The Role And Underlying Molecular Mechanisms Of Mycobacterium Tuberculosis CAMP-induced Gene Rv1265 In Intracellular Survival

Tuberculosis remains the dominating cause of death among chronic infectious disease. There were reported that 1.4 million deaths caused by tuberculosis in 2013. With the emergence of extensive-drug resistance and mutiple-drug resistance TB, controlling TB infection is becoming more and more challenge. Therefore, discovery of new drug target and screen of new drugs are urgent. Although M. tuberculosis is extensively studied, there are still many conserve proteins with unknown function. Whether they play a role in the interaction between pathogen and host is required further study.M. tuberculosis cAMP and underlying regulatory network are crucial for its ability to survive and thrive in the presence of numerous stresses mounted by the host. Our studies mainly focus on the cAMP induced M. tuberculosis gene Rv1265, which was upregulated under hypoxia and during macrophage infection by exogenous cAMP. In this paper, we investigate the role of cAMP induced gene Rv1265 in pathogenesis of M. tuberculosis, especially the survival ability in macrophage.To explore the role of Rv1265 in pathogen-host interaction, a recombinant plasmid pALACE-Rv1265 was constructed and electroporated into M. smegmatis. The expression of Rv1265 in Ms_Rv1265 was successfully deteced by western blotting. As we expected, there was no bar in the strain Ms_Vec. Overexpression of Rv1265 in M. smegmatis had no significant impact on the growth curve of M. smegmatis. Subcellular localization of the recombinant strain Ms Rv1265, we found that Rv1265 was detected in the cell wall and cytoplasm. It implied that Rv1265 may alter the properties of recombinant strain Ms_Rv1265. So we isolated the fatty acid of Ms_Rv1265 and Ms_Vec. Then we measured the survival ability of Ms_Rv1265 treating with different low PH and SDS. Susceptibility of the two recombinant strains to SDS was compared following 4 hours exposure to 0.1% SDS. To determine whether Rv1265 was essential for M. tuberculosis pathogenesis, we detected the intracellular survival of recombinant strain within macrophage. Ms_Rv1265 and Ms_Vec were used to infect THP-1 defferentiated macrophage or Raw264.7 cell at MOI of 10. The CFU determination at six hour showed that there was remarkable difference in infection rate between the two strains. In addition, we tested the macrophage viability infected with Ms_Rv1265 and Ms_Vec release by LDH release. The transcription level of both pro-infammatory cytokines （such as IL-1β,IL-6, IL-18, IL-12 P40, IL-19, TNF-α） and anti-infammatory cytokines （IL-10） were detected. To determine the pathway responsible for cytokine alteration, specific signaling inhibitors（such as p38 MAPK inhibitor SB202190, NF-kB inhibitor TPCK, ERK 1/2 pathway inhibitor PD98059） was used. To further explore the role of Rv1265, we construct a mutant strain △MSMEG₅010 （a homologous gene Rv1265 in M. tuberculosis）.Our data had shown that Rv1265 can successfully express in M. smegmatis. Sub-cellular localization showed that Rv1265 localize in cell wall and cytoplasm of recombinant strain, which implicated that Rv1265 was a cell envelope-associated protein. Gas chromatography mass spectrometry （GS-MS） analysis found that some compound of Ms_Rv1265 was significant higher than the Ms_Vec, especially C10:0, MeC13:0, MeC14:0, C15:0, MeC15:0, C16:1We7（c）, MeC16:0,3MeC16:0, C24:1W9（c）, C26:0. Among them, C26:0 was a result of thermal decomposition of mycolic acid, it suggested that the amount of mycolic acid in MS-Rv1265 may be increase. Principle component analysis （PCA） of the data generated by GC-MS can divide the Ms_Rv1265 and Ms_Vec into two different species due to the different metabolite profiles. The survival of the recombinant Ms_Rv1265 was enhanced within macrophages and under acid and SDS exposure. Macrophages infected with Ms_Rv1265 increased the production of pro-inflammatory cytokines IL-1β, IL-6 and IL-12 P40 and anti-inflammatory cytokine IL-10 through activation of NF-κB and ERK1/2 pathway. These findings indicated that cAMP induced gene Rv1265 enhanced the survival of mycobacterium within macrophage by manipulating the cytokine profile of macrophage accompanied by cell necrosis.