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

摘要

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 associ- ated with pathogenicity and with the formation of a poly-L-gluta- mate/glutamine cell wall Structure. Treatment of virulent M tuber culosis with 10 μM antisense PS-ODNs reduced glutamine synthetase activity and expression by 25-50/100 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 glu- tamine 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 anti- sense PS-ODNs also reduced the amount of poly-L-glutamate/glu- tamine in the cell wall by 24/100. 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 ex- periments 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.