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

Abstract

In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state.

FIG. 1.

DosR regulon induction by DosT or DosS. Shown is the percent induction observed by microarray analysis of the DosR regulon genes of each mutant compared to those of the wild type (wt). (A) Anaerobic dormancy model sampled at day 6, 1 day after the onset of anaerobiosis. (B) Anaerobic GasPak chamber sampled at 4 and 24 h with single- and double-sensor mutants. Standard deviation is indicated. The ΔdosT mutant is horizontally striped, the ΔdosS mutant is black, the ΔdosS/T mutant is cross-hatched, and the ΔdosS/T mutant with constitutively plasmid-expressed dosT is diagonally striped. (C) Both DosT and DosS are able to respond to the NO stimulus provided by the NO donor diethylenetriamine/NO adduct via induction of the DosR regulon. The NO donor was added to log-phase cultures at a concentration of 0.1 mM, and they were incubated for 1 h. Shown is the percent induction observed by microarray analysis of the DosR regulon genes of each mutant compared to that of the wild type. Standard deviation is indicated. The white bar indicates data from the ΔdosT mutant strain, and the black bar indicates data from the ΔdosS mutant strain.

FIG. 2.

Survival defects of various strains in the anaerobic dormancy model or in an anaerobic GasPak chamber sampled for CFU counting at the days indicated. (A) In the anaerobic dormancy model, day 4 is entry into hypoxia and day 6 is 1 day after the onset of anaerobiosis (data not shown.) Statistical significance of differences: *, P < 0.01; **, P < 0.001; ***, P < 0.0001. P values were determined for differences between H37Rv and the mutant strains. Standard deviation is indicated. H37Rv is white, the ΔdosT mutant strain is horizontally striped, the ΔdosS mutant strain is black, the ΔdosS/T mutant strain with constitutively plasmid-expressed dosT is diagonally striped, the ΔdosS/T mutant strain is cross-hatched, the complemented ΔdosS mutant strain is checked, and the complemented ΔdosT mutant strain is gridded. (B) Oxygen utilization in the GasPak chamber by H37Rv, shown by surveying methylene blue decolorization via OD₆₀₀ determination.

FIG. 3.

Real-time PCR analysis of dosT and dosS mutant strains during aerobic growth and day 6 of the anaerobic dormancy model. dosT mutant strain levels remain relatively constant during anaerobiosis with or without the presence of DosR, and dosS mutant strain levels serve as a control for DosR regulon induction. Transcript levels are normalized to those of Rv0239. Standard deviation is indicated. White bars represent the transcript level of dosT and black bars represent the level of dosS in the noted strains. No significant difference in the dosT copy number exists between H37Rv aerobic growth and anaerobic dormancy at day 6 or ΔdosR mutant strain aerobic growth and anaerobic dormancy at day 6.