The Mycobacterium tuberculosis DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy

ABSTRACT Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.

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.

Mycobacterium bovis BCG Vaccine Strains Lack narK2 and narX Induction and Exhibit Altered Phenotypes during Dormancy

ABSTRACT Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world's population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T cells of M. tuberculosis-infected individuals, especially those harboring latent infections. Differences in the expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for the induction of two dormancy genes: narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains.

Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control

In response to environmental anoxia, embryos of the brine shrimp Artemia franciscana enter a dormant state during which energy metabolism and development are arrested. The intracellular acidification that correlates with this transition into anaerobic dormancy has been linked to the inhibition of protein synthesis in quiescent embryos. In this study, we have addressed the level of control at which a mechanism mediated by intracellular pH might operate to arrest protein synthesis. Two independent lines of evidence suggest that there is an element of translational control when protein synthesis is arrested in dormant embryos. First, as determined by in vitro translation techniques, there were no significant quantitative differences in mRNA pools in dormant as compared to actively developing embryos. In addition, fluorography of the translation products showed that there are no large qualitative changes in mRNA species when embryos become dormant. These data suggest that there was no net degradation of mRNA pools in dormant embryos and that protein synthesis may therefore be controlled more strongly at translation than at transcription. Second, polysome profile studies showed that dormant embryos possess reduced levels of polysomes relative to those found in cells or active embryos. The disaggregation of polysomes is an indication that the initiation step in protein synthesis is disrupted and is further evidence that the mechanism involved in protein synthesis arrest in dormant Artemia involves translational control.

Arrest of cytochrome-c oxidase synthesis coordinated with catabolic arrest in dormant Artemia embryos

We have examined cytochrome-c oxidase (COX) biosynthesis in brine shrimp (Artemia franciscana) embryos during preemergence development (PED), as well as its inhibition under anaerobic dormancy, to determine whether transitions in intracellular pH (pHi) have a regulatory influence on anabolic processes. Under control aerobic conditions (embryo pHi greater than or equal to 7.9), incorporation of radiolabeled amino acids shows that substantial biosynthesis of COX occurs during 12 h of PED (500% increase when corrected for enzyme turnover). This anabolic process is blocked under anoxia, a condition known to foster intracellular acidification (pHi less than or equal to 6.8). The arrest of COX synthesis is quantitatively identical when embryos are incubated aerobically during artifical acidification with CO2 (pHi = 6.8). The data suggest that pHi, directly or indirectly, is a regulator of protein synthesis in Artemia embryos during anaerobic dormancy. Previous work has established a fundamental role for pHi in the arrest of carbohydrate catabolism under anoxia. Thus there appears to be a coordinated suppression of energy-producing and energy-utilizing events as Artemia embryos enter quiescence that involves pHi as the common intracellular signal.

Anhydrobiosis in Embryos of the Brine Shrimp Artemia: Characterization of Metabolic Arrest During Reductions in Cell-Associated Water

Upon entry into the state of anhydrobiosis, trehalose-based energy metabolism is arrested in Artemia embryos (cysts). We have compared changes in the levels of trehalose, glycogen, some glycolytic intermediates and adenylate nucleotides in hydrated embryos observed under conditions of aerobic development with those occurring after transfer to 50moll−1 NaCl. This treatment is known to reduce cellassociated water into a range previously referred to as the ametabolic domain. The trehalose utilization and glycogen synthesis that occur during development of fully hydrated cysts are both blocked during desiccation. Upon return to 0.25 moll−1 NaCl both processes are resumed. Analysis of glycolytic intermediates suggests that the inhibition is localized at the trehalase, hexokinase and phosphofructokinase reactions. ATP level remains constant during the 6-h period of dehydration, as does the adenylate energy charge. An additional dehydration experiment was performed in 5.0moll−1 NaCl containing 50mmoll−1 ammonium chloride (pH9-0). The resulting level of gaseous ammonia in the medium has been shown to maintain an alkaline intracellular pH (pHi) in the embryos. The metabolic response to dehydration under these conditions was very similar to the previous dehydration series. Thus, these results are taken as strong evidence that the metabolic suppression observed during dehydration does not require cellular acidification, in contrast to the pronounced inhibitory role of low pHi during entry of hydrated embryos into the quiescent state of anaerobic dormancy. The arrest of carbohydrate metabolism seen during anhydrobiosis indeed appears to be a strict function of embryo water content.