Context-specific inhibition of translation in mycobacterium.

Abstract

Mycobacterium tuberculosis (Mtb) has exclusively colonized host tissues by unexplored adaptive mechanisms of protein mistranslation. Errors in protein synthesis can give rise to diverse proteins that confer a selective advantage in Mtb. As a translation initiation inhibitor, kasugamycin (ksg) antibiotics led to increased discrimination of misacylated tRNAs in Mtb or reduced translational errors. Ksg was shown to inhibit translation of canonical mRNAs lacking Shine Dalgarno (SD) sequences, but not leaderless mRNAs, along with inducing ribosomal stalling near start codons, potentially mediated by a preceding guanine nucleotide. Structural studies revealed two binding sites of ksg that either interact with the first two nucleotides in the E-site and last nucleotide in the P site, or completely occludes the E site. I hypothesize that ksg modulates the translation of specific genes and impacts mistranslation in a context-specific manner that will be uncovered by high resolution genomic and structural approaches. Ribosomal profiling was conducted in Mtb-H37Rv with and without 10X minimal inhibitory concentration (4000 µg/mL) of ksg at 30 minutes or 6 hrs. Translation of (SD)-less transcripts with 5’ UTRs were inhibited by ksg, but not conical or leaderless mRNAs. Interestingly, lack of 16S ribosomal RNA (rRNA) methylation by GidB results in increased discrimination of mischarged tRNAs, which mimics the effect of mistranslation induced by ksg. Given these similar physiological events on translation that may influence ribosomal structure, we perfomed Cryo-EM of ribosomes from M. smegmatis strains that were from a wild-type (WT) or HWS19 background, a strain with extremely high rates of translational error, both with and without gidB deletion. Despite identical genetically encoded ribosomes, lack of gidB in HWS19 background led to a more ‘open’ ribosomal conformation than in a wild-type background. Our research addresses the pressing need for a better understanding of Mtb pathogenesis and virulence.