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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - MtbTransReg (Translational regulation in the persistence and drug susceptibility of Mycobacterium tuberculosis)

Teaser

Summary

Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB), one of the humankindâ€™s deadliest diseases. The World Health Organisation estimated that there were 6.3 million new cases of TB and 1.3 million TB deaths in 2016. Furthermore, it is estimated that one-third of the worldâ€™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. Amongst these, 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 â€˜specialised ribosomesâ€™, 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.

Work performed

To address our goals, we first tested our hypothesis that leaderless and SD genes are translated with different efficiencies under different growth conditions. In order to do so, we have constructed over 15 M. tuberculosis translational reporter strains representative of leaderless and SD gene pairs, including both luminescent and fluorescent reporters. These strains have been used to screen against a first panel of stresses that are thought to be relevant during M. tuberculosis infection. These have included nutrient starvation, exposure to nitric oxide and hydrogen peroxide, growth in different carbon sources (like cholesterol) and intracellular growth in human monocyte-derived macrophages. These expression screens have revealed differences in the translation efficiencies of leaderless and SD reporters. For example, upon nutrient starvation, translation of the leaderless reporter is more robustly maintained than the SD reporter. These results have allowed us to select polarising phenotypes that are giving a preferential translation of the two types of transcripts, and are feeding into the advancement and the generation of hypotheses within the project.
To gain more insight into the molecular mechanisms governing translation of leaderless and SD genes in M. tuberculosis, we have engineered some of the reporter strains previously mentioned, to selectively capture translating ribosomes by incorporating a stalling sequence. We have also developed and optimised protocols for ribosome isolation and for the implementation of the relatively new technique of ribosome profiling in this pathogen. We have performed ribosome profiling experiments both during exponential growth and upon nutrient starvation. Analysis of the sequence data is yielding valuable information about translation initiation sites, pausing sites and codon usage, and will ultimately shed light onto the extent of ribosomal heterogeneity in M. tuberculosis.

Final results

In this project, a fundamental aspect of biology â€“ the initiation of translation â€“ has 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. During the first 30 months of the project, we have greatly advanced in the generation of reporter strains that are allowing the quantification of translation efficiencies under different growth conditions. Together with the developments coming from the ribosome profiling experiments and the selective capture of ribosomal populations, we expect to gain a deeper insight into the way that translation without a SD signal is regulated in M. tuberculosis, and the ways it differs from the well-characterised E. coli model. Furthermore, since many of the antibiotics used to treat tuberculosis target the ribosome, results from future experiments to determine the effect that ribosome-targeting drugs have on the translation of leaderless and SD genes, will have direct relevance to tuberculosis drug discovery.