Mycobacterium bovis non-coding small RNA and applications thereof

Abstract

The application discloses a Mycobacterium bovis non-coding small RNA, and belongs to the field of biology. Mycobacterium bovis , M.bovis The nucleotide sequence of the non-coding small RNA is shown as SEQ ID NO:1. Research finds that the survival ability of a BCG strain with overexpressed ncBCG201 is obviously enhanced under carbon starvation culture conditions, the survival ability is obviously decreased under membrane pressure culture conditions, the overexpressed strain shows a slower growth speed in a logarithmic growth phase, and the biofilm forming capacity is obviously reduced. The above results show that the non-coding small RNA can reduce the pathogenicity and resistance of the Mycobacterium bovis, and has a good application prospect in preparation of a bovine tuberculosis vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a bovine mycobacterium ( Mycobacterium bovis , M.bovis Non-coding small RNAs and their applications. Background Technology

[0002] Tuberculosis is caused by the tuberculous complex (Mycobacterium tuberculosis) Mycobacterium tuberculosis Complex , MTBC Tuberculosis is a chronic wasting disease caused by infection. Almost all vertebrates, including humans, are susceptible, and it is one of the deadliest single-pathogen infections and one of the most prevalent infectious diseases globally. Global control of tuberculosis is urgently needed.

[0003] The unclear pathogenic mechanism is a major reason restricting the prevention and control of tuberculosis. Mycobacterium bovis ( Mycobacterium bovis , M.bovis ) is Mycobacterium tuberculosis ( Mycobacterium tuberculosis , M.tb The bovine variant of Mycobacterium bovis, whose in vitro attenuated vaccine strain, BCG, shares 99.9% genomic similarity with Mycobacterium tuberculosis, exhibits overlapping infection profiles and is commonly used as a model bacterium for Mycobacterium tuberculosis. Therefore, in-depth analysis of the virulence-related factors and pathogenic mechanisms of Mycobacterium bovis is a crucial foundation for the development of novel drugs for tuberculosis.

[0004] Over a long evolutionary process, *Mycobacterium bovis* has continuously adapted to various adverse environments through different regulatory mechanisms under a range of stresses, thus achieving long-term co-evolution with its host and continuously improving its drug tolerance. Therefore, in-depth exploration of the molecular mechanisms by which *Mycobacterium bovis* adapts to adversity and identification of key factors influencing its infection, pathogenesis, spread, and drug resistance are important approaches to developing novel diagnostic reagents, vaccines, and drugs for tuberculosis.

[0005] Approximately 93% of bacterial genomic DNA can be transcribed into RNA, of which only a small fraction can be translated into protein. The remainder, which cannot be translated into protein, belongs to non-coding RNA (ncRNA). Non-coding RNA can be divided into large non-coding RNA and small non-coding RNA. Among them, small non-coding RNA (sRNA) is a class of RNAs with a length of 20–300 nt. These RNAs play important roles in regulating gene expression, cell cycle regulation, and maintaining genome stability.

[0006] Given the current severe situation of drug-resistant tuberculosis, the search for sRNAs in the Mycobacterium tuberculosis complex that have the ability to regulate stress, influence bacterial pathogenicity (including adhesion, invasion, intracellular survival, and growth rate), and alter bacterial drug resistance will help improve our understanding of the pathogenesis of tuberculosis and host defense mechanisms, and provide a basis for the diagnosis and clinical treatment of tuberculosis. Summary of the Invention

[0007] The purpose of this invention is to provide a novel non-coding small RNA for Mycobacterium bovis that can regulate the stress adaptation ability of Mycobacterium bovis, alter the pathogenicity and resistance of the bacteria, and has the potential to become a novel diagnostic reagent, vaccine and drug target for tuberculosis.

[0008] To achieve the above objectives, the State Key Laboratory of Agricultural Microbiology and the National Laboratory for Animal Tuberculosis (Wuhan) of Huazhong Agricultural University, where the applicant is located, performed whole transcriptome sequencing on the total RNA of intracellular and extracellular bacteria after Mycobacterium bovis (BCG Tokyo strain) infected macrophages THP-1. Using six stress models, differentially expressed ncRNAs in intracellular and extracellular bacteria were screened, and it was found that the expression of 14 sRNAs changed significantly under different stress models.

[0009] One of the non-coding small RNAs, named ncBCG427, was patented on September 30, 2021, with patent publication number CN 113862267 A.

[0010] Another non-coding small RNA provided by this invention, which the applicant named ncBCG201, exists only in extracellular bacteria and its expression changes significantly under six stress conditions. This ncRNA has been identified as a novel sRNA located between the genes of BCG.

[0011] Furthermore, using the classic type strain of Mycobacterium bovis, Mycobacterium bovis Bacille Calmette-Guérin, Tokyo strain, BCG Using [a specific gene] as a template, an ncBCG201 overexpression strain was constructed. The survival rate of the overexpression strain was significantly enhanced under carbon starvation conditions and significantly decreased under membrane stress conditions, indicating that the ncBCG201 gene does indeed have a regulatory effect on the environmental stress of Mycobacterium bovis. Through culture, the ncBCG201 overexpression strain exhibited a slower growth rate during the logarithmic growth phase and entered the plateau phase earlier, while other growth characteristics such as single colony area, wrinkling, and cell morphology did not show significant changes. Biofilm formation ability measurements showed that the biofilm formation ability of the ncBCG201 overexpression strain was significantly reduced.

[0012] Given that biofilms are a major reason why bacteria develop resistance to antibiotics, harsh environments, and host immune defense mechanisms, and why bacterial diseases are difficult to eradicate, the non-coding small RNA provided by this invention has great potential for application in reducing the pathogenicity and resistance of Mycobacterium bovis.

[0013] The present invention further provides a recombinant plasmid containing the non-coding small RNA, which is used to construct recombinant Mycobacterium bovis overexpressing the non-coding small RNA.

[0014] The present invention also provides a recombinant bovine mycobacterium that overexpresses the non-coding small RNA, which can be used to prepare a bovine tuberculosis vaccine.

[0015] The present invention also provides a method for reducing resistance in Mycobacterium bovis by using transgenic technology to overexpress the non-coding small RNA in Mycobacterium bovis.

[0016] The discovery and functional determination of ncBCG201 in this invention have extremely important reference value for elucidating the pathogenic mechanisms of Mycobacterium bovis and even Mycobacterium tuberculosis complex, as well as for developing novel diagnostic reagents, vaccines and drugs. Attached Figure Description

[0017] Figure 1 Location distribution of ncBCG201 in the genome (A) and prediction of its secondary structure (B).

[0018] Figure 2 Detection of ncBCG201 expression level in BCG overexpression. ncBCG201 was significantly highly expressed ( p <0.001).

[0019] Figure 3 Survival of ncBCG201 overexpressing BCG under iron starvation, carbon starvation, acidification stress, oxidative stress, membrane stress, and simulated granulomatous stress conditions. The survival of ncBCG201 was significantly enhanced under carbon starvation conditions and significantly decreased under membrane stress conditions.

[0020] Figure 4 Determination of the growth rate of bacteria overexpressing ncBCG201. ncBCG201 can slow down bacterial growth.

[0021] Figure 5 Colony morphology and size determination of ncBCG201 overexpressing BCG. (A) Colony morphology; (B) Colony area. No significant changes were observed in the area and wrinkles of single ncBCG201 colonies.

[0022] Figure 6Determination of single cell length in ncBCG201 cells overexpressing BCG. (A) Cell morphology; (B) Cell length. No significant changes were observed in the length of single ncBCG201 cells.

[0023] Figure 7 Biofilm assay of BCG overexpression using ncBCG201. ncBCG201 significantly reduced biofilm formation capacity. p <0.01). Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions or reference books such as *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory, 2017), or according to the methods recommended in the manufacturer's operating manual. Materials in the embodiments that do not specify their source are all commonly used materials well known in the art, which can be constructed based on literature reports or obtained commercially.

[0025] The Mycobacterium bovis BCG Tokyo strain [American Type Culture Collection (ATCC): 35737] was a gift from Professor Liu Junyan of Wuhan University; the sRNA ncBCG201 overexpression plasmid was constructed in the laboratory previously.

[0026] Example 1: Secondary structure prediction of ncBCG201 and construction of overexpression strains

[0027] 1. Genomic location distribution and secondary structure prediction of ncBCG201

[0028] The RNAfold tool was used to predict the secondary structure of ncRNAs. A minimum free energy prediction model was set up to obtain the loop position and structural information of the binding site between ncRNA and target mRNA. The results showed that ncBCG201 is located in the intergenic region between the JTY-3720 and JTY-3721 genes in BCG, with a loop size ranging from 4 to 14 bp. Figure 1 Furthermore, it has a high GC content within the loop, enabling it to bind more precisely and firmly to the target mRNA. Its sequence is shown in SEQ ID NO:1.

[0029] 2. Construction of ncBCG201 overexpression strain

[0030] Five µL portions of the previously constructed sRNA ncBCG201 overexpression plasmid pMV261-PrrnB-ncBCG201 and the empty vector pMV261-PrrnB (Vector) were added to 200 µL of BCG electroporation competent cells thawed on ice. After gentle mixing, the mixture was transferred to a pre-chilled electroporation cuvette and placed on ice for 10 min. The cuvette was dried, and electroporation was performed using a program of 2.5 kV, 1000 Ω, and 25 µF. Immediately after electroporation, 1 mL of 7H9 medium (without Tween-80) was added to the cuvette, mixed, and transferred to a 15 mL centrifuge tube. The cells were then incubated at 37°C for 12 h to revive. After cell resuscitation, the cells were centrifuged at 5000 r / min for 5-10 min, 4 / 5 of the supernatant was discarded, and the remaining 100-200 µL of culture medium was used to resuspend the cells and spread onto 7H11 solid plates containing the corresponding antibiotic. After static incubation at 37℃ for 15-20 days, single clones were picked and the expression level of ncBCG201 was detected by real-time quantitative PCR (quantitative real-time polymerase chain reaction) with sigA as an internal control. The results showed that ncBCG201 was highly expressed in BCG ( p <0.001)( Figure 2 The primer sequences for qRT-PCR are shown in Table 1.

[0031] Table 1 qRT-PCR primer sequences

[0032]

[0033] 3. Phenotypic determination of ncBCG201 overexpression strains

[0034] The constructed overexpression strain was cultured for two weeks. When the OD600nm reached 0.6, the bacterial solution was resuspended and dispersed. Then, according to CN 113862267 A, the bacterial solution was cultured under six stress conditions: iron starvation, carbon starvation, acidification stress, oxidative stress, membrane stress, and simulated granuloma stress. The absorbance of the bacterial solution at 600 nm was measured every day. The empty vector expression strain was used as a control to compare the survival ability of the strain under different stress conditions.

[0035] The results are as follows Figure 3As shown, under the six stress models tested, the survival rate of ncBCG201 overexpressing strains was significantly enhanced under carbon starvation culture conditions, while the survival rate was significantly reduced under membrane pressure culture conditions. During infection, membrane pressure from alveolar surfactant and other substances acts like a neutral detergent, which can damage the M. tb cell membrane (Bychenko O, Skvortsova Y, Ziganshin R, et al. Mycobacterium tuberculosis Small RNAMTS1338 Confers Pathogenic Properties to Non-Pathogenic Mycobacterium smegmatis[J]. Microorganisms, 2021,9(2).). These results indicate that the ncBCG201 gene has a certain regulatory effect on the environmental stress of Mycobacterium bovis.

[0036] Example 2: Determination of growth characteristics of ncBCG201 overexpression strain

[0037] The ncBCG201 overexpression strain was statically cultured in 7H9 solution, and the absorbance at 600 nm was measured every 12 h. The results showed that ncBCG201 exhibited a slower growth rate during the logarithmic growth phase and entered the plateau phase earlier, with a higher OD value after the plateau phase. 600 nm The results were consistently significantly lower than those of the empty vector control. Figure 4 This phenomenon suggests that ncBCG201 may inhibit the growth of mycobacteria by regulating metabolic pathways.

[0038] After culturing the ncBCG201 overexpression strain on solid medium 7H11 for 15-20 days, the morphology of single colonies was recorded and the size of single colonies was measured under a stereomicroscope. The results showed no significant changes in single colony size, wrinkle morphology, or surface smoothness between the empty vector strain and the overexpression strain. Figure 5 )

[0039] After static culture of ncBCG201 strain in 7H9 for 15 days, single cell observation using scanning electron microscopy (SEM) revealed no significant changes in cell length or morphology of the overexpressing strain. Figure 6 ).

[0040] Example 3: Determination of biofilm formation ability of ncBCG201 overexpression strain

[0041] The recombinant and empty vector mycobacteria were inoculated into 5 mL of fresh 7H9 medium containing kanamycin resistance and cultured at 37°C and 180 r / min in a constant temperature shaker until the logarithmic growth phase (OD50). 600nm= 0.6~0.8), centrifuge at 2000 r / min for 5-10 min, collect the bacterial cells, resuspend and disperse in fresh 7H9, and adjust OD. 600nm Approximately equal to 1, the recombinant mycobacteria and empty vector mycobacteria were diluted 1:100 in fresh Sauton medium containing kanamycin resistance and inoculated into 12-well plates. The plates were sealed with sterile membrane and incubated statically at 37°C. After 20 days, the biofilm thickness of each strain was recorded. Three replicates were set up for each strain.

[0042] Carefully remove any liquid and bacteria outside the biofilm. After drying the 12-well plate in a biosafety cabinet, add 500 μL of 1% crystal violet per well and stain for 10 min. Wash three times with sterile double-distilled water, 5 min each time, and then dry. Add 1 mL of 95% ethanol per well and incubate for 10 min. After complete dissolution, dilute three times and measure the OD. 570nm Three replicate wells were set up. The difference between the biofilm formation thickness and the absorbance value was compared.

[0043] Mycobacterium tuberculosis can form biofilms, especially within the host's immune cells or certain microenvironments, a property that helps it resist attacks from the host's immune system and antibiotic treatment. The bacteria in the biofilm are encapsulated in a polymeric polysaccharide matrix, which not only provides a physical barrier but also reduces the bacteria's metabolic activity, making them more resistant to antibiotics and immune responses. Results showed that the biofilm-forming ability of the ncBCG201 overexpression strain was significantly reduced, and fewer folds floated on the surface of the culture medium. Figure 7 The host's immune system and drugs can more easily recognize and attack exposed bacterial antigens, and mycobacteria can be more easily recognized and eliminated.

Claims

1. A Mycobacterium bovis (Mycobacterium tuberculosis) non-coding small RNA, the nucleotide sequence of which is shown in SEQ ID NO:

1. Mycobacterium bovis , M. bovis ) non-coding small RNA, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. A recombinant plasmid comprising the non-coding small RNA of claim 1.

3. A recombinant Mycobacterium bovis overexpressing the non-coding small RNA of claim 1.

4. Use of the recombinant Mycobacterium bovis of claim 3 in the preparation of a vaccine against bovine tuberculosis, said recombinant Mycobacterium bovis having reduced ability to survive under membrane stress conditions, reduced ability to form biofilm and being more susceptible to recognition and attack by the host immune system or drugs.

5. A method of reducing the resistance of Mycobacterium bovis for non-diagnostic or therapeutic purposes, characterized in that: overexpressing the non-coding small RNA of claim 1 using transgenic techniques.

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