[Effects of isocitrate lyase from Mycobacterium tuberculosis on the survival of Mycobacterium smegmatis in macrophage and mechanism thereof].
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.
Li
Jun-Ming
JM
Department of Clinical Laboratory, First Affiliated Hospital of Nanchang University, Nanchang, China. lisir361@163.com
Wan
La-Gen
LG
Zhu
Dao-Yin
DY
Li
Na
N
He
Yong-Lin
YL
Yang
Chun
C
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000096:biosynthesis; D007522:Isocitrate Lyase / Q000235:genetics / Q000378:metabolism*; D008262:Macrophage Activation; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology*; D051379:Mice; D050296:Microbial Viability; D008856:Microscopy, Fluorescence; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D007371:Interferon-gamma; D007522:Isocitrate Lyase