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

Abstract

Mycobacterium tuberculosis remains one of the world's most important pathogens. Responsible for millions of new cases of tuberculosis annually (1), it is the leading cause of death from a single infectious agent. The emergence of multidrug-resistant M. tuberculosis underscores the need for new approaches to combat this pathogen (1).We recently have identified glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] as an important determinant of M. tuberculosis pathogenesis (2-4). An enzyme that plays a central role in nitrogen metabolism in every cell, glutamine synthetase is 1 of 10 proteins released in large quantity into the bacterium's extracellular milieu, whether the bacterium is growing axenically or intraphagosomally in human mononuclear phagocytes, the primary host cells (2). Of great interest, extracellular release of glutamine synthetase is unique to pathogenic mycobacteria and correlated with the presence of a poly-L-glutamate͞ glutamine (P-L-glx) component in the cell wall of these organisms, suggesting that not only is the enzyme involved in the synthesis of this heteropolymer but also that its presence is significant to virulence (4). Treatment of bacteria with L-methionine-Ssulfoximine, an irreversible inhibitor of glutamine synthetase, results in a decrease in extracellular glutamine synthetase activity, a marked reduction in the amount of cell wall P-L-glx, and inhibition of bacterial growth both in broth culture and in human macrophages (4).Antisense oligodeoxyribonucleotides (ODNs), which can base pair with a gene's transcript, constitute a new technology for the control of gene expression in prokaryotes and eukaryotes, including mammalian cells (5, 6). In addition, this technology shows promise as a means for developing new chemotherapeutic agents against human diseases, including antibiotics against such pathogens as Plasmodium falciparum, Toxoplasma gondii, and HIV (7-10). An antisense PS-ODN for treatment of cytomegalovirus retinitis in AIDS patients became the first antisense ODN-based drug approved for human use in the U.S. (11).In this report, we have utilized modified antisense ODNs, in which all internucleoside linkages are phosphorothioates (PSODNs), to study the expression and function of M. tuberculosis glutamine synthetase and the feasibility of using antisense PSODNs as antimicrobial agents against this pathogen. We find that glutamine synthetase-specific antisense PS-ODNs substantially inhibit the expression and activity of M. tuberculosis glutamine synthetase, the amount of the P-L-glx structure in the mycobacterial cell wall, and bacterial replication. These observations are consistent with entry of the antisense PS-ODNs into the bacterial cytoplasm. Materials and Methods PS-ODN Selection and Preparation.Three target sites for the binding of the antisense PS-ODNs were chosen (Table 1). One site, located near the 5Ј end of the glutamine synthetase mRNA, corresponds to codons 4-9 of the glutamine synthetase mRNA translation product, starting from the N-termi...

Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism

“…In addition, the level of extracellular GS in different mycobacterial strains has been shown to be correlated with their virulence (30,31). Inhibiting GS has a strong impact on Mycobacterium growth in many experimental systems, including human macrophages and guinea pigs (32). Our work provides novel insight into the molecular mechanisms underlying the GlnA1 and pathogenicity in actinomycetes and suggests that drugs targeting the GlnR-GlnA1 circuit might be useful for controlling populations of pathogenic mycobacteria.…”

Inhibition of aac(6′)-Ib -mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

“…Several groups have experimented with a variety of strategies to induce uptake of oligonucleotides and analogs into the cytosol. These strategies included liposome encapsulation, modification of the oligonucleotide by introduction of structures like hairpins, or attachment of cell-permeabilizing peptides (18,(24)(25)(26)(27)(28)(29)(30). We are presently carrying out assays using these approaches to induce penetration of LNA/DNA cooligomers into E. coli and other bacteria.…”

Differential Expression of Mycothiol Pathway Genes: Are they Affected by Antituberculosis Drugs?

“…M. tuberculosis H37Rv (atcc 25618) was cultured in BACTEC 12B medium (17) at 378C to growth index (GI) of 500 and 0.1 ml re-cultured in BACTEC 12B medium in the presence of 10 uM antisense oligonucleotide. Oligonucleotides were allowed to enter mycobacteria through passive diffusion (18). The GI of cultures was monitored every 24 h by BACTEC.…”

“…The probes generated were radioactively labelled through the incorporation of a-32 P-dCTP by random primed labelling (Stratagene). Southern blot analysis of PvuII digested M. tuberculosis DNA was done according to standard protocols (18).…”