title: Effects of Mycobacterium tuberculosis ESAT6-CFP10 Protein on Cell Viability and Production of Nitric Oxide in Alveolar Macrophages.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Xie
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Han
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Laixing
authors.initials: L
authors.lastname: Liu
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Zuye
authors.initials: Z
authors.lastname: Gu
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Yang
authors.affiliation: Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Shandong, China.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yang
keywords: Cell Proliferation; ESAT6-CFP10 Fusion Protein; Immune Protection; Macrophages; Mycobacterium tuberculosis