The aerobic energy metabolism of the juvenile Fasciola hepatica

doi:10.1016/0166-6851(81)90052-9

Molecular and Biochemical Parasitology

Volume 3, Issue 4, August 1981, Pages 205-214

https://doi.org/10.1016/0166-6851(81)90052-9 Get rights and content

Abstract

Juvenile Fasciola hepatica were isolated immediately after in vitro emergence from the metacecarial cysts and incubated with uniformly labelled glucose. Under aerobic conditions, carbon dioxide was the main end product of glucose breadkown. In the absence of oxygen, glucose was fermented mainly to propionate and acetate in a molar ratio of 2 : 1, with lactate as a minor product. This anaerobic end-product pattern closely resembles that of the adult liver fluke. In the presence of oxygen and 1 mM cyanide, lactate accumulated. The difference between anaerobic glucose breakdown and that in the presence of cyanide is explained by an inhibitory effect of cyanide on the malic enzyme (EC 1.1.1.40) of the juvenile mitochondria. A substantial Pasteur effect is calculated from these incubations. The oxygen consumption of the juveniles was completely cyanide-sensitive. From these results it is concluded that in aerobic conditions the juvenile liver flukes have an aerobic energy metabolism. Since they can survive prolonged periods of anaerobiosis, they should be called facultative anaerobes.

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Cited by (38)

Insights into Fasciola hepatica Juveniles: Crossing the Fasciolosis Rubicon
2021, Trends in Parasitology
Citation Excerpt :
Glucose catabolism is the best described metabolic difference between FhNEJ and the adult worm. Adult flukes are fully anaerobic, and the main product of glucose cytosolic degradation at this stage is acetate, while FhNEJ mainly use the aerobic route when freshly excysted and later switch to a facultative anaerobic profile that keeps specific aerobic paths of glucose breakdown both in the presence and absence of oxygen [27–29]. Therefore, it could be concluded that F. hepatica juveniles are gradually forced to switch from an aerobic metabolism to the anaerobic glycolysis that is fully developed in adult worms, in which the level of oxygen reaching internal tissues by diffusion is lower than in juvenile stages due to their larger size and the essentially anaerobic environment of the bile ducts.
Unraveling the molecular interactions governing the first contact between parasite and host tissues is of paramount importance to the development of effective control strategies against parasites. In fasciolosis, a foodborne trematodiasis caused mainly by Fasciola hepatica, these early interactions occur between the juvenile worm and the host intestinal wall a few hours after ingestion of metacercariae, the infectious stage of the parasite. However, research on these early events is still scarce and the majority of studies have focused on the adult worm. Here, we review current knowledge on the biology and biochemistry of F. hepatica juveniles and their molecular relationships with the host tissues and identify the research needs and gaps to be covered in the future.
Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead
2019, International Journal for Parasitology
Citation Excerpt :
The acidified supernatant was neutralised by the addition of 6 M NaOH. The labelled metabolic end products in the supernatant were analysed by anion exchange chromatography on a Dowex 1X8, 100–200 mesh column (Serva) (60 × 1.1 cm) in chloride form (Tielens et al., 1981). The column was eluted successively with 200 ml of 5 mM HCl and 130 ml of 0.2 M NaCl.
Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [¹⁴C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no ¹⁴CO₂ production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.
Lipids Are the Preferred Substrate of the Protist Naegleria gruberi, Relative of a Human Brain Pathogen
2018, Cell Reports
Citation Excerpt :
In aerobic incubations the gas phase was air. For anaerobic incubations the medium was degassed, flasks were flushed for 5 min with nitrogen and prior to closing the bottles, ascorbate oxidase (100 U) was added to the anaerobic incubations to remove any remaining oxygen (Tielens et al., 1981). To determine the effects of respiratory chain inhibitors on the cell growth of N. gruberi NEG-M, trophozoites were seeded in PYNFH medium at a density of 30,000 trophozoites per ml.
Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here, we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents an additional metabolic strategy in protists.
Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism
2004, Journal of Biological Chemistry
Citation Excerpt :
After removal of carbon dioxide, the acidified supernatant was separated from the cells by centrifugation (4 °C, 10 min at 500 × g) and neutralized by the addition of 40 μlof6 m NaOH. Analysis of the labeled end products occurred by anion-exchange chromatography on a Dowex 1X8, 100–200 mesh column (Serva), 60 × 1.1 cm in chloride form (44). The column was eluted successively with 200 ml of 5 mm HCl, 130 ml of 0.2 m NaCl, and 130 ml of 0.5 m NaCl.
Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Δasct/Δasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of ¹³C-enriched and ¹⁴C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei.
Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation
2003, Journal of Biological Chemistry
Citation Excerpt :
The simultaneous presence of KCN and SHAM resulted within 1 h in cell death of both the wild type and aconitase knockout procyclic trypanosomes (not shown). In addition, the complete removal of oxygen from the incubation, (using ascorbate and ascorbate oxidase as was described previously (31)), also resulted in rapid death of the organisms (not shown). Apparently, procyclic T. brucei cells are completely dependent on the use of oxygen by the respiratory chain for their energy generation and survival.
The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitrodifferentiation from wild type and mutant (Δaco::NEO/Δaco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO₂. Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
Respiratory Chain of the lung fluke paragonimus westermani - facultative anaerobic mitochondria
1994, Archives of Biochemistry and Biophysics
The respiratory chain of adult Paragonimus westermani, a lung fluke, was characterized in isolated mitochondria. The fluke mitochondria exhibited cyanide- and antimycin A-sensitive succinate oxidase activity at a rate of 16.8 nmol O₂ min⁻¹ mg⁻¹ protein. The succinate oxidation was shown to be stimulated by ADP and linked to the formation of membrane potential. The specific activities of oxidoreductases composing the succinate oxidase system, i.e., succinate-ubiquinone and succinate-cytochrome c oxidoreductase (complex II and complex II-III, respectively) and cytochrome c oxidase (complex IV), were compared in mitochondria from adult Paragonimus, bovine heart (an aerobic tissue), and muscle of adult Ascaris suum which possesses an anaerobic respiratory chain. The activity values of complex II-III and complex IV were high, middle, and low for bovine heart, Paragonimus, and A. suum, respectively, whereas the activity of complex II was comparable among the three sources. The cytochrome contents of Paragonimus mitochondria as determined by difference absorption spectrophotometry ranged between those in Ascaris and bovine mitochondria for types c and aa₃ cytochromes. Paragonimus mitochondria exhibited a high activity of NADH-fumarate reductase; the specific activity was about 18-fold higher in fluke submitochondria than in bovine heart submitochondria. Quinone analysis by HPLC and mass spectrometry showed that the fluke mitochondria contain both rhodoquinone-10 and ubiquinone-10 at concentrations of 0.572 and 0.321 nmol mg⁻¹ mitochondrial protein, respectively. These data clearly show that mitochondria from adult P. westermani, unlike adult Ascaris mitochondria, possess both cyanide-sensitive succinate oxidase and NADH-fumarate reductase systems, indicating that the fluke mitochondria are facultatively anaerobic.

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Molecular and Biochemical Parasitology

Abstract

References (22)

Studies on the carbohydrate metabolism of the liver fluke Fasciola hepatica

Biochim. Biophys. Acta

The metabolism of phosphoenolpyruvate and pyruvate in the adult liver fluke Fasciola hepatica

Biochim. Biophys. Acta

Pathways of acetate and propionate production in adult Fasciola hepatica

Int. J. Parasitol.

The formation of propionate and acetate as terminal processes in the energy metabolism of the adult liver fluke Fasciola hepatica

Int. J. Biochem.

The chemotherapy of fascioliasis

Br. Vet. J.

Liquid scintillation counting of aqueous samples using Triton-containing scintillants

Anal. Biochem.

Protein measurement with the Folin reagent

J. Biol. Chem.

‘Malic’ enzyme

Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity in adult Fasciola hepatica (Trematoda)

Int. J. Parasitol.

Catabolic metabolism in Trypanosoma cruzi

Int. J. Parasitol.

Fasciola hepatica: Cytochrome c oxidoreductases and effects of oxygen tension and inhibitors

Exp. Parasitol.

Cited by (38)

Insights into Fasciola hepatica Juveniles: Crossing the Fasciolosis Rubicon

Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead

Lipids Are the Preferred Substrate of the Protist Naegleria gruberi, Relative of a Human Brain Pathogen

Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism

Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

Respiratory Chain of the lung fluke paragonimus westermani - facultative anaerobic mitochondria