Summary Tuberculosis (TB) is a significant public health problem worldwide. Although the number of deaths due to TB has decreased in recent years thanks to improved diagnostics and early treatment, the sustained increase in antibiotic resistance and the shortage of new effective drugs against Mycobacterium tuberculosis threatens to undermine TB control efforts. Our studies focus on exploiting M. tuberculosis (Mtb) sensitivity to iron dysregulation to generate new therapeutic strategies that prevent Mtb virulence and potentiate antibiotic action. Iron is an essential micronutrient required by M. tuberculosis to establish a productive infection. However, excess iron can be very toxic due to the propensity of this metal to catalyze the production of reactive oxygen species, which can damage all macromolecules. Like all iron- dependent cells, Mtb must balance intracellular iron levels as it encounters diverse iron environments in the host. Evidence from animal studies indicates that the ability to maintain iron homeostasis is essential for Mtb to proliferate and cause disease. In turn, our previous studies established that Mtb depends on the global transcriptional regulator, IdeR, to control iron homeostasis. Our preliminary studies characterized a natural antisense transcript (IdeR-AS) capable of inducing iron dysregulation when expressed in trans in Mtb. Although this RNA alters the expression of genes controlled by IdeR, it does not seem to act by altering IdeR levels, and its mode of action is not understood. We hypothesize that IdeR-AS modulates IdeR activity. Our goals for this proposal are to investigate IdeR-AS mode of action and evaluate the synergy between IdeR-AS and antibiotics. We expect that deciphering how this RNA influences iron regulation would guide efforts to target iron homeostasis in Mtb.