Cardiolipin-deficient cells depend on anaplerotic pathways to ameliorate defective TCA cycle function

Previous studies have shown that the cardiolipin (CL)-deficient yeast mutant, crd1Δ, has decreased levels of acetyl-CoA and decreased activities of the TCA cycle enzymes aconitase and succinate dehydrogenase. These biochemical phenotypes are expected to lead to defective TCA cycle function. In this study, we report that signaling and anaplerotic metabolic pathways that supplement defects in the TCA cycle are essential in crd1Δ mutant cells. The crd1Δ mutant is synthetically lethal with mutants in the TCA cycle, retrograde (RTG) pathway, glyoxylate cycle, and pyruvate carboxylase 1. Glutamate levels were decreased, and the mutant exhibited glutamate auxotrophy. Glyoxylate cycle genes were up-regulated, and the levels of glyoxylate metabolites succinate and citrate were increased in crd1Δ. Import of acetyl-CoA from the cytosol into mitochondria is essential in crd1Δ, as deletion of the carnitine-acetylcarnitine translocase led to lethality in the CL mutant. β-oxidation was functional in the mutant, and oleate supplementation rescued growth defects. These findings suggest that TCA cycle deficiency caused by the absence of CL necessitates activation of anaplerotic pathways to replenish acetyl-CoA and TCA cycle intermediates. Implications for Barth syndrome, a genetic disorder of CL metabolism, are discussed.     

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO₂ using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO₂ to carboxylic acids.     

The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1

Methanotrophs are a group of bacteria that use methane as sole carbon and energy source. Type I methanotrophs are gamma-proteobacterial methanotrophs using the ribulose monophosphate cycle (RuMP) cycle for methane assimilation. In order to facilitate metabolic engineering in the industrially promising Type I methanotroph Methylomicrobium buryatense 5GB1, flux analysis of cellular metabolism is needed and ¹³C tracer analysis is a foundational tool for such work. This biological system has a single-carbon input and a special network topology that together pose challenges to the current well-established methodology for ¹³C tracer analysis using a multi-carbon input such as glucose, and to date, no ¹³C tracer analysis of flux in a Type I methanotroph has been reported. In this study, we showed that by monitoring labeling patterns of several key intermediate metabolites in core metabolism, it is possible to quantitate the relative flux ratios for important branch points, such as the malate node. In addition, it is possible to assess the operation of the TCA cycle, which has been thought to be incomplete in Type I methanotrophs. Surprisingly, our analysis provides direct evidence of a complete, oxidative TCA cycle operating in M. buryatense 5GB1 using methane as sole carbon and energy substrate, contributing about 45% of the total flux for de novo malate production. Combined with mutant analysis, this method was able to identify fumA (METBUDRAFT_1453/MBURv2__60244) as the primary fumarase involved in the oxidative TCA cycle, among 2 predicted fumarases, supported by ¹³C tracer analysis on both fumA and fumC single knockouts. Interrupting the oxidative TCA cycle leads to a severe growth defect, suggesting that the oxidative TCA cycle functions to not only provide precursors for de novo biomass synthesis, but also to provide reducing power to the system. This information provides new opportunities for metabolic engineering of M. buryatense for the production of industrially relevant products.     

Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO₂⁻) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO₂⁻-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO₂⁻ tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO₂⁻. Nitrite decreased the cellular NADH/NAD⁺ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO₂⁻-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO₂⁻. These findings demonstrate a link between NO₂⁻ tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO₂⁻-tolerating mechanism in this strain.     

Green tea polyphenols boost gut-microbiota-dependent mitochondrial TCA and urea cycles in Sprague–Dawley rats

Green tea polyphenols (GTPs) were found to boost mammal energy conversion by modulating gut-microbial community structure, gene orthologs and metabolic pathways. Here we examined the metabolites present in the gut-microbiota-dependent mitochondrial tricarboxylic acid (TCA) cycle and urea cycle using hydrophilic interaction liquid chromatography (HILIC)-heated electrospray ionization (HESI)-tandem liquid chromatogram mass spectrometry (LC–MS). Six groups (n=12) of Sprague–Dawley rats (6-mo, ~250 g) were administered with water containing 0%, 0.5%, and 1.5% GTPs (wt/vol or g/dL). Gut-content samples were collected at 3- and 6-mo. Untargeted metabolomics detected 2177 features, with 91 features demonstrating significant dose- and time-dependencies on the GTPs treatment. Targeted metabolomics analysis revealed remarkable changes of 39 metabolites in the mitochondrial TCA cycle and urea cycle, including argininosuccunic acid (0.9-fold vs control), dihydrouracil (1.14-fold vs control), fumaric acid (1.19-fold vs control), malic acid (2.17-fold vs control), citrulline (1.86-fold vs control), and succinic acid (0.4-fold vs control). The untargeted metabolomics data were mined using bioinformatics approaches, such as analysis of variance-simultaneous component analysis (ASCA), enrichment pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping analysis. The results of 16S rRNA survey, metagenomics analysis, and metabolomics analysis were extrapolated and integrated using databases of Integrated Microbial Genomes and Microbiomes (IMG/M) and KEGG. Our analysis demonstrates that GTPs enhance energy conversion by boosting mitochondrial TCA cycle and urea cycle of gut-microbiota in rats. This metabolic modulation is achieved by enriching many gene orthologs, following the increase of beneficial microbials in families C. Ruminococcaceae, C. Lachnospiraceae and B. Bacteroidaceae.     

An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Background

In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/Principal Findings

We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/Significance

Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.     

Coordination of energy and carbon metabolism in lysine producing Brevibacterium strains

The present paper deals with the coordination of energy metabolism, glucose consumption rate, glycolytic and TCA cycle enzyme activities in the lysine-producing bacterium Brevibacterium flavum. It is shown, that inhibition of the elctron transport chain causes changes of the following sequence:

at first, TCA cycle enzymes are activated;

secondly, TCA cycle enzyme activity decreases, and glycolytic enzyme activities as well as glucose transport rate increase; there is a slight increase in Q_o2 and a considerable one of O₂ consumption in cyanide-resistant respiration pathway;

thirdly, TCA cycle enzyme activities and glucose transport rate decrease.

It is supposed, that coordination of carbon and energy metabolism in B. flavum depends on intracellular ATP concentration or energy charge value.     

An ATP and Oxalate Generating Variant Tricarboxylic Acid Cycle Counters Aluminum Toxicity in Pseudomonas fluorescens

Although the tricarboxylic acid (TCA) cycle is essential in almost all aerobic organisms, its precise modulation and integration in global cellular metabolism is not fully understood. Here, we report on an alternative TCA cycle uniquely aimed at generating ATP and oxalate, two metabolites critical for the survival of Pseudomonas fluorescens. The upregulation of isocitrate lyase (ICL) and acylating glyoxylate dehydrogenase (AGODH) led to the enhanced synthesis of oxalate, a dicarboxylic acid involved in the immobilization of aluminum (Al). The increased activity of succinyl-CoA synthetase (SCS) and oxalate CoA-transferase (OCT) in the Al-stressed cells afforded an effective route to ATP synthesis from oxalyl-CoA via substrate level phosphorylation. This modified TCA cycle with diminished efficacy in NADH production and decreased CO₂-evolving capacity, orchestrates the synthesis of oxalate, NADPH, and ATP, ingredients pivotal to the survival of P. fluorescens in an Al environment. The channeling of succinyl-CoA towards ATP formation may be an important function of the TCA cycle during anaerobiosis, Fe starvation and O₂-limited conditions.     

Comparative study of metabolism of the purple photosynthetic bacteria grown in the light and in the dark under anaerobic and aerobic conditions

For three species of anoxygenic phototrophic alphaproteobacteria differing in their reaction to oxygen and light, physiological characteristics (capacity for acetate assimilation, activity of the tricarboxylic acid (TCA) cycle enzymes, respiration, and the properties of the oxidase systems) were studied. Nonsulfur purple bacteria Rhodobacter sphaeroides, Rhodobaca bogoriensis, and aerobic anoxygenic phototrophic bacteria Roseinatronobacter thiooxidans were the subjects of investigation. All of these organisms were able to grow under aerobic conditions in the dark using the respiratory system with cytochrome aa ₃ as the terminal oxidase. They differed, however, in their capacity for growth in the light, bacteriochlorophyll synthesis, and regulation of activity of the TCA cycle enzymes. Oxygen suppressed bacteriochlorophyll synthesis by Rha. sphaeroides and Rbc. bogoriensis both in the dark and in the light. Bacteriochlorophyll synthesis in Rna. thiooxidans occurred only in the dark and was suppressed by light. The results on acetate assimilation by the studied strains reflected the degree of their adaptation to aerobic growth in the dark. Acetate assimilation by light-grown Rha. sphaeroides was significantly higher than by the dark-grown ones. Unlike Rha. sphaeroides, acetate assimilation by Rbc. bogoriensis in the light under anaerobic and aerobic conditions was much less dependent on the growth conditions. Aerobic acetate assimilation by all studied bacteria was promoted by light. In Rha. sphaeroides, activity of the TCA cycle enzymes increased significantly in the cells grown aerobically in the dark. In Rbc. bogoriensis, activity of most of the TCA cycle enzymes under aerobic conditions either decreased or remained unchanged. Our results confirm the origin of modern chemoorganotrophs from anoxygenic phototrophic bacteria. The evolution from anoxygenic photoorganotrophs to aerobic chemoorganotrophs included several stages: nonsulfur purple bacteria → nonsulfur purple bacteria similar to Rbc. bogoriensis → aerobic anoxygenic phototrophs → chemoorganotrophs.     

Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under anaerobic and microaerobic conditions

The Arc system is a two-component regulatory system composed of ArcA and ArcB in Escherichia coli. In the present study, the effects of arcA and arcB genes knockout on the TCA cycle activation in E. coli were investigated for the anaerobic and microaerobic conditions. Under anaerobic condition, the TCA cycle was up-regulated along with high lactate production, together with up-regulation of LDH for arcB mutant as compared with the parent strain. Due to down-regulation of aceE, aceF and lpdA genes which code for PDHc and low activity of Pfl in arcB mutant, the glycolysis as well as oxidative pentose phosphate pathway was down-regulated under anaerobic condition. The TCA cycle enzymes were further up-regulated when nitrate was added by modifying the redox state along with lower lactate production for arcB mutant. Different from the case of anaerobic condition, the glycolysis was activated under microaerobic condition, which may be partly due to the increased activity of PDHc encoded by aceE, F and lpdA genes. Under microaerobic condition, the TCA cycle genes together with their corresponding enzymes were up-regulated for arcB mutant as compared with the parent strain. These characteristics were further enhanced in arcA mutant as compared with the case of arcB mutant. The up-regulation of the TCA cycle together with down-regulation of cydB gene expression caused higher redox state in the arcA/B mutants, which in turn repressed the TCA cycle. Then the TCA cycle could be further increased by the addition of nicotinic acid (NA).     

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments

Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor.Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O₂ in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F_oF₁-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS.Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.     

Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum

Mass spectrometric analysis of O₂ and CO₂ exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH₄Cl resulted in nearly a 250% increase in the rate of TCA cycle CO₂ efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O₂ consumption in both light and dark. Anaerobiosis inhibited the CO₂ release caused by NH₄Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O₂ consumption in the light. This implied that the Mehler reaction did not play a significant role in O₂ consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions.     

Metabolic changes accompanying the loss of fumarate hydratase and malate–quinone oxidoreductase in the asexual blood stage of Plasmodium falciparum

In the glucose-rich milieu of red blood cells, asexually replicating malarial parasites mainly rely on glycolysis for ATP production, with limited carbon flux through the mitochondrial tricarboxylic acid (TCA) cycle. By contrast, gametocytes and mosquito-stage parasites exhibit an increased dependence on the TCA cycle and oxidative phosphorylation for more economical energy generation. Prior genetic studies supported these stage-specific metabolic preferences by revealing that six of eight TCA cycle enzymes are completely dispensable during the asexual blood stages of Plasmodium falciparum, with only fumarate hydratase (FH) and malate–quinone oxidoreductase (MQO) being refractory to deletion. Several hypotheses have been put forth to explain the possible essentiality of FH and MQO, including their participation in a malate shuttle between the mitochondrial matrix and the cytosol. However, using newer genetic techniques like CRISPR and dimerizable Cre, we were able to generate deletion strains of FH and MQO in P. falciparum. We employed metabolomic analyses to characterize a double knockout mutant of FH and MQO (ΔFM) and identified changes in purine salvage and urea cycle metabolism that may help to limit fumarate accumulation. Correspondingly, we found that the ΔFM mutant was more sensitive to exogenous fumarate, which is known to cause toxicity by modifying and inactivating proteins and metabolites. Overall, our data indicate that P. falciparum is able to adequately compensate for the loss of FH and MQO, rendering them unsuitable targets for drug development.     

Carbon Flow of Heliobacteria Is Related More to Clostridia than to the Green Sulfur Bacteria

The recently discovered heliobacteria are the only Gram-positive photosynthetic bacteria that have been cultured. One of the unique features of heliobacteria is that they have properties of both the photosynthetic green sulfur bacteria (containing the type I reaction center) and Clostridia (forming heat-resistant endospores). Most of the previous studies of heliobacteria, which are strict anaerobes and have the simplest known photosynthetic apparatus, have focused on energy and electron transfer processes. It has been assumed that like green sulfur bacteria, the major carbon flow in heliobacteria is through the (incomplete) reductive (reverse) tricarboxylic acid cycle, whereas the lack of CO₂-enhanced growth has not been understood. Here, we report studies to fill the knowledge gap of heliobacterial carbon metabolism. We confirm that the CO₂-anaplerotic pathway is active during phototrophic growth and that isoleucine is mainly synthesized from the citramalate pathway. Furthermore, to our surprise, our results suggest that the oxidative (forward) TCA cycle is operative and more active than the previously reported reductive (reverse) tricarboxylic acid cycle. Both isotopomer analysis and activity assays suggest that citrate is produced by a putative (Re)-citrate synthase and then enters the oxidative (forward) TCA cycle. Moreover, in contrast to (Si)-citrate synthase, (Re)-citrate synthase produces a different isomer of 2-fluorocitrate that is not expected to inhibit the activity of aconitase.     

Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine,l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro

Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-oxoglutarate dehydrogenase complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate dehydrogenase complex (hOADHc) from the final degradation pathway of l-lysine, l-hydroxylysine and l-tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (k_cat/K_m) were displayed by the two complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subcomplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both complexes produced superoxide/H₂O₂ from O₂ in the reductive half reaction suggesting that hE1o, and hE1a (within their complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two complexes on the different metabolic pathways.     

Cytochrome and Alternative Pathway Respiration during Transient Ammonium Assimilation by N-Limited Chlamydomonas reinhardtii

Mass spectrometric analysis of gas exchange in light and dark by N-limited cells of Chlamydomonas reinhardtii indicated that ammonium assimilation was accompanied by an increase in respiratory carbon flow to provide carbon skeletons for amino acid synthesis. Tricarboxylic acid (TCA) cycle carbon flow was maintained by the oxidation of TCA cycle reductant via the mitochondrial electron transport chain. In wild-type cells, inhibitor studies and ¹⁸O₂ discrimination experiments indicated that respiratory electron flow was mediated entirely via the cytochrome pathway in both the light and dark, despite a large capacity for the alternative pathway. In a cytochrome oxidase deficient mutant, or in wild-type cells in the presence of cyanide, the alternative pathway could support the increase in TCA cycle carbon flow. These different mechanisms of oxidation of TCA cycle reductant were reflected by the much greater SHAM sensitivity of ammonium assimilation by cytochrome oxidase-deficient cells as compared to wild type.     

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.