Physiological and functional characterisation of RafS, a ribosome associated factor of Mycobacteria

Abstract

Tuberculosis (TB) is a leading cause of death from a single infectious agent and infects one third of the world’s population in a latent form. Latent TB is characterised by presence of TB antigens but a lack of symptoms of TB. Latent TB is associated with the persistent form of Mycobacterium tuberculosis, and is a reservoir from which symptomatic infection arises. Non-replicating persistence (NRP) is postulated to be a reversible state characterised by lack of replication, decreased metabolic activity and increased antimicrobial resistance. To achieve viable persistence, NRP cells have been postulated to require stabilisation of cellular structures needed for stress tolerance and for the transition from NRP to active replication. This study investigates the hypothesis that ribosome stabilisation assists in mycobacterial stress tolerance and persistence. RafS is a novel mycobacterial ribosome associated factor and putative ribosome stabilisation factor. The physiological roles and functional characteristics of RafS are investigated in this study. The role of RafS in M. smegmatis (Msm) and M. tuberculosis (Mtb) physiology were investigated. Competitive survival assays between wild type and ΔrafSMtb illustrated that RafSMtb confers a competitive advantage during survival under nutrient limitation. RafSMsm and RafHMtb were found to significantly inhibit in vitro translation. Furthermore, RafSMsm and RafHMtb inhibited in vitro translation of mRNA with and without Shine Dalgarno sequences. It was determined that RafSMsm is dispensable for growth and survival in several conditions and also for mature biofilm and pellicle formation. Also, RafSMsm is dispensable for tolerance of heat, acid and antibiotic stress. Ribosomal profiling indicated no significant effect of rafSMsm deletion on ribosomal subunit association in log phase and stationary phase rich media cultures. These findings are discussed in the context of mycobacterial growth, survival, stress tolerance and persistence mechanisms.