Unknown,Transcriptomics,Genomics,Proteomics

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MRNA degradation in Mycobacterium tuberculosis with DosR ectopically induced

ABSTRACT: After induction of DosR from a tet regulated promoter (uninduced controls were treated with an equivalent ammount of the solvent, DMSO), transcription was arrested using 50 ug/ul of rifampin leaving RNA degradation as the sole vector responsible for mRNA abundance. mRNA half-lives were calculated from the decay of signal intensity from T0. Three replicates for each condition (DosR induced and uninduced) were sampled after 0, 10, 20, 30, and 60 minutes of mRNA decay for a total of 30 arrays.

ORGANISM(S): Mycobacterium tuberculosis H37Rv

SUBMITTER: Tige Rustad

PROVIDER: E-GEOD-36344 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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MRNA degradation in Mycobacterium tuberculosis with DosR ectopically induced

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