African trypanosomes are parasitic protozoa that cause sleeping sickness and nagana. Trypanosomes are not only of scientific interest because of their clinical importance, but also because these protozoa contain several very unusual biological features, such as their specially adapted mitochondrion and the compartmentalization of glycolytic enzymes in glycosomes. The energy metabolism of Trypanosoma brucei differs significantly from that of their hosts and changes drastically during the life cycle. Despite the presence of all citric acid cycle enzymes in procyclic insect-stage T. brucei, citric acid cycle activity is not used for energy generation. Recent investigations on the influence of substrate availability on the type of energy metabolism showed that absence of glycolytic substrates did not induce a shift from a fermentative metabolism to complete oxidation of substrates. Apparently, insect-stage T. brucei use parts of the citric acid cycle for other purposes than for complete degradation of mitochondrial substrates. Parts of the cycle are suggested to be used for (i) transport of acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, (ii) degradation of proline and glutamate to succinate, (iii) generation of malate, which can then be used for gluconeogenesis. Therefore the citric acid cycle in trypanosomes does not function as a cycle.

Unusual biological features of trypanosomatids

Trypanosoma brucei is a unicellular eukaryote that causes sleeping sickness in humans and nagana in livestock. These African trypanosomes are part of the family of Trypanosomatidae, which belongs to the protozoan order Kinetoplastida. This order includes species that parasitize a wide range of hosts, ranging from humans to plants. Trypanosomatidae are not only of scientific interest because of their clinical importance, but also because these protozoa possess a number of unusual biological features. Trypanosomatids are unusual with respect to the subcellular compartmentation of their energy metabolism, because a large part of the glycolytic pathway is sequestered within peroxisome-like organelles called glycosomes [1]. Trypanosomatid species also demonstrate distinctive traits in the mechanism of controlling gene expression, such as non-regulated polycistronic transcription by RNA polymerase II, trans-splicing of precursor mRNA molecules and RNA polymerase I transcription of not only rRNA but also of mRNA [2]. In addition, the mitochondrion of trypanosomatids contains many extraordinary features (Table 1). (i) All members of the order of Kinetoplastida are characterized by a single, large mitochondrion per cell. (ii) The trypanosomal mitochondrion contains a special structure, the kinetoplast, which comprises a giant network of thousands of concatenated circular DNAs. This kinetoplast DNA consists of thousands of minicircles (0.5–10 kb) that are heterogeneous in sequence and 40–50 maxicircles (20–40 kb) which encode typical mitochondrial gene products, such as rRNAs and respiratory chain subunits [3]. Therefore kinetoplast DNA is structurally the most complex mitochondrial DNA found in Nature and its replication requires a very complex machinery that differs from that of other eukaryotes [4]. (iii) Because trypanosomatids contain a single mitochondrion per cell, replication of the kinetoplast DNA and mitochondrial division are linked to cell division, which generally starts with the replication of the basal body and flagellum, followed by the division of the kinetoplast [5]. (iv) Finally, most mitochondrial mRNAs of trypanosomatids are subject to elaborate and precise post-transcriptional RNA-processing that inserts hundreds and deletes tens of uridylates. This RNA-editing process remodels mRNA to create initiation and termination codons, as well as the protein coding sequences that are subsequently translated, thereby defying the dogma that a DNA nucleotide sequence always determines the corresponding protein sequence [6,7]. RNA editing in trypanosomatids of mitochondrial mRNAs requires a very complex machinery, comprising a multiprotein editosome and multiple small guide RNAs to specify insertions and deletions.

Table 1
Special features in structure and biogenesis of mitochondria in trypanosomatids

Data for classical mammalian mitochondria are derived from Scheffler [31]. See text for references of data for trypanosomatids.

 Classical mammalian mitochondria Mitochondria in trypanosomatids 
Number of mitochondria per cell 100–10000 
DNA structure Circular, 16–17 kb mtDNA in total Many concatenated circular DNAs: 5000–10000 minicircles (0.5–10 kb each) 40–50 maxicircles (20–40 kb each) 
RNA editing No editing Major editing of transcripts: insertion and deletion of uridylates 
Protein translation tRNAs are mitochondrion encoded tRNAs nuclear encoded and imported from the cytosol 
Biogenesis Division not directly linked to cell division Division directly linked to cell division 
 Classical mammalian mitochondria Mitochondria in trypanosomatids 
Number of mitochondria per cell 100–10000 
DNA structure Circular, 16–17 kb mtDNA in total Many concatenated circular DNAs: 5000–10000 minicircles (0.5–10 kb each) 40–50 maxicircles (20–40 kb each) 
RNA editing No editing Major editing of transcripts: insertion and deletion of uridylates 
Protein translation tRNAs are mitochondrion encoded tRNAs nuclear encoded and imported from the cytosol 
Biogenesis Division not directly linked to cell division Division directly linked to cell division 

Taken together, these unique features indicate that the Kinetoplastida diverged relatively early in evolution from other eukaryotic lineages [8] and that especially their mitochondrion differs from those in other eukaryotes.

Adaptations in energy metabolism during the life cycle of T. brucei

African trypanosomes undergo a complex life cycle when they move from the bloodstream of their mammalian host to the blood-feeding insect vector, the tsetse fly (Glossina spp.). They encounter many different environments during their life cycle and respond to these by significant morphological and metabolic changes, including adaptation of their energy metabolism.

The long-slender bloodstream form T. brucei has a very simple type of energy metabolism, as it is entirely dependent on degradation of glucose into pyruvate by glycolysis. Glucose is degraded to 3-phosphoglycerate inside the glycosomes and this intermediate is then further degraded in the cytosol to pyruvate, the excreted end-product. The redox balance in the glycosome is maintained via a glycerol 3-phosphate shuttle and the alternative oxidase present in the mitochondrion [9]. Transformation of bloodstream form T. brucei into the procyclic insect stage is accompanied by striking changes in energy metabolism [10,11]. Upon transformation into the procyclic insect stage, the glycosomal metabolism is extended and part of the produced phosphoenolpyruvate is imported from the cytosol and subsequently converted into succinate via PEPCK (phosphoenolpyruvate carboxykinase), malate dehydrogenase, fumarase and a soluble glycosomal NADH:fumarate reductase [12]. In this procyclic insect stage, the end-product of glycolysis, pyruvate, is not excreted but is further metabolized inside the mitochondrion in which it is mainly degraded to acetate. Acetate production occurs by acetate:succinate CoA-transferase and involves a succinate/succinyl-CoA cycle that generates extra ATP [13,14]. In addition to carbohydrate degradation, amino acids, mainly proline and threonine, are important substrates for the production of ATP in procyclic insect-stage T. brucei [1517].

The factors that trigger the differentiation of trypanosomes from the bloodstream form into the insect form in vivo are not known, but in vitro this transformation can be induced by the addition of cis-aconitate [18,19] or by depletion of glucose [20]. This suggests that substrate availability and/or energy metabolism is not only changed upon transformation, but that it is also involved in the induction of differentiation, although the underlying mechanisms are not yet fully understood.

Unusual features of the citric acid cycle in procyclic insect-stage T. brucei

Until recently, the mitochondrion of procyclic insect-stage trypanosomes was believed to metabolize pyruvate to carbon dioxide by pyruvate dehydrogenase and subsequent citric acid cycle activity. However, this presumed flux through the citric acid cycle is only poorly supported by direct experimental data and is mainly based on the detected presence of all the enzyme activities involved [10,15,21]. Therefore its contribution to the energy metabolism was recently investigated by a more comprehensive metabolic study in which aconitase (a citric acid cycle enzyme) knockout cells were also used [16]. These investigations, which were performed under standard in vitro culture conditions, showed that pleiomorphic insect-stage T. brucei do not degrade glucose via the citric acid cycle to CO2, but produce acetate, succinate and alanine instead. Furthermore, no difference in pattern or amounts of excreted end-products was detected between wild-type and aconitase-knockout insect-stage T. brucei. This demonstrated that complete citric acid cycle activity is not involved in ATP production in insect-stage T. brucei under standard in vitro culture conditions [16]. Many microorganisms switch between a fermentative metabolism, when carbohydrates are abundant, to the complete oxidation via the citric acid cycle, when fermentable substrates are limited. Therefore it was recently investigated whether procyclic insect-stage T. brucei use the citric acid cycle for ATP production when the amount of fermentable substrates (glucose) is limited, a situation more resembling their natural environment in the gut of the fly [22]. However, also in the nearabsence of carbohydrates, insect-stage trypanosomes did not shift from a fermentative metabolism to the use of the citric acid cycle activity for complete oxidation of substrates.

What can be the reason that in procyclic insect-stage trypanosomes the citric acid cycle does not function in its usual way, as a complete cycle degrading acetyl-CoA to carbon dioxide? All genes for the eight enzymes of the citric acid cycle are present in the T. brucei genome and the protein expression of all the citric acid cycle enzymes is reported to be induced upon transformation of the bloodstream to the insect form [10,22]. The most likely explanation in terms of kinetics would be that the activity of one or more enzymes of the cycle is just too low, compared with the activities of those enzymes diverting metabolites from the cycle. The entry of acetyl-CoA into the citric acid cycle by citrate synthase meets these criteria the most, because (i) citrate synthase, aconitase and isocitrate dehydrogenase have a low specific activity compared with the other citric acid cycle enzymes [10] and (ii) insect-stage trypanosomes contain an acetate:succinate CoA-transferase that pulls acetyl-CoA towards acetate production.

If the citric acid cycle is not used as such, then what could be the function in procyclic insect-stage T. brucei of the expressed citric acid cycle enzymes? A clear catabolic function is known for the α-ketoglutarate to succinate part of the citric acid cycle in insect-stage T. brucei, because it is used for the degradation of proline (Figure 1) [16,17]. In addition, a recent report proposed anabolic functions for other parts of the citric acid cycle [22]. This report suggested that although citrate formation occurs at a rather low rate, it is used for fatty acid biosynthesis, which is known to occur in insect-stage T. brucei [23]. Because both glucose and threonine were found to be substrates for fatty acid biosynthesis in the proliferating insect-stage T. brucei [22], it implies that these substrates are first converted into acetyl-CoA, a process that occurs inside the mitochondrion. Subsequently, this acetyl-CoA has to be transferred from the mitochondrion to the cytosol for the biosynthesis of fatty acids. This transport has not been investigated in T. brucei, but in all other systems studied, this transport proceeds via citrate. Therefore it is likely that the first enzyme of the citric acid cycle, citrate synthase, is used in procyclic insect-stage T. brucei mainly for anabolic purposes, the formation of citrate for the biosynthesis of fatty acids. For this reaction to occur, the last enzyme of the cycle, malate dehydrogenase, has to participate in the formation of oxaloacetate that is needed in the citrate synthase reaction.

Schematic representation of the unusual functions of the citric acid cycle enzymes in the mitochondrion of procyclic insect-stage T. brucei

Figure 1
Schematic representation of the unusual functions of the citric acid cycle enzymes in the mitochondrion of procyclic insect-stage T. brucei

Substrates are shown by ovals and end-products are boxed. The shaded broad arrows in the background of the citric acid cycle represent functions of those parts of the cycle that are discussed in this review and that are active in insect-stage trypanosomes. The shaded broad arrow from pyruvate and oxaloacetate to citrate indicates the flux via this part of the citric acid cycle, used in the transport of acetyl-CoA units from the mitochondrion to the cytosol. The shaded broad arrow from α-ketoglutarate to succinate represents that part of the cycle that is used for the degradation of proline and glutamate to succinate. The shaded broad arrow from succinate to malate indicates the part of the cycle that is used during gluconeogenesis (see text for further explanations). Abbreviations: AA, amino acid; CI, II, III and IV, complex I, II, III and IV of the respiratory chain; c, cytochrome c; Glu, glutamate; α-KG, α-ketoglutarate; OA, oxoacid; Q, ubiquinone. Adapted from [22] with permission. © 2005 The American Society for Biochemistry and Molecular Biology.

Figure 1
Schematic representation of the unusual functions of the citric acid cycle enzymes in the mitochondrion of procyclic insect-stage T. brucei

Substrates are shown by ovals and end-products are boxed. The shaded broad arrows in the background of the citric acid cycle represent functions of those parts of the cycle that are discussed in this review and that are active in insect-stage trypanosomes. The shaded broad arrow from pyruvate and oxaloacetate to citrate indicates the flux via this part of the citric acid cycle, used in the transport of acetyl-CoA units from the mitochondrion to the cytosol. The shaded broad arrow from α-ketoglutarate to succinate represents that part of the cycle that is used for the degradation of proline and glutamate to succinate. The shaded broad arrow from succinate to malate indicates the part of the cycle that is used during gluconeogenesis (see text for further explanations). Abbreviations: AA, amino acid; CI, II, III and IV, complex I, II, III and IV of the respiratory chain; c, cytochrome c; Glu, glutamate; α-KG, α-ketoglutarate; OA, oxoacid; Q, ubiquinone. Adapted from [22] with permission. © 2005 The American Society for Biochemistry and Molecular Biology.

The part of the citric acid cycle in which succinate is converted into malate is used by insect-stage T. brucei in reversed direction for mitochondrial succinate production [24]. After mitochondrial import of malate, it is converted into succinate by the subsequent action of fumarase and a soluble NADH:fumarate reductase. This pathway thereby provides the mitochondrion of insect-stage T. brucei with an alternative electron sink, as NADH can not only be reoxidized by the NADH:ubiquinone oxidoreducatases, but also by this soluble fumarate reductase during succinate production from glucose. In addition, Van Weelden et al. [22] proposed that this part of the citric acid cycle is used in its unusual direction by the insect-stage T. brucei for gluconeogenesis, when glucose is absent from their environment (Figure 1). Malate is then produced from succinate by succinate dehydrogenase and fumarase, and transported from the mitochondrion to the cytosol after which it can be used for gluconeogenesis. Insect stage T. brucei contain and express all enzymes needed for gluconeogenesis, including fructose-1,6-bisphosphatase and PEPCK [25,26]. Furthermore, insect-stage T. brucei are known to be able to proliferate in the absence of carbohydrates [17,22,27] and therefore they have to be able to perform gluconeogenesis, because they need carbohydrates for the biosynthesis of nucleotides and for the glycoconjugates present on their surface proteins.

All together these proposed new functions in the mitochondrial metabolism of procyclic insect-stage T. brucei require six out of the eight enzymes of the citric acid cycle. Only the mitochondrial aconitase and isocitrate dehydrogenase have no clear metabolic function in procyclic insect-stage T. brucei yet. This correlates with their relatively low expression level [10], and with the absence of a specific phenotype in the energy metabolism of the aconitase-knockout mutant in insect-stage T. brucei [16]. Furthermore, trypanosomal isocitrate dehydrogenase is NADP dependent, which suggests that it is involved in the production of NADPH rather than in citric acid cycle activity connected to NADH production.

Special features of the respiratory chain in procyclic insect-stage T. brucei

The mitochondrion of procyclic insect-stage T. brucei contains a branched electron-transport chain (Figure 2). It contains four complexes that donate electrons to the ubiquinone pool, two NADH:ubiquinone oxidoreductases (complex I as well as a rotenone-insensitive enzyme) [28,29], complex II (succinate dehydrogenase) and glycerol-3-phosphate dehydrogenase. Although the last two complexes are present, metabolic studies suggest that both succinate dehydrogenase and glycerol-3-phosphate dehydrogenase do not function as major entry sites for electrons in the respiratory chain of insect-stage cells during standard in vitro culture conditions [16,22,24]. Reduced ubiquinol can be reoxidized by the transfer of electrons to either the AOX (plant-like alternative oxidase), which does not translocate protons, or to the cytochrome-containing complexes III (cytochrome c reductase) and IV (cytochrome c oxidase). These complexes III and IV are similar to those of a classical respiratory chain, producing a proton motive force by translocation of protons (Figure 2).

Schematic representation of the major components of the mitochondrial respiratory chain in procyclic insect-stage T. brucei

Figure 2
Schematic representation of the major components of the mitochondrial respiratory chain in procyclic insect-stage T. brucei

Boxes indicate electron–transport chain complexes that are either involved in proton translocation (shaded and indicated by H+→) or not (white). Ovals represent the electron transporters ubiquinone (UQ) and cytochrome c (cyt. c). Electron transport is indicated by broken arrows. The vertical bar represents a scale for the standard redox potentials in mV. Abbreviations: CI, II, III and IV, complex I, II, III and IV of the respiratory chain; DHAP, dihydroxy-acetonephosphate; G3P, glycerol 3-phosphate; Fum, fumarate; NDH2, rotenone-insensitive NADH:ubiquinone oxidoreductase; Succ, succinate; UQH2, ubiquinol.

Figure 2
Schematic representation of the major components of the mitochondrial respiratory chain in procyclic insect-stage T. brucei

Boxes indicate electron–transport chain complexes that are either involved in proton translocation (shaded and indicated by H+→) or not (white). Ovals represent the electron transporters ubiquinone (UQ) and cytochrome c (cyt. c). Electron transport is indicated by broken arrows. The vertical bar represents a scale for the standard redox potentials in mV. Abbreviations: CI, II, III and IV, complex I, II, III and IV of the respiratory chain; DHAP, dihydroxy-acetonephosphate; G3P, glycerol 3-phosphate; Fum, fumarate; NDH2, rotenone-insensitive NADH:ubiquinone oxidoreductase; Succ, succinate; UQH2, ubiquinol.

As described above, the mitochondrial energy metabolism of insect-stage T. brucei mainly depends on degradation of pyruvate to acetate and degradation of amino acids to succinate and/or acetate. Both catabolic pathways produce ATP by substrate level phosphorylation, because both pathways demonstrate succinyl-CoA to succinate conversion by succinyl-CoA synthetase. However, these pathways also produce large amounts of NADH (Figure 1), which has to be reoxidized either by NADH:fumarate reductase or by one of the NADH:ubiquinone oxidoreductases and the subsequent respiratory chain activity. Simultaneous addition of salicylhydroxamic acid and cyanide results in rapid death of the procyclic insect-stage T. brucei [16,30], which shows that NADH:fumarate reductase is a poor electron-sink and cannot compensate for the simultaneous loss of both the AOX and cytochrome c oxidase activity. These results demonstrate that electron-transport via the branched electron-transport chain is essential in insect-stage T. brucei and indicates that the NADH:fumarate reductase has a limited function in maintenance of the mitochondrial redox-balance.

From all these metabolic studies, it became clear that procyclic insect-stage T. brucei cells are very flexible in their energy metabolism and that they can adjust their metabolism to changing conditions in the environment.

Mechanistic and Functional Studies of Proteins: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by S. Crosthwaite (Manchester, U.K.), M. Ginger (Oxford, U.K.), K. Gull (Oxford, U.K.), A. Lee (Southampton, U.K.), H. McWatters (Oxford, U.K.), J. Mottram (Glasgow, U.K.), P. Rich (University College London, U.K.), C. Robinson (Warwick, U.K.) and H. van Veen (Cambridge, U.K.).

Abbreviations

     
  • AOX

    plant-like alternative oxidase

  •  
  • PEPCK

    phosphoenolpyruvate carboxykinase

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