Mechanism Of Mycobacterium Tuberculosis On Extensively Taking In Macrophage Transcripts

Tuberculosis(TB)is a chronic,wasting and zoonotic infectious disease which is mainly caused by Mycobacterium tuberculosis(M.tb)and sometime Mycobacterium bovis(M.bovis).M.tb is an intracellular bacterium that mainly infects immune cells such as macrophages and dendritic cells.Since it has been co-evolved with host immune cells for thousands of years,forming a complete set of interaction mechanisms that can effectively invade macrophages without being eliminated.However,the mechanisms of M.tb on evading the host's immune regulationand maintaining persistent infection are not completely clear.In this study,the infection model were M.tb and macrophage THP-1 cell line.THP-1 cells were innoculated with M.tb1458 clinical strain and vaccine strain BCG at MOI=10 and incubated for 12 h.Then the medium was replaced by fresh medium with gentamicin to kill the extracellular bacilli and infected cells further cultured for 6h and 24 h when the cell samples and intracellular bacilli M.tb and BCG were collected respectively.Then the cellular and intracellular bacterial samples were subjected to high-throughput RNA sequencing and bioinformatics analysis.The results were partially confirmed at cellular and molecular levels.The main findings were described as follows:1.Detection of extraction quality of intracellular bacteriaBased on the results of preliminary high-throughput sequencing in the laboratory,it was shown that intracellular M.tb and BCG contained a large number of host transcripts.In order to eliminate the contamination of cells and other bacteria,ourresearch adopted a variety of methods to comprehensively determine that the extracted 6h and 24 h intracellular bacteria and their total RNA samples were pure.(1)Morphological observation: both acid-fast staining and electron microscope confirmed that the extracted intracellular M.tb and BCG were pure without any organelles contamination of macrophages and other bacterial contamination,at 6h and 24 h after infection.(2)Quality identification of intracellular bacteria RNA: intracellular M.tb and BCG as well as total cellular RNA were extracted by different intensities,and detected the genes of bacteria and host by absolute fluorescence quantitative method.It was confirmed that the extracted RNA of intracellular bacteria M.tb and BCGwithout any cellular RNA contamination at 6h and 24 h after infection.2.High-throughput RNA sequencing and bioinformatics analysis of intracellular bacteria and macrophages(1)Cell RNA sequencing: in order to eliminate the contamination of host ribosomal RNA,the RNA samples were removed host ribosomal RNA,and built the library for sequencing.(2)Bioinformatics analysis: analysis of sequencing data showed that intracellular bacteria(M.tb and BCG)contained a large amount of host RNA,including m RNA,sno RNA,linc RNA,antisense RNA and mi RNA.Further analysis found that intracellular bacteria uptaken host RNA was favorable,and virulent strain M.tb had a stronger ability to absorb host transcripts than vaccine strain BCG.Linc RNA and sno RNA were more easily uptakenby intracellular M.tb and BCG,and bothof them were verified by RT-PCR.In addition,intracellular host m RNA uptakenby M.tband BCG is mainly primary m RNA which without selective splicing.WGCNA method is applied to gather the THP 1 cells and intracellular bacteria detected in M.tb and BCG host transcription level,found that both M.tb and BCG strains can absorb a large number of host genes,Target genes were functionally clustered in pathways of ubiquitination dependent protein degradation,lysosome and proteasome,and Fc?R mediated phagocytosis.This phenomenon was also verified by RT-PCR.These findings not only confirmed the extensive RNA transfer from host cells to intracellular M.tb and BCG during M.tb and BCG infection,but also showed that the uptake of host m RNA transcripts by intracellular M.tb and BCG was selective,which provided new evidence to clarify the anti-macrophage defense mechanism of mycobacterium tuberculosis.3.Validation of intracellular bacteria uptaken host RNAThrough fluorescence in situ hybridization and laser confocal microscope observation,it was confirmed that there are 6 host RNAs could be detected in the intracellular bacteria,while the two negative control host RNAs were not detected in the bacteria,and the detection results were vividly displayed with dynamic 3D images.Meanwhile,absolute fluorescence quantitative method was also used to verify the presence of 8 host RNAs in intracellular bacteria,while 8 negative control host RNAs were not detected in bacteria.4.Proteomic analysis of intracellular bacteria M.tb and BCGProteomics study of intracellular bacteria M.tb and BCG Extract intracellular bacterium proteins of M.tb and BCG,detected the protein by i TRAQ quantitative proteomic technology and bioinformatics analysis,the results showed that: the host protein is less than 1% of total protein,and it's mainly keratin and found no RNA binding protein,Therefore the bacteria did not directly ingest the host protein.Further analysis showed that there was a significant difference between intracellular M.tb and BCG,M.tb uptaken-protein was more involved in glyceride metabolism and ribosomal pathway.Combined with the transcriptome sequencing results,the proportion of host transcripts taken by intracellular bacteria was 45.99-70.69%,it means transcription of host in the intracellular bacteria cannot be translated in directly.In summary,this study confirmed that virulent strain M.tband vaccine strains BCG,can selectively uptake host transcripts when they infect macrophages,but do not translate.The virulent strain M.tb was more capable of absorbing host transcripts than the vaccine strain BCG.linc RNA and sno RNA are more easily absorbed by intracellular M.tb and BCG than other kinds of RNA,.This discovery provides a new basis for the explanation of the interaction between M.tb and the host,and clarifies the pathogenesis of mycobacterium tuberculosis,and provides a new idea for the prevention and control of M.tb.