DosS Responds to a Reduced Electron Transport System To Induce the Mycobacterium tuberculosis DosR Regulon

Abstract

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.Mycobacteria are strict aerobes, but Mycobacterium tuberculosis encounters microaerobic to anaerobic environments during the course of infection. Oxygen-limited microenvironments occur in mature granulomas, which are known to be avascular, inflammatory, and necrotic (1,5,20,40,56). Recent reports have detected mycobacterial DNA in visibly normal lung tissue (17), as well as adipose tissue (32). Interestingly, adipose tissue has been associated with hypoxia as well as the presence of nitric oxide (NO), both of which inhibit respiration (38,51,57,60). Further, M. tuberculosis is able to survive for long periods of time in a nonreplicating and nonrespiring state (15, 55).The DosR regulon is expressed in response to hypoxia, NO, and carbon monoxide (CO) and is thought to be important for early adaptation to these stimuli as well as long-term survival in the host (2,25,26,46,47,53,58). Despite induction by other gases, the presence of oxygen itself inhibits induction of the regulon (26,41,43,48). The DosR regulon is regulated by the response regulator DosR (DevR; Rv3133c) and was recently shown to be positively regulated by PhoP (Rv0757) to a basal level during aerobic growth (16). DosR is activated by two sensor histidine kinases, DosT (Rv2027c) and DosS (DevS; Rv3132c). The activation of DosR occurs through autophosphorylatio...

The Mycobacterium DosR regulon structure and diversity revealed by comparative genomic analysis

“…Abnormal metabolism might constitutively reduced ETS, accordingly the overexpression of the regulon through DosS. Duplications of some other gene within this strains, specifically the nuo gene cluster encoding the type I oxidoreductase, and the succinate dehydrogenase gene, lend additional creditability to this hypothesis [Honaker et al, 2010]. Both enzyme clusters are involved in the ETS.…”

“…In brief, DosS and DosT of Mtb play different role in hypoxia: DosT is a hypoxia sensor which can respond to the decrease of oxygen tension more sensitively and strongly than DosS, whose function is a redox sensor. Under aerobic conditions, DosR regulon could be induced by DosS not DosT after the addition of ascorbate, a powerful cytochrome c reductant, indicating that DosS can respond to redox signal even in high oxygen tension [Honaker et al, 2010]. This might results from their different autooxidation rates due to the different hemes containing GAF-A.…”

“…These two kinases contain several similar domains: histidine kinase domain and an ATP-binding domain in the C-terminal, and two tandem GAF (cGMP, adenylyl cyclase, FhlA) domains (GAF A and GAF B) in the N-terminal. GAF domains are regulatory domains which can bind small molecules and widely distributed among prokaryotes and eukaryotes [Honaker et al, 2010]. The threedimensional structures of GAF-A and GAF-B have been determined: a b-type heme is embedded into the GAF-A domain, which is composed of five strand anti-parallel b-sheet and four ahelices.…”

“…(2) menaquinone [Honaker et al, 2010]: the expression levels of DosR regulon are proportional to the amount of menaquinone. Menaquinone is central to the electron transport system (ETS) and provides a mechanistic link between the respiratory state of the bacilli and DosS signaling.…”

“…Interestingly, DosR regulon expression in W/Beijing strain is constitutive under normal conditions, somewhat relevant to a natural frameshift mutation in the DosT gene [Fallow et al, 2010]. However, role of dosT mutation in the constitutive DosR regulon phenotype is controversial [Honaker et al, 2010]. DosT mutation can attenuate DosR regulon expression under certain conditions.…”