Regulation of mycobacterial adaptive mistranslation

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) uses regulation of specific translation error rates – mistranslation – to survive a variety of stressors including antibiotics and the host environment. Both too high and too low mistranslation rates can be detrimental to the pathogen. However, it is not known how

Mtb regulates translational error. Understanding the regulation of mistranslation by Mtb will allow for the development of targeted therapies that interfere with Mtb’s tolerance to antibiotics and survival within the host. The source of adaptive mistranslation in Mtb is the indirect tRNA aminoacylation pathway: used for the cognate

synthesis of aminoacylated glutamine and asparagine tRNAs, and which results in high levels of mischarged glutamine and asparagine tRNAs. This pathway is present in most pathogens with the exception of Escherichia coli. Using forward genetics and small molecule screens, we have identified drugs and genetic mutations that

are implicated in Mtb’s regulation of high mistranslation rates. The aminoglycoside kasugamycin (Ksg) can decrease specific mistranslation rates in Mtb but has very limited anti-microbial activity and has a very high minimum inhibitory concentration (MIC) against Mtb growth in vitro. When given at subMIC concentrations to

Mtb-infected mice, Ksg can, as a single agent, attenuate Mtb survival; and when given in combination, can substantially potentiate anti-TB drugs. Similarly, deletion of the 16S rRNA methyltransferase GidB allows low/moderate levels of mistranslation but prevents runaway catastrophic mistranslation. Both of these

perturbations affect the ribosome. This is intriguing because prior studies on ribosomes had suggested that ribosomes do not discriminate against mischarged tRNAs. A potential explanation may be that those prior studies were performed exclusively with ribosomes from E. coli – which, unlike Mtb, doesn’t routinely encounter

mischarged tRNAs. To understand the mechanisms by which the Mtb translational apparatus can discriminate against mischarged tRNAs we will use biochemical, structural and kinetic approaches. We will solve the structure of Mtb ribosomes +/- Ksg or +/- GidB-mediated methylation to identify how these ribosomal perturbations affect

ribosomal recognition of, and discrimination of mischarged tRNAs. Using single molecule fluorescence resonance energy transfer (smFRET), we will identify the steps in the translation cycle in which ribosomes can proofread against mischarged tRNA-mediated mistranslation. Together, these studies will a) allow mechanistic

insight into an important non-genetic and non-transcriptional mechanism by which Mtb survives diverse stressors, b) provide greater understanding of Mtb ribosome function and the translation cycle, which is likely to be substantially different from model organisms, and c) permit the future development of structure- and

biochemical-informed therapies that target Mtb adaptive mistranslation.