M. tuberculosis MazF toxins: target identification and stress adaptation



 DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) has adapted to survive a wide range of assaults-from our immune response to antimicrobial therapeutics-intended to eradicate the organism. However, the molecular switches that enable Mtb to endure these stresses, to slow replication or to become dormant as a latent tuberculosis infection (LTBI) are not known. Emerging studies on the molecular underpinnings of stress survival in Escherichia coli generally point to a major role for toxin-antitoxin (TA) systems, which are operons comprising adjacent genes encoding two small proteins, a toxin and its cognate antitoxin that inhibits toxin activity in the TA protein-protein complex. Their expression has been implicated in Mtb stress survival and/or the switch to the non-replicating persistent state characteristic of LTBI. However, several bottlenecks have impeded progress toward rigorous testing of this provocative association. This proposal enlists a strong team to develop and apply a new technology, 5' RNA-seq, to overcome these obstacles as they apply to the nine member MazE (antitoxin) - MazF (toxin) family in Mtb. Our goal is to apply 5' RNA-seq technology toward comprehensive detection of MazF-mt targets in the Mtb transcriptome. We will then apply it to Mtb cultures grown under unstressed conditions or after exposure to stresses that are relevant to TB latency. Finally, we will also study how cleavage of rRNAs by Mtb MazF toxins modifies ribosome function. These approaches should lead to an accurate snapshot of RNAs targeted by MazF toxins under these metabolic states, reveal how RNA cleavage alters translation and identify the environmental signals that trigger toxin activation in Mtb.