Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis.
BACKGROUND
Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.


METHODS
MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.


RESULTS
RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.


CONCLUSIONS
MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of 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 330006, China. lisir361@163.com
Li
Na
N
Zhu
Dao-yin
DY
Wan
La-gen
LG
He
Yong-lin
YL
Yang
Chun
C
D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000378:metabolism; D007522:Isocitrate Lyase / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology; D050296:Microbial Viability; D008856:Microscopy, Fluorescence; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics*; D009569:Nitric Oxide / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D009569:Nitric Oxide; D007371:Interferon-gamma; D007522:Isocitrate Lyase