A transcriptional repressor co-regulatory network integrates iron homeostasis and mycobacterial stress tolerance to oxidative stress

A hallmark of Mycobacterium tuberculosis survival and proliferation within the host is attributable to the effective stress response of the pathogen against hostile environmental conditions. Although controlled iron (Fe) uptake and maintenance of Fe homeostasis are closely associated with intracellular survival of mycobacteria, the underlying mechanism that accounts for the interdependence of mycobacterial Fe regulation and oxidative stress response remains unknown. In this study, we discovered that Fe accumulation connects intra-bacterial redox environment via the phoP locus. Gene expression analyses coupled with genetic, biochemical, and transcriptomic studies uncover a distinct mechanism that utilizes Lsr2 and the virulence regulator PhoP to modulate transcriptional control of the major Fe storage protein-encoding gene bfrB. Remarkably, regulated expression of bfrB determines bacterial tolerance to oxidative stress and its intracellular survival. Although both regulators could independently influence bfrB expression, the most fundamental insights are derived from the inhibition of Lsr2 binding at the bfrB regulatory region of the phoP-KO mutant strain owing to Lsr2-PhoP protein-protein interactions. Building on these results, we proposed a model suggesting how Lsr2-PhoP interaction, or lack thereof, contributes to stress-specific regulation of bfrB expression. Collectively, these results uncover a key mechanism integrating Fe accumulation to oxidative stress response, an essential pathogenic determinant of M. tuberculosis.IMPORTANCEWhile the availability of Fe remains essential for Mycobacterium tuberculosis infection and replication of the pathogen in human macrophages, Fe may be harmful as it catalyzes the generation of toxic reactive oxygen species from products of aerobic respiration to induce oxidative stress. Although interdependence of mycobacterial Fe regulation and oxidative stress response has been reported, the mechanism connecting Fe acquisition and oxidative stress response is yet to be fully understood. Results reported in this study identify and elaborate on a new mechanism of regulation of expression of the major mycobacterial Fe storage protein, which determines bacterial tolerance to oxidative stress and impacts intracellular survival of the pathogen. We demonstrate that the virulence-associated phoP locus controls Fe accumulation and contributes to the intra-bacterial redox environment. The results uncover a distinctly different transcriptional control mechanism of the major Fe storage protein requiring a co-repressor-dependent regulatory circuit, including nucleoid-associated protein Lsr2 and the virulence regulator PhoP. Together, these results unveil a unique mechanism of stress-specific gene expression, which integrates Fe homeostasis to oxidative stress response, an essential pathogenic determinant of M. tuberculosis.