Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Abstract

A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe 3؉ ) form, whereas DosT exists in the O 2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O 2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O 2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O 2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.carbon monoxide ͉ dormancy ͉ nitric oxide ͉ oxygen ͉ persistence T uberculosis (TB) is a major global health burden, and current estimates suggest that one-third of the world's population (Ϸ2 billion) is latently infected with TB (1). Latency is important largely because persistent Mycobacterium tuberculosis (Mtb) are in a state of ''drug unresponsiveness'' wherein the bacilli are resistant to existing antimycobacterial drugs. A major question in the TB field is: ''what are the mechanisms that allow Mtb to persist in human tissues for decades without replicating, to then abruptly resume growth and cause disease?'' Addressing that question is essential to the development of effective therapeutic intervention strategies. Recent evidence implicates NO as an environmental trigger of mycobacterial persistence (2-5). The latter findings are particularly interesting in light of the fact that inducible NO synthase (iNOS) and therefore NO production is crucial for protection of mice against Mtb (6), and that human macrophages in Mtb-infected tissues express iNOS (2, 7). Another factor associated with latent TB is hypoxia (8). The role of oxygen tension in TB is receiving wide attention, especially because it was demonstrated that rapid withdrawal of oxygen is lethal to Mtb, whereas a gradual depletion allows time for adaptation and bacterial survival (8). Interestingly, a significant overlap exists between the gene expression profiles of Mtb cells treated with NO and that of bacilli cultured under hypoxic conditions (3-5). ...

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses

“…We confirmed that vit C mediates the generation of hypoxia, which triggers a rapid induction of the DevR regulon in a primarily DosT-dependent manner. These observations are consistent with (1) earlier reports of hypoxia generation by vit C (Scarpa et al, 1983;Taneja et al, 2010), (2) the known function of DosT as an oxygen sensor (Kumar et al, 2007;Sousa et al, 2007) and (3) the role of the DosT oxygen sensor in rapid induction of the DevR regulon (Honaker et al, 2010;Taneja et al, 2010). However, our findings are not in agreement with those of Honaker et al (2010), which implicated DevS, and not DosT, in vit Cmediated induction of the DevR regulon.…”

Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS

“…The NO-bound protein displayed a strong absorbance at 419 nm with two broad bands at 545 and 576 nm. Exposure of wild-type Fe(II) DosS to O 2 resulted in rapid formation of the Fe(III) state, as reported by others (26). However, on pretreating the buffer with Chelex 100 to remove metal ions, the Fe(II)-O 2 complex was formed in the presence of O 2 with a Soret maximum at 421 nm (27).…”

Nitrite produced byMycobacterium tuberculosisin human macrophages in physiologic oxygen impacts bacterial ATP consumption and gene expression

“…DOS regulon expression is coordinated by the heme-containing kinases DosS and DosT, which function as redox and hypoxia sensors, respectively. Hypoxia, carbon monoxide, and nitric oxide are physiologic ligands that induce DOS regulon expression by modulating the oxidation and ligation states of the iron atoms in DosS and DosT (49). Nitrite may repress DOS regulon expression by oxidizing the iron of these sensor kinases in a manner that prevents their regulation by other physiologic ligands.…”