On the stability of mycobacterial ribosomes during stasis

Abstract

Latent tuberculosis is estimated to account for over 99% of Mycobacterium tuberculosis infections globally. Bacteria are believed to enter a non-­replicating persistent (NRP) state to counter the effect of oxygen and nutrient limitation during latent infection. While NRP bacteria are believed to retain some metabolic activity, it is not known how the stability and functionality of the biosynthetic apparatus is maintained. Using the Wayne hypoxia model, we focused on mycobacterial ribosomes and found that, in contrast to enteric bacteria, no higher order structures (e.g. ribosomal dimers) are formed upon entry into stasis. We devised a strategy incorporating microfluidic, proteomic and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during NRP. We compared the stability of wild-type ribosomes to those of mutants in the transcriptional regulator DosR, which cannot survive prolonged oxygen starvation. While stability was comparable under conditions of active growth and normoxic stasis, ΔdosR mutants showed a marked decrease in levels of 70S ribosomes and 30S ribosomal subunits under hypoxia. Microfluidic analyses were consistent with these observations and pointed to a progressive degradation of rRNA during prolonged hypoxia, with evidence of discrete rRNA cleavage. Proteomic analysis also supported a gradual degradation of ribosomes and led to the identification of S30AE proteins as possible ribosome stabilisation factors. There is an S30AE domain protein is the DosR regulon and it may contribute to the observed destabilisation of the ribosome during hypoxia in ΔdosR mutants. Macromolecular stability is further compromised during infection by the presence of reactive oxygen and nitrogen species. We used mass spectrometry to assess the impact of oxidative stress on mycobacterial nucleic acids. We found that RNA was more susceptible to damage. Since only limited RNA synthesis occurs in NRP, the ability of cells to preserve intact ribosomes could be crucial for long-­term survival.