[Inhibitory effects of 10-23 deoxyribozyme targeting ICL gene on the expression of isocitrate lyase and the infection of Mycobacterium tuberculosis in macrophages].
OBJECTIVE
To investigate the inhibitory effects of the 10-23 deoxyribozyme (DRZ) targeting ICL gene on the expression of isocitrate lyase (ICL) and the survival of Mycobacterium tuberculosis in macrophages.


METHODS
Five 10-23 DRZ targeting ICL genes (DZ1-DZ5) were designed according to the predicted secondary structure of Mycobacterium tuberculosis ICL mRNA. Their cleavage activity and specificity were identified in cell free conditions. Then Mycobacterium tuberculosis pretreated with DZ4 with or without subinhibitory concentration of isoniazid (INH) were used to infect THP-1 cells. The bacterial burden of the infected THP-1 cells was monitored at indicated times after infection. The effect of 10-23 DRZ on the growth of Mycobacterium tuberculosis in vitro was also assayed by plating Mycobacterium tuberculosis treated with INH alone or DZ4 plus INH on M7H10 agar directly.


RESULTS
Four of the five designed 10-23 DRZ, DZ1, DZ3, DZ4 and DZ5 could cleavage ICL mRNA efficiently and specifically. Treating Mycobacterium tuberculosis with 5 micromol/L DZ4 plus subinhibitory concentration of INH decreased the expression of ICL dramatically, by 34.9% - 46.7% (10 microg/L INH, P < 0.01) or 21.9% - 36.9% (5 microg/L INH, P < 0.01) when compared with corresponding concentration of INH alone. The survival of Mycobacterium tuberculosis in THP-1 cells was decreased significantly when Mycobacterium tuberculosis was pretreated with 5 micromol/L DZ4 plus subinhibitory concentration of INH. 4 or 7 days after infection, the bacteria burden in macrophages was decreased from 126.5 x 10(4) CFU, 307.5 x 10(4) CFU to 54.6 x 10(4) CFU, 114.3 x 10(4) CFU (when 10 microg/L INH used) or from 133.0 x 10(4) CFU, 325.4 x 10(4) CFU to 71.7 x 10(4) CFU, 174.4 x 10(4) CFU (when 5 microg/L INH used). 10-23 DRZ showed no obvious effect on the growth of Mycobacterium tuberculosis in M7H10 agar.


CONCLUSIONS
INH at the subinhibitory concentration can improve the entry of 10-23 DRZ in Mycobacterium tuberculosis. In the presence of subinhibitory concentration of INH, 10-23 DRZ targeting ICL gene can strongly inhibit the expression of ICL and decrease the survival of Mycobacterium tuberculosis in macrophages.
Li
Jun-ming
JM
Department of Microbiology and Immunology, Chongqing University of Medical Sciences, 400016 Chongqing, China.
Li
Na
N
Zhu
Dao-yin
DY
Yi
Zheng-jun
ZJ
Liu
Ye-hua
YH
Yang
Chun
C
He
Yong-lin
YL
D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D005798:Genes, Bacterial; D006801:Humans; D007522:Isocitrate Lyase / Q000235:genetics / Q000378:metabolism*; D007538:Isoniazid / Q000494:pharmacology; D008264:Macrophages / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; C402582:RNA-cleaving DNA 10-23; D007522:Isocitrate Lyase; D007538:Isoniazid