Objective: To investigate the effects of isocitrate lyase (ICL) from Mycobacterium tuberculosis (MTB-icl) on the survival of Mycobacterium smegmatis (MS) in macrophage and illuminate the possible mechanisms.
Methods: MTB-icl gene was amplified by PCR and cloned into Ecoli-Mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmid pUV15-icl expressing ICL-GFP. The recombinant shuttle plasmid pUV15-icl and blank plasmid pUV15 were induced into MS of the line 1-2c so as to obtain rMS-pUV15-icl and rMS-pUV15. Shuttle plasmid rMS-pUV15-IG expressing ICL-green fluorescent protein (GFP) was constructed. rMS-pUV15-IG and MS 1-2c were used to infect the murine macrophages of the line RAW264.7, fluorescence microscopy was used to observe the expression of ICL-GFP. The expression of ICL in the MS swallowed by the macrophages was verified by RT-PCR and Western blotting. Another macrophages RAW264.7 were cultured and infected with rMS-pUV15-icl and rMS-pUV15 respectively. 0, 24, and 48 hours later macrophages were collected and the number of MS colonies was calculated. The interferon (IFN)-gamma and nitrogen oxide (NO) concentrations in the culture supernatants of macrophages infected by rMS-pUV15-icl and rMS-pUV15 were measured by ELISA and Griess assay respectively. The apoptotic rate of the macrophages was assayed by in situ TUNEL technique.
Results: Western blotting showed that the MTB ICL protein expression of the rMS-pUV15-icl was significantly higher than that of rMS-pUVI5. Fluorescence microscopy showed green fluorescence in the RAW264.7 cells infected with rMS-pUV15-IG, but not ion the RAW264.7 cells infected with MS 1-2c. 0 h after the infection of the macrophages there was not significant difference in the MS amount in the macrophages between the rMS-pUV15-isl and rMS-pUV15 groups, and 24 h and 48 h later the MS amounts of the rMS-pUV15-icl group were (32.78 +/- 2.90) x 10(3) and (23.33 + 2.34) x 10(3) respectively, both significantly higher than those of the rMS-pUV15 group [(14.67 +/- 2.45) x 10(3) and (2.28 +/- 0.25) x 10(3) respectively, both P < 0.01]. There were not significant differences in the concentrations of IFN-gamma and NO in the culture supernatants between the 2 groups (both P >0.05). 48 h after the infection the apoptotic rate of the rMS-pUV15-icl group was significantly lower than that of the rMS-pUV15 group (P <0.01). Conclusion MTB-ICL promotes the intracellular survival of MS. Suppressing the apoptosis of the host macrophage may be one of the important mechanisms involved in this increased intracellular survival.