Nitrate Enhances the Survival of Mycobacterium tuberculosis during Inhibition of Respiration

255

330

Mycobacterium tuberculosis is a chronic, facultative intracellular pathogen that spends the majority of its decades-long life cycle in a non-or slowly replicating state. However, the bacterium remains poised to resume replicating so that it can transmit itself to a new host. Knowledge of the metabolic adaptations used to facilitate entry into and exit from nonreplicative states remains incomplete. Here, we apply 13 C-based metabolomic profiling to characterize the activity of M. tuberculosis tricarboxylic acid cycle during adaptation to and recovery from hypoxia, a physiologically relevant condition associated with nonreplication. We show that, as M. tuberculosis adapts to hypoxia, it slows and remodels its tricarboxylic acid cycle to increase production of succinate, which is used to flexibly sustain membrane potential, ATP synthesis, and anaplerosis, in response to varying degrees of O 2 limitation and the presence or absence of the alternate electron acceptor nitrate. This remodeling is mediated by the bifunctional enzyme isocitrate lyase acting in a noncanonical role distinct from fatty acid catabolism. Isocitrate lyase-dependent production of succinate affords M. tuberculosis with a unique and bioenergetically efficient metabolic means of entry into and exit from hypoxia-induced quiescence. Q uiescence, or exit from cell cycle, is a physiologic prerogative of all cells, executed irreversibly by some upon terminal differentiation and reversibly by others as they adapt to changing conditions (1). For Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), quiescence has emerged as a hallmark of its pathogenicity. M. tuberculosis infects approximately one in every three people worldwide and is the leading bacterial cause of death. Following infection, M. tuberculosis enters a clinically asymptomatic state of non-or slowly replicating physiology that often lasts decades, if not the lifetime, of the infected host, and exhibits a form of nonheritable resistance to nearly all TB drugs that has hindered mass eradication strategies (2). Clinical TB arises when M. tuberculosis reenters cell cycle and provokes an inflammatory response that inflicts host tissue damage and enables it to transmit itself to a new host. However, some of the M. tuberculosis in active TB is nonreplicating. This is thought to impose the need for chemotherapies that are longer and more complex than for virtually any other bacterial infection (2-7). However, biochemical knowledge of quiescent M. tuberculosis remains highly incomplete.Relieved of the requirement to double biomass, quiescent cells have generally been perceived to have minimal metabolic activity. However, quiescent cells often occupy ecological niches that are highly dynamic and face the challenge of preserving both their viability and their ability to reenter cell cycle. Fibroblasts induced into quiescence by contact inhibition metabolized glucose through all branches of central carbon metabolism at a rate similar to those of proliferating cells (8). Such studies ha...

Section: Metabolic Essentiality Of Succinate Dehydrogenase Activity Inmentioning

confidence: 99%

Multifunctional essentiality of succinate metabolism in adaptation to hypoxia in Mycobacterium tuberculosis

Eoh

Rhee

2013

255

330

“…Nitrate is a physiologic component of human body fluids, where it arises from the diet and from autooxidation of the NO produced not only by iNOS (NOS2) but also by the constitutively expressed enzymes NOS1 and NOS3. Studies in which nitrate was furnished in vitro revealed that Mtb's ability to respire nitrate allows it to better withstand acid, nitrosative stress (27), or sudden anaerobiosis (28). The evolutionary success of "modern" Mtb strains, which are more prevalent than "ancestral" strains, has been attributed to their enhanced nitrate reductase activity (29,30).…”

mentioning

confidence: 99%

Nitrite produced byMycobacterium tuberculosisin human macrophages in physiologic oxygen impacts bacterial ATP consumption and gene expression

Cunningham‐Bussel¹,

Zhang

Nathan³

2013

In high enough concentrations, such as produced by inducible nitric oxide synthase (iNOS), reactive nitrogen species (RNS) can kill Mycobacterium tuberculosis (Mtb). Lesional macrophages in macaques and humans with tuberculosis express iNOS, and mice need iNOS to avoid succumbing rapidly to tuberculosis. However, Mtb's own ability to produce RNS is rarely considered, perhaps because nitrate reduction to nitrite is only prominent in axenic Mtb cultures at oxygen tensions ≤1%. Here we found that cultures of Mtbinfected human macrophages cultured at physiologic oxygen tensions produced copious nitrite. Surprisingly, the nitrite arose from the Mtb, not the macrophages. Mtb responded to nitrite by ceasing growth; elevating levels of ATP through reduced consumption; and altering the expression of 120 genes associated with adaptation to acid, hypoxia, nitric oxide, oxidative stress, and iron deprivation. The transcriptomic effect of endogenous nitrite was distinct from that of nitric oxide. Thus, whether or not Mtb is hypoxic, the host expresses iNOS, or hypoxia impairs the action of iNOS, Mtb in vivo is likely to encounter RNS by producing nitrite. Endogenous nitrite may slow Mtb's growth and prepare it to resist host stresses while the pathogen waits for immunopathology to promote its transmission.

mentioning

confidence: 99%

“…M. tuberculosis uses anaerobic electron sinks such as nitrate and possibly fumarate (9,10). In contrast to the pathogen, M. smegmatis lacks a functional nitrate reductase (10,11), and it is unclear if one of the putative succinate dehydrogenase/fumarate reductase operons (9, 12) acts as a fumarate reductase. However, it can scavenge oxygen using a cytochrome bd complex that is upregulated under hypoxia (13,14) and can enhance its metabolic plasticity using three phylogenetically distinct [NiFe]-hydrogenases for both hydrogen oxidation and possibly hydrogen production (15).…”

mentioning

confidence: 99%

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

Berney

Greening

Conrad

et al. 2014

116

126

Oxygen availability is a major factor and evolutionary force determining the metabolic strategy of bacteria colonizing an environmental niche. In the soil, conditions can switch rapidly between oxia and anoxia, forcing soil bacteria to remodel their energy metabolism accordingly. Mycobacterium is a dominant genus in the soil, and all its species are obligate aerobes. Here we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, adopts an anaerobe-type strategy by activating fermentative hydrogen production to adapt to hypoxia. This process is controlled by the two-component system DosR-DosS/DosT, an oxygen and redox sensor that is well conserved in mycobacteria. We show that DosR tightly regulates the two [NiFe]-hydrogenases: Hyd3 (MSMEG_3931-3928) and Hyd2 (MSMEG_2719-2718). Using genetic manipulation and high-sensitivity GC, we demonstrate that Hyd3 facilitates the evolution of H 2 when oxygen is depleted. Combined activity of Hyd2 and Hyd3 was necessary to maintain an optimal NAD + /NADH ratio and enhanced adaptation to and survival of hypoxia. We demonstrate that fermentativelyproduced hydrogen can be recycled when fumarate or oxygen become available, suggesting Mycobacterium smegmatis can switch between fermentation, anaerobic respiration, and aerobic respiration. Hydrogen metabolism enables this obligate aerobe to rapidly meet its energetic needs when switching between microoxic and anoxic conditions and provides a competitive advantage in low oxygen environments.