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Translational regulation in the persistence and drug susceptibility of Mycobacterium tuberculosis

Periodic Reporting for period 4 - MtbTransReg (Translational regulation in the persistence and drug susceptibility of Mycobacterium tuberculosis)

Reporting period: 2019-12-01 to 2021-11-30

Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB), one of the humankind’s deadliest diseases. The World Health Organisation estimated that there were 10 million new cases of TB and 1.5 million TB deaths in 2020. Furthermore, it is estimated that one-quarter of the world’s population harbours the bacterium in the form of an asymptomatic infection referred to as latent TB. This reflects the complex life cycle of the bacteria that can involve prolonged periods of non-replicating persistence prior to the active disease process that is required for onward transmission. During this process, the bacteria overcomes numerous stresses presented by the immune system of the host. Still today, the mechanisms underlying persistence are poorly understood, and the emergence of drug-resistant bacteria makes the development of effective new treatments an urgent challenge. Understanding the ability of M. tuberculosis to switch between replicating and non-replicating states during infection and disease is central in the search for improved treatments.

Translation, the process by which the sequence of nucleotides in a gene directs the synthesis of proteins, is an intricate process involving many cellular components. The ribosome has been traditionally considered as a conserved nucleoprotein with the only role of mediating this process. Genes contain specific signals that optimise their interaction with ribosomes, known as leader sequences, these include the Shine-Dalgarno (SD) sequence required for canonical translation initiation in bacteria. There is recent evidence that suggests that ‘specialised ribosomes’, which are modified ribosomal particles, can modify the proteome profile by preferential translation of particular gene subsets, particularly in response to stress. M. tuberculosis differs from other important human pathogens in expressing a large number of leaderless genes, which do not have the SD sequence required for canonical translation initiation. In the model bacteria Escherichia coli, only a few leaderless genes have been described, and they are selectively translated by specialised ribosomes upon stress conditions. We have previously shown in M. tuberculosis that under conditions of nutrient starvation, the abundance of leaderless genes increases, suggesting that translation of leaderless genes may be an important component of the adaptive response of this pathogen.

The MtbTransReg project aims at understanding what is the role of selective translation of leaderless and SD genes in the context of adaptation to stress and drug resistance in M. tuberculosis. It is divided in three main objectives. The first objective aims at identifying differences in translation efficiencies during different growth conditions. The second objective is focused on determining what are the molecular mechanisms underlying differences in translational efficiencies. Finally, the third objective is devoted at establishing relationships between translational regulation and drug susceptibility in M. tuberculosis.
The state-of-the art in mycobacterial translational regulation was reviewed and published in NAR in 2018 (https://doi.org/10.1093/nar/gky574). During this project we have created a panel of M. tuberculosis translational reporter strains representative of leaderless and Shine-Dalgarno gene pairs that have been used to study differences in translation. These reporter strains have shown robust leaderless translation during exponential growth, and also shown that leaderless translation is more stable than Shine–Dalgarno translation during adaptation to stress conditions. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine–Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable. Work that has been published in Frontiers Microbiology in 2021 (https://doi.org/10.3389/fmicb.2021.746320) and widely presented at national and international conferences during the lifetime of the project (EMBO Tuberculosis 2016, Royal Society 2018, FEMS 2019, ESM 2019). We have also used ribosome profiling to characterise for the first time the translational landscape of virulent M. tuberculosis during conditions of exponential growth and nutrient starvation. We find widespread translation of non-canonical transcripts lacking bacterial translation initiation signals used for canonical initiation. Our data points towards different translation initiation mechanisms compared to canonical Shine-Dalgarno transcripts. Furthermore, during conditions of nutrient starvation, patterns of ribosome recruitment to start codons vary, suggesting that regulation of translation in this pathogen is more complex than originally though. This data represents a rich resource for others seeking to understand translational regulation in bacterial pathogens (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-8835/) and was published in Cell Reports in 2021 (https://doi.org/10.1016/j.celrep.2021.108695). The work has been presented and disseminated at both national and international meetings ( Translation UK 2017, Royal Society 2018, Translation UK 2018, EMBO Risosome 2019, ESM 2019, ESM 2021). Furthermore, we have engineered some of the reporter strains previously mentioned, to selectively capture translating ribosomes by incorporating a stalling sequence. This work has been presented at several national and international conferences (UK Biochemical Society 2017, EMBO Ribosome 2019).
In this project, a fundamental aspect of biology has been identified that M. tuberculosis differs from the canonical E. coli model in a way that has relevance to the understanding of pathogenesis and with potential applications to drug discovery. We have been able to construct a wide set of M. tuberculosis translational reporters representative of leaderless and Shine-Dalgarno genes that can be used to assess translation differences and that can be further used to monitor changes in translation during infection. These reporter strains have been used to study translation under conditions of exponential growth and under in vitro conditions of stress (nutrient starvation, nitric oxide and macrophage infection), showing that the effect that stress has on canonical translation is more profound than that of leaderless translation. We have also engineered a novel approach to selectively capture translating ribosomes. By creating translational reporters that stall the ribosome we have been able to purify ribosome-nascent chains with ribosomes still attached that can be used to study ribosomal composition. Finally, we have been able to apply for the first time the technique of ribosome profiling to study translation at a global level in a virulent strain of M. tuberculosis. All together, the approaches here developed are helping towards our understanding into the way that translation without a SD signal is regulated in M. tuberculosis, and the ways it could potentially differ from the well-characterised E. coli model. Furthermore, since many antibiotics used to treat tuberculosis target the ribosomes, these approaches can be applied to determine the effect that ribosome-targeting drugs have on the translation of leaderless and SD genes, having direct relevance to tuberculosis drug discovery.
Graphical abstract summarising results from the ribosome profiling experiments