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.