| M.tuberculosis,M.bovis and M.leprae are the common pathogens to elicit the occur of tuberculosis(TB).Especially,M.tuberculosis causes disease in human,it mainly elicits tuberculosis in lung,but also could infect other organs and organizations.Based on the data of the Center for Disease Control and Prevention(CDC),there was1.8 million people losing their lives because of TB and nearly 1/3 people latently infected by M.tuberculosis in our world in 2015.In recent years,the increasing emergence of mutiple-drug resistant strains and extensive-drug resistant strains because of the extensive and substantial usage of antibiotics brought new challenges.In addition,nontuberculosis mycobacterial(NTM)strains were newly reported to cause server diseases in lung and other organs.Thus,it is very important to explore the pathogenesis of M.tuberculosis and the immunologic mechanism of host to provide strategies for the prevention and treatment of TB.Rv0177 encoding a conserved hypothetical protein is located in the 13 genes co-transcriptional mce1(mammalian cell entry 1)operon in M.tuberculosis.This protein was shown to be up-regulated on infection in macrophages,be essential in vivo survival and had no homologous proteins in human and gut microorganisms in meta-analysis and bioinformatics analysis.We speculated that it might be a virulent factor or an effective drug target.In our study,we constructed the recombinant pNIT_rv0177 plasmid harboring a Myc-tag.The pNIT and pNIT_rv0177 plasmids were electroporated into the nonpathogenic M.smegmatis mc2155,respectively.Then we performed the PCR amplification to verify the positive recombinant strain and theWestern blotting to determine the expression of Rv0177 protein in M.smegmatis.We separated the whole lysates of M.smegmatis into the cell wall fractions and cytoplasmic/membrane factions after the centrifugation at different speed,and identified the Rv0177 protein localization on the cell wall.We found the similar growth rate at the exponential phage in MS_Vec and MS_Rv0177.Nevertheless,the Rv0177 protein modified the biofim formation and significantly enhanced the sliding motility of MS_Rv0177.Moreover,in the transmission electron microscopy(TEM)analysis,we observed compact surface,fewer “holes” of MS_Rv0177;in the accumulation of cellular ethidium bromide(EB)assay,we identified the overexpresssion of Rv0177 protein decreased the envelope permeability of the recombinant strains.In addition,we found that MS_Rv0177 was shown some resistant growth in pH=3.0 and pH=5.0 7H9 medium,5mM H2O2.There was no significant difference among fatty acid(C7:0 ~C26:0)compositions by GC-MS analysis;and also no divergent glycopeptidolipids(GPLs)by the thin-layer chromatography(TLC)analysis in these both recombinant strains.To explore the interaction of mycobacterium and host,we used MS_Vec and MS_Rv0177 to infect RAW264.7 macrophages and PMA-differentiated THP-1macrophages.We found the same invasion ability of MS_Rv0177 and MS_Vec for macrophages,and MS_Rv0177 was significant susceptible to survival in RAW264.7and THP-1 cells than MS_Vec.Interestingly,we checked MS_Rv0177 could down-regulate the expression of IL-6 and up-regulate the expression of MCP-1 in RAW264.7 and THP-1 cells.At the same time,we observed MS_Rv0177 enhanced the expression of MCPIP and induced ER stress in RAW264.7 macrophages.In addition,we tested the viability of macrophages by the MTT and LDH assay and determined cell apopotosis by the AnnexinV-FITC/PI kits infected with the recombinant strains.Further,to determine the signal pathway affected by the Rv0177 protein,we used the special signaling inhibitors(NF-?B inhibitor TPCK,JNK inhibitor SP 600125 and p38 inhibitor SB 202190)to treatment for 1h,prior to infection with MS_Rv0177 and MS_Vec.Then we revealed that Rv0177 could activate the JNK pathway associated with the upregalation of MCP-1,MCPIP and the induction of ER stress. |
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