Preliminary Study On The Interaction Between Macrophages Protein And Cas6 Protein In Mycobacterium Tuberculosis

Tuberculosis is a chronic disease caused by Mycobacterium tuberculosis.Mycobacterium tuberculosis can invade a variety of internal organs.According to the World Health Organization's Reports,10 million people were newly infected by tuberculosis every year all across the world.As long-term use of drugs in tuberculosis patients,genotype mutations have been found in M.tuberculosis,leading to the drug resistance of M.tuberculosis,which increases the difficulties for the treatment of tuberculosis concurrently.Therefore,further studies on the basic research of tuberculosis,especially on its pathogenicity,are quite important for the diagnosis and treatment of tuberculosis.The CRISPR/Cas system is an acquired immune mechanism in prokaryotes that not only prevents itself from being invaded by foreign genes,but also affects the pathogenicity of bacteria.In this study,we analyzed the virulence of Cas6 protein and the pathways of macrophage apoptosis.To know the role of Cas6,an important component of CRISPR/Cas system in M.tuberculosis,we constructed the Cas6-MBP expression vector and made it express in Escherichia coli.The purified Cas6-MBP fusion protein confirmed by Western blot was used to infect macrophages.The results showed that Cas6 protein improved the bacteria virulence by inducing macrophages apoptosis.Then,to know the manner of macrophage apoptosis induced by Cas6,the intracellular localization pattern of Cas6 in macrophage was analyzed.We express Cas6-GFP vector in macrophages,and found that Cas6 was broadly compartmentalized in the cytosol,indicating the potential targets were in the macrophages cytosol.Adding to this,we identified the interaction between Cas6 protein and macrophage protein by Pull-down experiment and analyzed the correlation between protein components.The cytosolic protein interacted with Cas6 was identified by mass spectrometry,and the pathways of apoptosis induced by Cas6 were predicted based on the cytoplasmic protein which interact with Cas6.Four proteins including GRP78,ERP44,IF4A3 and 2AAA were found interacted with Cas6,most of them were predicted involved in apoptotic pathway.Taken together,this work provides valuable information for understanding the roles of CRISPR/Cas system in infection of macrophage in M.tuberculosis.