Mycobacterium tuberculosis (Mtb) has adapted to survive a wide range of assaults-from our immune response to antimicrobial therapeutics-intended to eradicate the organism. This unique ability to persist for long periods of time in its human host as a latent tuberculosis infection dominates the infection landscape relative to active tuberculosis cases. Despite ongoing efforts to understand latent tuberculosis infection, the molecular switches that enable Mtb to slow or stop replication and become dormant are poorly understood. However, the convergence of studies on bacterial toxin-antitoxin (TA) systems/modules demonstrating their general growth-regulating properties, coupled with the identification of a striking abundance of TA systems in the Mtb genome, suggest that their expression may facilitate the non-replicating persistent state of Mtb during latent tuberculosis infection. This proposal seeks to fill the gap in our understanding of the molecular basis of laten tuberculosis infection by investigating the structure and function of the large, uncharacterized family of Mtb VapC toxins through detailed study of three family members. The studies in this fellowship proposal integrate into the broader focus of the Woychik laboratory and address our hypothesis that the VapC toxin family in Mtb represents a new class of enzymes that recognize both RNA sequence and tertiary structure to specifically cleave tRNA and/or rRNA(s) and inactivate the translation machinery. The studies proposed in Aim 1 enlist a specialized genome scale method, 5'RNA-seq, to identify the RNA targets for three Mtb VapC toxins using an Escherichia coli host, those in Aim 2 validate the RNA targets and cleavage sites identified by 5'RNA-seq.
Mycobacterium tuberculosis, the bacterium that causes tuberculosis, has the unique ability to persist for long periods of time in its host as a latent infection. This latent state, in which the bacteria are thought to be dormant with markedly altered physiology, is pivotal to the survival of the bacteria in the stressful environments it encounters. Importantly, M. tuberculosis cells in the dormant state are generally refractory to antibiotics, most of which target processes occurring in actively replicating bacteria. The molecular switches that enable M. tuberculosis to slow or stop replication and become dormant remain unknown. This proposal seeks to fill the gap in our understanding of the molecular basis of latent tuberculosis infection by investigating the function of a large family of toxin-antitoxin systems, implicated in contributing to regulation of growth rate.