Functional dissection of tRNA-cleaving toxins in mycobacteria
Creators: name: Cristovao Barth Junior, Valdir
Publisher: name: No Publisher Supplied
Year: 2020

description: Tuberculosis (TB) is the leading cause of infectious disease-related deaths world-wide. The most common infectious agent of TB, Mycobacterium tuberculosis, is a highly resistant bacterium that evades the human immune response, presumably allowing it to persist in a dormant state in the lungs for decades. The factors that lead to its high persistence are not completely understood, but bacterial toxin-antitoxin (TA) systems have been implicated. Most TA systems do not have their mode of action elucidated, limiting our understanding of how they participate in bacterial persistence and latent TB. Therefore, the aim of this work was to identify the specific targets of VapC and MazF toxins and their physiological effects in M. tuberculosis and in the fast-growing model organism, Mycobacterium smegmatis. To do so, we applied 5’ RNA-seq to accurately detect toxin-cleaved RNAs differentiating them from other cellular RNAs by their distinct 5’ end left by the toxin. Knowing that VapCs and MazFs leave 5’ monophosphate and 5’ hydroxyl (OH) ends upon cleavage, respectively, our method analyzes enrichment of those cleavage markers in mycobacterial cells expressing these TA toxins. We first found by 5’ OH RNA-seq that MazF-mt9 is an isoacceptor-specific tRNAse which targets tRNALys-UUU in M. tuberculosis. 5’ OH RNA-seq also suggested that ribosomes were selectively stalling at lysine AAA codons due to the low levels of tRNALys-UUU , which we confirmed using Ribo-seq. Expressing MazF-mt9 in the model organism M. smegmatis generates a shift in translational output favoring protein synthesis from transcripts with low levels of AAA codons and lowering the levels of AAA-rich proteins. Therefore, we documented a possible new mechanism of post-transcriptional regulation by tRNAs that dictates gene expression by codon usage. We predict this resulting cellular AAA-depleted proteome may induce persistence against antibiotics and protection against the host’s immune system. In the second Chapter, we show that the only MazF toxin described in M. smegmatis genome (here named MazF-ms) is also a tRNALys-specific tRNA-cleaving toxin. Expression of MazF-ms in M. smegmatis generates similar effects as observed for MazF-mt9 in M. tuberculosis. Newly synthesized protein production is heavily dictated by the transcript’s Lys AAA codon content. The change in proteome favors genes involved in stress response and reduces expression of genes involved in cell division and DNA replication. In the third Chapter, we show that in vitro studies may be misleading. As we report for VapC-mt11, the toxin is highly specific when studied in vivo in its original host (M. tuberculosis) and loses specificity in in vitro assays. We propose the actual targets of VapC-mt11 are tRNAGln and tRNALeu, even though additional targets can be observed in vitro or when expressing in M. smegmatis. In the fourth and last Chapter, we show that VapC-mt4 is another tRNA-cleaving toxin that behaves promiscuously in in vitro assays. We report that VapC-mt4 targets the only tRNACys in vivo in M. tuberculosis. As observed with MazF-mt9, depletion of tRNACys also triggered selective ribosome stalling in Cys codons and we were able to identify stalling sites by 5’ OH RNA-seq. This selective ribosome stalling allowed the identification of dozens of putative small unannotated Cys-containing open reading frames (ORFs), some of which have been confirmed experimentally by quantitative mass spectrometry. Overall, our results indicate that tRNA cleavage is a common feature in mycobacterial TA toxins, which may be used to prompt responses to facilitate survival in stressful situations. Also, we demonstrate that our 5’ OH RNA-seq method may be used to shed light on hidden ORFs in mycobacterial genomes.