Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/glutamine cell wall structure, and bacterial replication
Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/glutamine cell wall structure, and bacterial replication
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DOI：
10.1073/pnas.97.1.418
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发表时间：
2000-01-04
影响因子：
11.1
通讯作者：
Horwitz, MA
中科院分区：
文献类型：
--
作者：
Harth, G;Zamecnik, PC;Horwitz, MA
迫切需要新的抗生素来对抗新出现的结核分枝杆菌耐药菌株的大流行。我们研究了硫代修饰的反义寡核苷酸(PS-ODN)对谷氨酰胺合成酶基因的影响。谷氨酰胺合成酶是一种与致病性相关的酶，它的输出与多聚L-谷氨酸/谷氨酰胺细胞壁结构的形成有关。用10亩M反义PS-ODN处理强毒力结核分枝杆菌，可使谷氨酰胺合成酶活性和表达降低25%-50%。使用两个或三个不同的PS-ODN和PS-ODN在mRNA上的特定靶点。用PS-ODN处理表达结核分枝杆菌谷氨酰胺合成酶的重组耻垢分枝杆菌，选择性地抑制重组酶，但不抑制2-4个核苷酸错配的内源酶。用反义PS-ODN处理结核分枝杆菌，细胞壁中多聚L-谷氨酸/谷氨酰胺的量也减少了24%。最后，反义PS-ODN使结核分枝杆菌的生长减少0.7logs(1PS-ODN)到1.25logs(3PS-ODN)，但对污垢分枝杆菌的生长没有影响，因为耻垢分枝杆菌不输出谷氨酰胺合成酶，也不具有聚L-谷氨酸/谷氨酰胺(P-L-Glx)细胞壁结构。实验表明，反义PS-ODN进入结核分枝杆菌细胞质，并与其同源靶点结合。虽然需要更有效的ODN技术，但本研究证明了将反义ODN用于抗结核分枝杆菌抗生素制剂的可行性。
New antibiotics to combat the emerging pandemic of drug-resistant strains of Mycobacterium tuberculosis are urgently needed. We have investigated the effects on M. tuberculosis of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of glutamine synthetase, an enzyme whose export is associated with pathogenicity and with the formation of a poly-L-glutamate/glutamine cell wall structure. Treatment of virulent M. tuberculosis with 10 mu M antisense PS-ODNs reduced glutamine synthetase activity and expression by 25-50% depending on whether one. two, or three different PS-ODNs were used and the PS-ODNs' specific target sites on the mRNA. Treatment with PS-ODNs of a recombinant strain of Mycobacterium smegmatis expressing M. tuberculosis glutamine synthetase selectively inhibited the recombinant enzyme but not the endogenous enzyme for which the mRNA transcript was mismatched by 2-4 nt, Treatment of M. tuberculosis with the antisense PS-ODNs also reduced the amount of poly-L-glutamate/glutamine in the cell wall by 24%. Finally, treatment with antisense PS-ODNs reduced M. tuberculosis growth by 0.7 logs (1 PS-ODN) to 1.25 logs (3 PS-ODNs) but had no effect on the growth of M, smegmatis, which does not export glutamine synthetase nor possess the poly-L-glutamate/glutamine (P-L-glx) cell wall structure. The experiments indicate that the antisense PS-ODNs enter the cytoplasm of M. tuberculosis and bind to their cognate targets. Although more potent ODN technology is needed, this study demonstrates the feasibility of using antisense ODNs in the antibiotic armamentarium against M. tuberculosis.