Project description:The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions and its induction is controlled by two histidine kinases, DosS and DosT. Recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. This report demonstrates that DosS responds to a reduced electron transport system (ETS), although this does not rule out a role for oxygen in silencing signaling. 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 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 the menaquinone pool is a limiting factor in regulon induction. Taken together these data indicate 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, rescue inhibition of menaquinone synthesis, and is less toxic. The menaquinone pool is central to the ETS and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Aerobically growing logarithmic phase DosS and DosT mutant strains were analyzed after treatment with the cytochrome c reductant ascorbate to examine the effect on DosR signaling caused by reducing the electron transport system. Experiments were repeated in triplicate (dosT mutant) or duplicate (dosS mutant).

Project description:Background: Conflicting results have been reported about the role of the two-component sensor and transcriptional regulator DosS/DosR, controlling the expression of the dormancy DosR regulon, for in vivo virulence of M. tuberculosis. Here, we have used a new approach to further analyze the relevance of the dosRS system, by driving DosR (Rv3133c) expression under the control of a constitutive promoter (phsp60). Methodology/Principal Findings: M. tuberculosis H37Rv constitutively expressing the transcriptional regulator DosR (Mtb::DosR) was compared to wild type M. tuberculosis (Mtb+/+) for in vitro growth kinetics and expression of the target genes of the DosR dormancy regulon, for in vivo virulence and for immunogenicity in mice. Under aerobic conditions, hsp60-driven DosR induced the expression of 28 out of 39 tested DosR regulon genes. In vitro growth characteristics were comparable for both strains, but Mtb::DosR showed an attenuated in vivo phenotype in immunocompetent mice, as indicated by reduced bacterial replication, reduced pulmonary immunopathology, reduced cachexia and significantly prolonged survival time as compared Mtb+/+. In immunodeficient SCID mice, Mtb::DosR was fully virulent. RT-qPCR analysis revealed a strong and comparable pulmonary TNF-?? and IL-23 expression following intratracheal infection, whereas IL-12 and IL-17 expression was slightly higher with wild type Mtb+/+. Finally, mice persistently infected with Mtb::DosR for 8 months showed five to tenfold higher lung IFN-?? responses against ten of the 48 DosR regulon encoded antigens (Rv1733c, Rv1734, Rv1738, Rv1996, Rv1997, Rv2029c, Rv2623, Rv2627c, Rv2628 and Rv3127) than mice actively infected with Mtb+/+. In spleen however, DosR regulon encoded antigen specific IFN-?? responses were similar in both groups. Conclusions/Significance. Collectively, these results suggest that increased DosR regulon encoded antigen specific pulmonary T cell responses are responsible for the attenuated phenotype of Mtb::DosR and that infection with Mtb::DosR could be used as a new animal model for studying key aspects of latent tuberculosis. Set of arrays that are part of repeated experiments

Project description:Background: Conflicting results have been reported about the role of the two-component sensor and transcriptional regulator DosS/DosR, controlling the expression of the dormancy DosR regulon, for in vivo virulence of M. tuberculosis. Here, we have used a new approach to further analyze the relevance of the dosRS system, by driving DosR (Rv3133c) expression under the control of a constitutive promoter (phsp60). Methodology/Principal Findings: M. tuberculosis H37Rv constitutively expressing the transcriptional regulator DosR (Mtb::DosR) was compared to wild type M. tuberculosis (Mtb+/+) for in vitro growth kinetics and expression of the target genes of the DosR dormancy regulon, for in vivo virulence and for immunogenicity in mice. Under aerobic conditions, hsp60-driven DosR induced the expression of 28 out of 39 tested DosR regulon genes. In vitro growth characteristics were comparable for both strains, but Mtb::DosR showed an attenuated in vivo phenotype in immunocompetent mice, as indicated by reduced bacterial replication, reduced pulmonary immunopathology, reduced cachexia and significantly prolonged survival time as compared Mtb+/+. In immunodeficient SCID mice, Mtb::DosR was fully virulent. RT-qPCR analysis revealed a strong and comparable pulmonary TNF-?? and IL-23 expression following intratracheal infection, whereas IL-12 and IL-17 expression was slightly higher with wild type Mtb+/+. Finally, mice persistently infected with Mtb::DosR for 8 months showed five to tenfold higher lung IFN-?? responses against ten of the 48 DosR regulon encoded antigens (Rv1733c, Rv1734, Rv1738, Rv1996, Rv1997, Rv2029c, Rv2623, Rv2627c, Rv2628 and Rv3127) than mice actively infected with Mtb+/+. In spleen however, DosR regulon encoded antigen specific IFN-?? responses were similar in both groups. Conclusions/Significance. Collectively, these results suggest that increased DosR regulon encoded antigen specific pulmonary T cell responses are responsible for the attenuated phenotype of Mtb::DosR and that infection with Mtb::DosR could be used as a new animal model for studying key aspects of latent tuberculosis.

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The unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy

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