Effects of Mycobacterium tuberculosis ESAT6-CFP10 Protein on Cell Viability and Production of Nitric Oxide in Alveolar Macrophages

BACKGROUND: Mycobacterium tuberculosis is the major pathogen of tuberculosis, which affects approximately one-third of the world’s population. The 6 kDa early secreted antigenic target (ESAT6) and the 10 kDa culture filtrate protein (CFP10), which are secreted by the ESX-1 system in M. tuberculosis, can contribute to mycobacterial virulence. OBJECTIVES: The aim of this study was to research the effects of M. tuberculosis ESAT6-CFP10 protein on macrophages during a host’s was first and second exposures to M. tuberculosis. MATERIALS AND METHODS: In this study, the ESAT6 and CFP10 genes were amplified to create a fusion gene (ESAT6-CFP10) and cloned into the pET-32a(+) and pEGFP-N1 expression vectors, respectively. The recombinant pET-32a(+)-ESAT6-CFP10 plasmid was transformed into the Escherichia coli Origami strain, and the fusion protein was expressed and confirmed by SDS-PAGE and Western blot analysis. The recombinant pEGFP-N1-ESAT6-CFP10 plasmid was transfected into rat alveolar macrophage cells (NR8383). The cell line expressing the ESAT6-CFP10 protein was selected with RT-PCR and designated as NR8383-EC. Finally, the effects of the ESAT6-CFP10 fusion protein on the NR8383 cell line, as well as on the newly constructed NR8383-EC cells, were further assessed. RESULTS: The recombinant ESAT6-CFP10 protein was expressed in E. coli and in NR8383 rat alveolar macrophages. This protein affected the proliferation and nitric oxide (NO) generation of the NR8383 and NR8383-EC cells. Although NO generation was inhibited in both cell lines, proliferation was inhibited in NR8383 while it was increased NR8383-EC. CONCLUSIONS: The data indicate that ESAT6-CFP10 could support the survival of M. tuberculosis in the host through altering the host immune response. It also indicates that the host may gain some level of protection from a second exposure to M. tuberculosis, as evidenced by increased proliferation of NR8383-EC.