Elucidating Mechanisms of Post-Transcriptional Regulation in Mycobacterium smegmatis

Vargas Blanco, Diego A.

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Elucidating Mechanisms of Post-Transcriptional Regulation in Mycobacterium smegmatis

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One of the deadliest diseases in the world is tuberculosis, caused by the bacillus Mycobacterium tuberculosis. In contrast to most bacterial infections, treatment for tuberculosis requires of multidrug regimes that can extend from six months to over a year. Importantly, the exceptional capability of M. tuberculosis to survive stress conditions, such as low energy environments and the host immune response, has been shown to confer drug tolerance and drug resistance, two factors that impact treatment length and limit the selection of available antimicrobials, particularly when only a reduced number of drugs are effective against M. tuberculosis. Therefore, understanding the biology behind the microbial stress response becomes fundamental for drug development. Mycobacteria employ diverse transcriptional and posttranscriptional mechanisms that allow them to survive stressful environments. One of these is global RNA stabilization, a conserved microbial stress response usually associated with non-growing states. However, while there is extensive research on transcriptional regulation as a response to stress, only limited information is available on regulation of mRNA degradation. Here we sought to address this gap. Because M. tuberculosis is a slow-growing bacteria, we conducted our studies on Mycobacterium smegmatis, a non-pathogenic and fast-growing relative. In Chapter 2, we show that it is possible to alter translation efficiency, transcript stability, and transcription rates in M. smegmatis by altering the 5’ UTR in reporter constructs. The relative efficiency of leadered vs leaderless translation depended on the nature of the 5’ UTR. Combined with a global proteome and transcriptome analysis, our results suggest that leaderless genes are globally translated with a similar range of efficiencies as leadered genes. In Chapter 3 we show that mRNA stabilization as a response to stress is a reversible mechanism. The stable transcriptome of M. smegmatis in hypoxia can be rapidly degraded upon re-exposure to oxygen. This discovery led us to stablish a connection between mRNA degradation and metabolic status. We investigated distinct mechanisms that could stabilize the mRNA pool in response to stress. We found that global stabilization could not be explained by RNA-degradation protein abundance, the stringent response, or changes in transcript abundance. However, we discovered that we could modulate mRNA degradation in growth-arrested M. smegmatis as a response to energy metabolism. These exciting findings provided evidence that mRNA degradation is not necessarily dependent on cell growth status, as previously conceived, and instead responds directly to energy metabolism status. These findings will help reorient studies on transcriptome stabilization, bringing us a step closer to identify the mechanism(s) responsible for mRNA stabilization under stress. Work in other bacteria has shown that ribosome occupancy and translation can regulate mRNA degradation, at least in a transcript-specific manner. Therefore, we wondered if increased ribosome occupancy was responsible for global mRNA stabilization during stress. We used diverse approaches to investigate the impacts of translation and ribosome occupancy on mRNA degradation. Our results, detailed in Chapter 4, show that changes in ribosome occupancy do not explain global mRNA stabilization in energy stress. Interestingly, while we explored ribosome occupancy, we serendipitously accumulated data consistent with the idea that transcription and translation may be physically coupled in mycobacteria. Understanding the universality of mRNA stabilization as part of the mycobacterial stress response has been a fascinating and challenging task, and an important one to undertake. As we aim to discover new antimicrobials, we must comprehend the biology of bacterial adaptation to stress. And because of increasing levels of antimicrobial resistance in M. tuberculosis, it is more important than ever to study different bacterial processes to reveal new drug targets. Here, we have compiled evidence that bring us closer to identifying the mechanism(s) by which mycobacteria can quickly stabilize their transcriptomes in response to stress.

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