Exploring The Mechanism Of Mycobacterium Tuberculosis Interfering Macrophage Autophagy

Tuberculosis(TB),caused by Mycobacterium tuberculosis(Mtb), is one of the most serious bacterial diseases in the world. There are many strategies can be used by host cells to eliminate Mtb, but Mtb have also evolved protective ways to combat the killing by host cells. It is indicated that the fate of intracellular Mtb is influenced by the conflict between host cell and bacilli. However, the genes of Mtb and host involved in this process are not well explained.In this study, we target Mtb, through construct a cyan or blue fluorescence expressing H37 Ra strain and a GphML-Raw 264.7 cell line that indicated autophagy, and a CRISPRi/CRISPRa system to inhibit or activate gene expression to reveal the releated genes that modulate survival of Mtb in host cells. The main contents in this study are including:1. The construction of CFP/BFP-H37 Ra strain. In order to visualize Mtb in host cells by fluorescence microscope, we transformed the plasmid pMV261-CFP/BFP into competent cells of H37 Ra. We obtained an H37 Ra strain expressing CFP or BFP.2. The construction of double fluorescence macrophage to indicate autophagy. Firstly, we mutated EGFP gene to GFPph gene, which is more sensitive to pH and then co-expressed with mCherry and LC3. The LC3-GFPph-mCherry fusion gene was inserted in the genome of Raw264.7 cells by CRISPR/Cas system. The cell line was confirmed by the sequencing and Western Blot. Finally, autophagy inducer was used to confirm autophagy, suggesting that this cell line could indicate autophagy effectively.3. The analysis of Mtb protein interfere the autophagy. To investigate if the Mtb proteins participate in the survival of Mtb in macrophages by interfering autophagy, 33 plasmids with codon optimized Mtb gene were incubated with GphML-Raw264.7 cells, and then rapamycin was applied to induce autophagy. The autophagy was examined at indicated time points by confocal microscopy. The results show that the green signals could be co-localized with red signals in Rv0847 treated cells, indicating that Rv0847 might interfere autophagosome fuse with lysosomes. Thus, Rv0847 might be involved in modulating survival of Mtb in host cells by interfering autophagy.4. The gRNA library of host autophagy genes. The CRISPRi/CRISPRa system was used to inhibit or activate gene expression. Firstly, the related plasmids were transfected into 293 T cells, the results show that could inhibit or activate gene expression effectively. Then, we obtained all genes up and down streams information that related with autophagy by NCBI. The best gRNA sequence was obtained using online software. These gRNAs were inserted into CRISPRi/CRISPRa plasmids to get gRNA library. With this study, we laid the foundation for the research of the host autophagy genes related to the intracellular survival of mycobacterium tuberculosis.