberculosis.

[0043] In some embodiments, diseases caused by Mycobacterium tuberculosis include tuberculosis. Tuberculosis is a chronic infectious disease that can affect all organs of the body, with pulmonary tuberculosis being the most common. Symptoms of pulmonary tuberculosis include cough, scanty sputum, hemoptysis, chest pain, and dyspnea. Systemic symptoms also include fatigue, hot flashes, night sweats, fever, poor stomach sodium, and menstrual irregularities.

[0044] In some embodiments, tuberculosis also includes gastric tuberculosis. Clinical manifestations vary widely, with some cases being asymptomatic or mild, others resembling chronic gastritis or gastric cancer, and most resembling ulcer disease. Patients experience upper abdominal discomfort or pain, often accompanied by acid reflux and belching, with the abdominal pain unrelated to eating. Vomiting associated with pyloric obstruction is often more severe in the afternoon and evening. The vomitus is the food consumed and does not contain bile. Occult blood may be negative, and abdominal distension is relieved after vomiting. In addition to gastric symptoms, systemic tuberculosis symptoms such as fatigue, weight loss, afternoon fever, and night sweats may also be present.

[0045] In some embodiments, tuberculosis also includes hepatic tuberculosis. The most common symptoms are fever and fatigue. Other symptoms include loss of appetite, nausea, vomiting, abdominal distension, and diarrhea. Fever often occurs in the afternoon, sometimes accompanied by chills and night sweats. The fever can range from low-grade to remittent, with a high fever reaching 39-41°C. Tuberculosis patients may experience recurring fevers over a long period of time.

[0046] In some embodiments, tuberculosis also includes intestinal tuberculosis. Clinical manifestations are often subtle in the early stages, with a slow onset and a long course. If coexisting with extraintestinal tuberculosis, its clinical manifestations can be masked and overlooked. Therefore, active extraintestinal tuberculosis, for example, presents with obvious gastrointestinal symptoms.

[0047] On the other hand, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in the preparation of a product for increasing the sensitivity of Mycobacterium tuberculosis to antibiotics.

[0048] In some embodiments, the antibiotics include: rifamycin antibiotics (such as rifampicin) and quinolone antibiotics (such as levofloxacin).

[0049] On the other hand, embodiments of the present invention provide use of an agent for inhibiting the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein in improving the sensitivity of Mycobacterium tuberculosis to antibiotics.

[0050] In addition, an embodiment of the present invention provides a composition, the active ingredients of which include: an antibiotic and an inhibitor, wherein the inhibitor includes an agent that inhibits the expression level of the Mycobacterium tuberculosis Rv2241 gene and / or the activity of the Rv2241 protein.

[0051] In some embodiments, the composition is a pharmaceutical composition used to inhibit or assist in inhibiting Mycobacterium tuberculosis and / or its biofilm.

[0052] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0053] Example 1

[0054] (1) Construction and identification of Rv2241 gene knockout mutants:

[0055] Using the principle of homologous recombination, primer pairs were designed to amplify the homologous recombination arms upstream, downstream, and on the inner left and right wings of the Rv2241 (aceE) gene. The genomic DNA of H37Rv was used as a template to amplify the homologous recombination arms and construct the recombination arms into the AE159 shuttle plasmid. The homologous recombination plasmid was introduced into H37Rv by means of temperature-sensitive phage-mediated transfection. The hygromycin resistance marker in the recombination element was used to screen positive recombinant clones, and the ΔaceE mutant strain ( Figure 1 ).

[0056] (2) Rv2241 gene knockout Mycobacterium tuberculosis mutants cannot form biofilms:

[0057] The biofilm formation experiment of the ΔaceE mutant of Mycobacterium tuberculosis found that the ΔaceE mutant could not form a biofilm structure in the contact layer between the liquid surface and the air, while the H37Rv wild strain could form a biofilm as shown in the figure ( Figure 2 A and B), suggesting that aceE gene deletion may affect the pathogenicity and drug-resistant phenotype of Mycobacterium tuberculosis.

[0058] (3) Inactivation of the Rv2241 gene reduces the ability of Mycobacterium tuberculosis to persist and grow under high-concentration drug pressure conditions:

[0059] The drug retention model was used to analyze the effect of the Rv2241 (aceE) gene on the persistence of Mycobacterium tuberculosis under high concentrations of antibiotics, and the survival of the wild-type and knockout strains in liquid culture medium containing high concentrations of rifampicin and levofloxacin was compared. Figure 3 ), after 7 days of culture, the survival rate of the knockout strain was significantly lower than that of the wild-type strain, indicating that the aceE gene is related to the persistence and survival of Mycobacterium tuberculosis under high-concentration drug pressure.

[0060] (4) The cell wall lipid composition of the Rv2241 gene knockout Mycobacterium tuberculosis mutant was significantly changed:

[0061] Using mass spectrometry analysis, the composition of several major lipids, including mycolic acid, phospholipids, and glycophospholipids, in the cell wall of the ΔaceE knockout strain and the H37Rv wild-type strain was compared. It was found that after the aceE gene was knocked out, the cell wall lipid composition of Mycobacterium tuberculosis changed significantly, including phosphatidylethanolamine (PE), phosphatidylinositol (PI), diphosphatidylglycerol (CL), phosphatidic acid (PA), phosphatidylglycerol (PG), and mycolic acid (MA). The content of almost all lipids was significantly reduced ( Figure 4 ), suggesting that the aceE gene may be involved in the synthesis of cell wall lipids, and inactivation of this gene would reduce the cell wall lipid content, thereby leading to biofilm formation defects in Mycobacterium tuberculosis.

[0062] (5) Similarity analysis between the amino acid sequence of the Rv2241 gene and homologous gene sequences of Escherichia coli, mouse, human and other species:

[0063] The gene sequences from different species were downloaded from the NCBI database for multiple sequence alignment analysis ( Figure 5 ), and found that the sequence conservation of this gene in different mycobacteria of the genus Mycobacterium was high, with the amino acid sequence similarity reaching more than 80%, but the similarity with the homologous genes of Escherichia coli, mice and humans was low, with the amino acid sequence similarities being approximately 48.8%, 4.3% and 4.1%, respectively, which means that this gene has the basic properties to serve as a drug target for the development of small molecule drugs.

[0064] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for increasing the sensitivity of Mycobacterium tuberculosis to antibiotics in an in vitro environment, characterized in that: The method is to knock out Mycobacterium tuberculosis by using the principle of homologous recombination Rv2241 gene, and the antibiotic is rifampicin or levofloxacin.

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