Periodic Reporting for period 3 - MtbTransReg (Translational regulation in the persistence and drug susceptibility of Mycobacterium tuberculosis)
Reporting period: 2018-06-01 to 2019-11-30
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 ‘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.
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.