Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

Shinya Watanabe,Michael Zimmermann,Uwe Sauer,Helena I Boshoff,Michael B Goodwin,Clifton E Barry,Vojo Deretic

doi:10.1371/journal.ppat.1002287

Abstract

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.

Highlights

A third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis [1]
The dense tissue formed by the granuloma severely limits the amount of oxygen available and yet somehow the bacteria manage to survive for many years
In this study we have examined TB bacteria artificially locked at specific oxygen tensions to understand how they maintain basic metabolic functions in the absence of oxygen

Summary

Introduction

A third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis [1]. This reservoir maintains the epidemic by ensuring the availability of future cases of reactivation disease. Any serious attempts at eradicating tuberculosis would require drastically reducing this burden of latent disease. The drug of choice for prophylaxis of latent disease is isoniazid [2]. Recent evidence from an analysis of host transcriptional responses of latently infected individuals, compared to healthy individuals and individuals with active disease, suggests that a subset of such people are, experiencing sub-clinical disease [4].

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Discussion

Conclusion