Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica☆
Introduction
Fasciola hepatica, the common liver fluke, is a parasitic flatworm that infects a large variety of herbivorous animals causing a disease called fascioliasis. Additionally, in the last decades it has become an important emerging, or re-emerging trematode infection in humans, with estimated numbers varying from 2.4 million to 17 million human infections worldwide [1], [2], [3].
Adult F. hepatica worms reside in the bile ducts of infected organisms and due to their size and the poor availability of oxygen in this environment, these helminths depend on an anaerobic mitochondrial metabolism using a pathway called malate dismutation [4]. Their fumarate-respiring anaerobic mitochondria use a specialized electron-transport chain to perform oxidative phosphorylation (Fig. 1). In this anaerobic energy metabolism carbohydrates are degraded via the glycolytic pathway to phosphoenolpyruvate. This phosphoenolpyruvate is then converted via oxaloacetate to malate, which is transported into the mitochondria. In a split pathway some of the malate is oxidized and some is reduced (dismutation). The oxidation of malate is catalyzed by malic enzyme and pyruvate dehydrogenase and results in the formation of acetyl-CoA, which is converted to acetate. The rest of the malate is reduced to succinate. This reduction of malate to succinate occurs in two reactions whereby the reduction of fumarate to succinate is the essential NADH-consuming reaction for maintaining redox balance. Fumarate reduction is linked to the electron-transport chain, proton-pumping by NADH-dehydrogenase (Complex I) and ATP-formation (Fig. 1). The NADH formed from NAD in the oxidative branch donates its electrons to the proton-pumping Complex I (enzyme complex 9 in Fig. 1) and via rhodoquinone and fumarate reductase (enzyme complex 8 in Fig. 1) these electrons are then used to reduce fumarate to succinate. In this way malate dismutation in anaerobically functioning mitochondria is coupled to ATP formation via oxidative phosphorylation, but without the use of oxygen as final electron acceptor [5]. In malate dismutation redox balance is maintained when twice as much succinate as acetate is formed. In F. hepatica, acetate is excreted as metabolic end product, whereas succinate is further metabolized and decarboxylated to the other excreted metabolic end product, propionate [6]. In the final reaction of their production, both acetate and propionate are formed in F. hepatica mitochondria by a similar enzymatic reaction catalyzed by a CoA-transferase. In both cases the CoA moiety is transfered to succinate, thereby converting acetyl-CoA and propionyl-CoA into acetate and propionate, respectively (Fig. 1). The succinyl-CoA formed during acetate production is converted back into succinate by succinyl-CoA synthetase, an enzyme that yields ATP and is also present in the Krebs cycle. The production of propionate from succinate also results in the formation of ATP (Fig. 1).
Most enzymatic reactions involved in malate dismutation are also present in aerobically functioning mitochondria and the enzymes catalyzing these reactions, such as fumarase, malic enzyme, pyruvate dehydrogenase and succinyl-CoA synthetase, are characterized and their genes identified in many organisms [5]. This is not the case for the acetate:succinate CoA-transferase (ASCT) reaction yielding acetate nor for the propionate:succinate CoA-transferase (PSCT) reaction yielding propionate. Eventhough the ASCT and the PSCT reactions in F. hepatica were the first acetate and propionate yielding CoA-transferase reactions described in anaerobic mitochondria [7], the enzymes catalysing them are still waiting to be characterized and their genes to be identified.
Recently two enzymes catalysing ASCT reactions in different parasites have been characterized: the ASCT of the aerobic mitochondria of Trypanosoma brucei and the ASCT of the anaerobic hydrogenosomes of Trichomonas vaginalis [8], [9]. Hydrogenosomes are ATP producing organelles that are evolutionary related to mitochondria [10], [11], [12], [13], [14], [15] and therefore it is intruiging that T. brucei ASCT and T. vaginalis ASCT are non-homologous proteins [9]. This suggests that the presence of ASCT activity in both hydrogenosomes and mitochondria is not caused by a direct evolutionary relation. Also in that respect, it is interesting to know what kind of CoA-transferase is present in the third type of energy-generating organelle, anaerobically functioning mitochondria, found for example in many parasitic helminths [5]. Unfortunately, purification of the CoA-transferase protein from F. hepatica was not successful up to now (S.W.H. van Weelden, PhD thesis, Utrecht, 2005).
For these reasons we searched an F. hepatica expressed sequence tag (EST) database for homologs of both the T. brucei ASCT and the T. vaginalis ASCT. No homologs of the mitochondrial T. brucei ASCT were found, but we discovered a sequence homologous to part of the hydrogenosomal T. vaginalis ASCT. This enabled us to isolate the complete Open Reading Frame (ORF) of the F. hepatica CoA-transferase, which contained the expected mitochondrial target signal. In this study we describe the sequence, expression, enzymatic functions and localization of this F. hepatica CoA-transferase, which is shown to catalyze the two different final reactions used to produce acetate and propionate, the two major metabolic end products in the anaerobic mitochondrial metabolism of F. hepatica.
Section snippets
Isolation of F. hepatica mRNA and cDNA synthesis
Adult F. hepatica worms were collected from slaughterhouse material and washed with a medium containing 10 mM Hepes (pH 7.6) 100 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 1.8 mM NaH2PO4, 0.5 mM CaCl2, 10 mM glucose and 25 mM NaHCO3 at 37 °C. After visual inspection, clean liver flukes were selected, snap frozen and ground in liquid nitrogen, after which the frozen, powdered worms were dissolved in TRIzol reagent (Invitrogen). Total RNA was extracted following the manufacturers instructions and cDNA was
Identification of F. hepatica ASCT
A database containing 15,000 F. hepatica ESTs [16], was searched for homologs of T. brucei ASCT and T. vaginalis ASCT. Neither template resulted in a good match within the F. hepatica EST database. However, when Artemia franciscana p49, a homolog of T. vaginalis ASCT, was used as a template, Fhep37e09, an incomplete sequence, was identified as a high scoring EST. The complete coding sequence corresponding to Fhep37e09 was identified from cDNA upon amplification using primers against the F.
References (35)
- et al.
Food-borne Trematodes: ignored or emerging?
Parasitol Today
(1994) - et al.
The energy metabolism of Fasciola hepatica during its development in the final host
Mol Biochem Parasitol
(1984) Energy generation in parasitic helminths
Parasitol Today
(1994)- et al.
The relationship of some intermediary metabolites to the production of volatile fatty acids by adult Fasciola hepatica
Comp Biochem Physiol B
(1971) - et al.
Pathways of acetate and propionate production in adult Fasciola hepatica
Int J Parasitol
(1978) - et al.
Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism
J Biol Chem
(2004) - et al.
Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization
J Biol Chem
(2008) - et al.
Anaerobic eukaryote evolution: hydrogenosomes as biochemically modified mitochondria?
Trends Ecol Evol
(1997) The missing link between hydrogenosomes and mitochondria
Trends Microbiol
(2005)- et al.
RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs
J Biol Chem
(1994)
Steady state kinetics
Mechanism and specificity of succinyl-CoA:3-ketoacid coenzyme A transferase
J Biol Chem
The formation of propionate and acetate as terminal processes in the energy metabolism of the adult liver fluke Fasciola hepatica
Int J Biochem
Predicting subcellular localization of proteins based on their N-terminal amino acid sequence
J Mol Biol
Homing in on helminths
Am J Trop Med Hyg
Epidemiology of fascioliasis in human endemic areas
J Helminthol
A common evolutionary origin for mitochondria and hydrogenosomes
Proc Natl Acad Sci USA
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Stage-specific transcriptomic analysis of the model cestode Hymenolepis microstoma
2021, GenomicsCitation Excerpt :In particular, there are two paralogs of SDHC in H. microstoma [70], one that is highly expressed in adults and in cysticercoids (HmN_003036360), and another paralog of moderate expression levels but which is up-regulated 37-fold in adult worms with respect to cysticercoids (HmN_003036350). On the other hand, an ASCT protein has been identified in vitro in the trematode Fasciola hepatica [71], and its ortholog in H. microstoma (HmN_003032760) is upregulated 2.4-fold in adult worms relative to cysticercoids (Fig. 5B top panel, Supplementary Table 6). Finally, a mitochondrial NAD(P) transhydrogenase activity has been proposed to be important for malate dismutation in H. diminuta, connecting NADPH generation via mitochondrial NADP+-specific malic enzyme, with NADH formation required for electron transport and fumarate reduction [72].
The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei
2020, Biochimica et Biophysica Acta - BioenergeticsCitation Excerpt :Thus, collectively, these data demonstrate that although TbASCT and TbACH catalyze distinct reactions, they share a common role in terms of acetate production to feed the de novo fatty acid biosynthesis [15,28], act redundantly, and are indispensable for growth of BSF trypanosomes as previously observed for the PCF [22]. Biochemical and structural biology studies of ASCT have thus far been hampered due to a difficulty to express and purify the recombinant enzyme because of its low expression level and solubility and high degree of instability [20,33,34]. Expression of recombinant TbASCT fused with a His6-SUMO tag at the N-terminus in this study is therefore critical for increasing the expression level as well as solubility of the enzyme [61–64], and the addition of glycerol [18,64] in all purification steps is necessary to stabilize the enzymatic activity.
Transcriptome analysis of the adult rumen fluke Paramphistomum cervi following next generation sequencing
2015, GeneCitation Excerpt :Additionally, gene encoding enzymes involved in the pathway that converts glucose to acetyl-CoA were transcribed at high levels (FPKM > 20) in P. cervi. This observation is consistent with a study showing glucose serving as an energy source in C. sinensis (van Grinsven et al., 2009; Huang et al., 2013). Given these data, how does the adult fluke obtain enough glucose in the rumen?
Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: A critical junction between aerobic and anaerobic metabolism
2012, Experimental ParasitologyNaegleria gruberi metabolism
2011, International Journal for ParasitologyCitation Excerpt :Indeed multiple isoenzymes of malate dehydrogenase, malic enzyme and NADH-dependent fumarate reductase were identified, with a mitochondrial transit peptide predicted for at least one of each. The acetyl-CoA produced in the oxidative branch may be converted via a cycle comprising acetate:succinate CoA-transferase (of family type 1B, homologous to the recently identified Fasciola hepatica enzyme by Van Grinsven et al., 2009; Tielens et al., 2010) and succinyl-CoA synthetase. The resulting acetate is excreted.