Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis

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 CO(2) incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope ((13)C) 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 (13)C-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.

Figure 1. Oxygen concentration directly affects the growth of M. tuberculosis.

(A) Growth curve of H₃₇Rv in fermentor culture maintained at various dissolved oxygen tensions. H₃₇Rv cultures were grown in six fermentors and growth was followed by measuring OD₆₅₀. 10 ml (50, 8, 2, 1 and 0.5 mmHg culture) or 50 ml (0.1 mmHg culture) of H₃₇Rv preculture (OD₆₅₀ = 0.26) was inoculated in 850 ml of Dubos medium in fermentors. They were preincubated for 3 days to reach each set point of dissolved oxygen concentrations after which the measurements were recorded as shown. (B) Growth rate of H₃₇Rv under defined oxygen tensions. Growth rate was calculated from the growth curve of H₃₇Rv culture in Dubos medium in a fermentor.

Figure 2. NADH/NAD⁺ and ATP concentration is a function of dissolved oxygen concentration.

(A) NADH and NAD⁺ and (B) ATP concentrations under various dissolved oxygen tensions. NADH/NAD⁺ and ATP concentrations were measured in chemostat cultures with a constant growth rate of 0.0077 h⁻¹ under 50.0, 8.19, 1.85, 1.44 and 0.42 mmHg of dissolved oxygen tensions. They were also measured in batch culture under 0.10 mmHg on Day 10.

Figure 3. Succinate accumulates in supernatants of H₃₇Rv culture under anaerobic condition.

(A) Succinate and lactate accumulations in NRP culture. H₃₇Rv was grown in Wayne tubes with septa and the supernatant was collected via syringe. Succinate and lactate concentrations were determined by LC/MS. (B) Succinate accumulation in supernatants under various dissolved oxygen tensions. Succinate concentrations were measured by LC/MS in supernatants of the H₃₇Rv cultures shown in Figure 1A.

Figure 4. Mass isotopomer ratios of secreted succinate following labeling with ¹³C isotopic substrates.

H₃₇Rv was grown in a Wayne model tube for 5 days to adapt hypoxic conditions and then exposed to (A) [1,4-¹³C₂]- aspartate, (B) U-¹³C aspartate and (C) ¹³C sodium bicarbonate in an anaerobic chamber for 24 h. The results of anaerobic conditions were compared with those from aerobically growing cultures.

Figure 5. Isotopic succinate accumulation in supernatant of NRP culture in 0.75% U-¹³C Glucose Dubos medium.

H₃₇Rv was grown in Dubos medium where glucose was replaced with U- ¹³C glucose using the Wayne model of hypoxic adaptation. The supernatant was collected every 5 days and secreted succinate was analyzed for ¹³C incorporation. These cells reach NRP-1 by day 10 and NRP-2 by day 15, coincident with a significant increase in the M+3 fraction of secreted succinate arising from reversal of the TCA cycle.

Figure 6. Dynamic isotopic intracellular metabolite measurements in anaerobically persisting cells during metabolism of U-¹³C glucose.

The schematic shows isotopomer distribution of intracellular glycolytic and TCA cycle intermediates during metabolism of U-¹³C glucose under anaerobic conditions following replacement of the glucose with U-¹³C glucose before metabolomics analyses over time. Under anaerobic conditions the isotopomer distribution of the intracellular metabolites support flux towards the reverse TCA cycle and mirror the isotopomer distribution of the secreted succinate.

Figure 7. Dynamic isotopic intracellular metabolite measurements in aerobic growing cells during metabolism of U-¹³C glucose.

Schematic illustration of glycolysis and the TCA cycle and graphs of isotopomer distribution during metabolism of U-¹³C glucose. H₃₇Rv was grown under aerobic conditions in Dubos medium where glucose was replaced with U-¹³C glucose followed by metabolomics analyses over time. Under aerobic conditions the isotopomer distribution supports forward flux through the oxidative TCA.

Figure 8. The mass isotopomer ratios of succinate in supernatant of knock out mutant strains labeling with ¹³C isotopic substrates.

Cultures of H₃₇Rv ΔfrdA, H₃₇Rv Δmez, Erdman Δicl1/Δicl2 and Erdman ΔpckA as well as their parent strains and complement strain of Erdman pckA were exposed to (A) 1,4-¹³C₂ aspartate, (B) U-¹³C glucose and (C) ¹³C sodium bicarbonate in an anaerobic chamber for 24 h. Ratios of succinate isotopomers were measured by LC/MS.

Figure 9. Proposed model for succinate production and NADH reoxidation in Dubos medium under anaerobic condition in M. tuberculosis.

Under aerobic conditions, glucose is metabolized to acetyl-CoA by glycolysis and pyruvate dehydrogenase with further oxidation by the TCA cycle. Labeling of TCA cycle intermediates by feeding of cells with [1,4-¹³C₂]-aspartate results in loss of both ¹³C atoms (green highlighting) through oxidative decarboxylation in the TCA cycle. Reduced cofactors (red arrows) are re-oxidized by respiratory complexes. Under anaerobic conditions, the direct incorporation of C3 metabolites from glycolysis into the reductive branch of the TCA cycle is favored resulting in re-oxidation of reduced cofactors. Succinate is secreted into extracellular milieu to maintain a proton motive force. Under these conditions, labeling of the C4 branch of the TCA cycle using [1,4-¹³C₂]-aspartate results in secretion of succinate labeled at the C1 and C4 positions with ¹³C.