The Mechanism Of Mtb Virulent Factor Rv3033 On Promoting The Survival Of Mtb In Macrophages

Tuberculosis(TB) is a permanently respiratory disease which has afflicted humans for years. It is a chronic infectious disease caused by mycobacterium tuberculosis. Since discovered in 1882 by Koch, mycobacterium tuberculosis has led to hundreds of millions of deaths worldwide. Mycobacterium tuberculosis is a kind of aerobic, slow growing bacillus with positive rich lipid acid staining. Macrophage is inquilinous niche of mycobacterium tuberculosis as well as the place where the body fights mycobacterium tuberculosis. For years, scientists have dedicated to investigations on the interactions between mycobacterium tuberculosis and macrophage, looking for more effective therapeutic methods for the treatment of tuberculosis. Apoptosis is an important way that macrophages defense against mycobacterial infections, contributing efficiently to mycobacterium tuberculosis removal by innate immune response. Moreover, apoptosis initiates efficient antigen cross-presentation leading to activation of adaptive immune response against the pathogen. In this kind of interdependent and mutually restricted environment, rival mycobacterium tuberculosis also takes means to defense the host, especially by its virulent proteins. Rv3033, a 19 kDa secreted protein, is a virulent factor of mycobacterium tuberculosis which contributes to the survival of mycobacterium tuberculosis in macrophages. How does Rv3033 promote the survival of mycobacterium tuberculosis has not been studied. Understanding the functions of Rv3033 could help us to reveal the mechanisms by which Mtb evades macrophage killing as well as to provide theoretical premise and basis for finding new drug targets.To explore the mechanism how mycobacterium tuberculosis virulent factor Rv3033 support Mtb survival in macrophage. Rv3033 eukaryotic expression plasmid pMSCV-eGFP-Rv3033 was constructed using retroviral vector. Retrovirus expressing Rv3033 was packed, and RAW264.7 cells were infected by the virus to build a macrophage cell line RAW-Rv3033 which stably expressed Rv3033 and its vector control cell line RAW-Vector. We infected RAW-Rv3033 and RAW-Vector with H37 Rv and BCG to observe the survival of mycobacterium tuberculosis in Rv3033 overexpressed macrophages. The results showed that overexpression of Rv3033 could promote survival of mycobacterium tuberculosis in macrophages. Compared the gene expression levels in RAW-vector and RAW-Rv3033 cells 12 h after BCG infection by high-throughput sequencing, we discovered 464 differentially expressed genes. Among them, 235 genes were overexpressed in Rv3033-RAW cells, and 229 genes were down-expressed. Analyzing these genes according to the biological processes they belong to, we found that genes involved in apoptosis make up the biggest section. We then checked the apoptosis levels in BCG infected Rv3033-RAW and vector-RAW cells, and found that overexpression of Rv3033 could inhibit BCG induced macrophage apoptosis by blocking ER stress. Eventually, Rv3033 protein was expressed in bacteria and purified, and Rv3033 interacting proteins were obtained by pull down experiments after incubating with macrophage cell lysate. Eight candidates of Rv3033 interacting proteins were identified by mass spectrometry, including Lrrfip1, Cofilin-1, Rhog, Rac2, HMGA1, Arhgap15, Arpc4, and Tropomodulin-3。The above results indicated that Rv3033 could promote survival of mycobacterium tuberculosis in macrophages, at least partially by inhibiting macrophage apoptosis induced by mycobacterium tuberculosis.