Energy Metabolism
Synonym
Bioenergetics
General Information
The generation of chemical energy (usually in the form of ATP) can be accomplished by either aerobic or anaerobic strategies. Aerobic generation of energy is defined as the complete oxidation of substrates to carbon dioxide and water via the combined action of the tricarboxylic acid (TCA) cycle and mitochondrial respiratory chain. In this pathway, in which oxygen acts as the final electron acceptor to reoxidize reduced coenzymes, the major portion of the chemical energy is produced by the process known as oxidative phosphorylation. In parasitic protozoa and helminths, the occurrence of this conventional type of energy metabolism is rather limited. Only the insect stages of certain protozoa and the free-living and some larval stages of helminths are supposed to possess a functional TCA cycle and aerobic mitochondrial respiratory system. A special category of organisms are the trypanosomatids, such as the bloodstream form of Trypanosoma brucei, whose energy metabolism is dependent on a plant-like alternative terminal oxidase. These organisms use oxygen as terminal electron acceptor but are incapable of coupling this oxidative process with phosphorylation (aerobic “fermentation”). Fermentations are defined as energy generating processes that produce their own oxidants to balance the production and consumption of coenzymes (NADH) without the use of oxygen as final electron acceptor. In some cases, this strategy is linked to an electron transport chain and oxidative phosphorylation (anaerobic “respiration”). Suitable substrates for fermentations are carbohydrates, because both oxidation and reduction of these compounds can occur. During lactate or ethanol producing fermentations these redox reactions are accomplished in the linear process of glycolysis. An alternative strategy, which is widely found in helminths involves a branched pathway (malate dismutation), in which a portion of the substrate is oxidized while another portion is reduced. Fermentations are widespread in endoparasites and are the sole or major ATP-producing routes in many protozoan and adult helminth parasites.
In all living cells, nutrient molecules are broken down to provide the energy required for the generation of ATP. This ATP can be synthesized from ADP via two basically different processes: substrate level phosphorylation and oxidative phosphorylation. Substrate level phosphorylation is the formation of ATP by the direct phosphorylation of ADP via the transfer of a phosphoryl group from a high-energy intermediate to ADP. Oxidative phosphorylation is the process in which ATP is formed when electrons are transferred from the reduced coenzymes NADH or FADH2 to oxygen via a series of electron carriers that make up the mitochondrial electron transport chain (also called respiratory chain). NADH and FADH2 (produced in glycolysis, fatty acid oxidation and the TCA cycle, from NAD+ and FAD, respectively) are energy-rich compounds. They contain high-energy electrons obtained from metabolic intermediates. Transfer of these electrons from the reduced coenzymes to oxygen releases a large amount of free-energy that can be used to produce ATP. The transport of electrons through the electron transport chain leads to the pumping of protons across the mitochondrial inner membrane by three electron-driven proton pumps: NADH-ubiquinone reductase (Complex I), cytochrome reductase (Complex III) and cytochrome oxidase (Complex IV). The resulting proton gradient across the inner mitochondrial membrane is then used for the generation of ATP when electrons flow back into the mitochondrial matrix via ATP synthase. Oxidative phosphorylation provides most of the energy in aerobically functioning parasites, but is also connected to malate dismutation, the anaerobic fermentation variant operative in most adult helminths, where instead of oxygen, fumarate acts as terminal electron acceptor of the electron transport chain.
Inhibitors of the electron transport chain are often used to study the energy metabolism of parasites. These compounds bind to a component of the chain and block the flow of electrons, which results in a decreased ATP production as electron transport and energy production are tightly coupled. Examples of frequently used inhibitors are: rotenone and amytal (inhibitors of Complex I), antimycin A (an inhibitor of electron flow through Complex III), and cyanide, azide or carbon monoxide (inhibitors of Complex IV). Oligomycin and other unrelated compounds block the proton channel of the ATP-synthase, thereby directly inhibiting ATP synthesis.
In the energy metabolism of endoparasites, several biochemical reactions exist which have either no parallel in other eukaryotes or are a result of a modification of rather universal pathways, but which usually do not have a function in energy generation. Energy metabolism of endoparasites is a fascinating and most extensively studied research area of parasite biochemistry. Its vital function and divergence from that of higher organisms makes the energy metabolism of parasites an interesting target for antiparasitic drug design.
Closely related to bioenergetics is the function of molecular oxygen in living organisms. Most of the oxygen consumed by animal cells is utilized by the cytochrome oxidase-linked mitochondrial respiration. In many parasitic protozoa and helminths this metabolic capability often does not exist or is considerably reduced. On the other hand, endoparasites unequivocally have an aerobic requirement, if not for energy generation, then for specialized oxidative reactions such as egg shell tanning in helminths. These other functions of oxygen vary greatly between different species and their developmental stages, and have not yet been very well studied.
Adaptations
The major functions of the metabolism of animals are (1) to catabolize organic substances and to couple these processes to the conservation of chemical energy; (2) to assemble distinct, low-molecular weight precursors that are derived either directly from external sources or via metabolic interconversion of absorbed nutrient molecules into species-specific polymeric components (nucleic acids, proteins, polysaccharides and lipids); and (3) to form and degrade the biomolecules required in specialized functions. Endoparasites are no exception to this universal concept but obviously, as a consequence of their parasitic way of life, they have evolved a variety of specific modifications, extensions or simplifications to the metabolic pattern observed in most other forms of life.
As in many habitats, the major nutritional requirements for parasites are supplied by the host. Many synthetic pathways in parasites were abandoned during their evolution. These reduced biosynthetic capabilities have resulted in the elaboration of efficient mechanisms for substrate absorption and in specific pathways for interconversion and modification of substrate molecules. Additional features relate to the sophisticated adaptive mechanisms which enable parasites to evade the host's immune response and other defence systems. In many cases, unique metabolic structures and processes are maintained by parasites to cope with the extreme physical and chemical conditions often prevailing within their host environments. Still other metabolic peculiarities may be related to the distinct morphological organization of parasites, such as the lack of a circulatory system in helminths or the absence of a digestive tract in cestodes. Next to the adaptations in metabolism related to their opportunistic way of living as a parasite, helminths show a second type of adaptations, i.e. those connected to the environmental changes that occur in their life cycle. During their complex developmental cycles, these organisms live alternating periods as free-living and parasitic stages. Usually, the free-living stages cannot obtain substrates from the environment. They have to live on the endogenous reserves that they obtained in their previous host. In the environment of parasitic stages, on the other hand, substrates are plentiful, and their only concern is to produce offspring and avoid being destroyed by the host's immune system. These changes in the environmental conditions are accompanied by metabolic adaptations. Unusual adaptive processes are found particularly in the strategies of energy metabolism, in the various routes of purine and pyrimidine salvage, and in the synthesis of numerous other molecular structures serving specialized functions.
Kinetoplastid Flagellates
The energy metabolism of kinetoplastid flagellates is better characterized than that of any other group of protozoan parasites. Most of the knowledge of the bioenergetics in these organisms has derived from studies on Trypanosoma brucei, T. cruzi and a few Leishmania species. This work has uncovered a variety of metabolic properties unique to the kinetoplastids, although large differences in metabolism also exist between the different species and stages of these organisms.
The blood-stream long slender trypomastigotes of T. brucei rely entirely on glycolysis for their energy production, with pyruvate being the sole end product (Fig. 1). In this pathway one mol of glucose is converted to two mol of pyruvate with a net synthesis of two mol of ATP. Glucose is first degraded to 3-phosphoglycerate within glycosomes, organelles unique to the order Kinetoplastida. The 3-phosphoglycerate formed is then further degraded in the cytosol to pyruvate. Unlike in anaerobic lactate fermentation, in which the reducing equivalents generated by glyceraldehyde 3-phosphate oxidation are finally transferred to pyruvate (resulting in the formation of lactate), in T. brucei molecular oxygen serves as terminal electron acceptor, resulting in the formation of water (Fig. 1). Reducing equivalents (NADH) produced in the glycosomes are transferred to the mitochondrion via the classical glycerol 3-phosphate shuttle. The mitochondrial FAD-linked glycerol 3-phosphate dehydrogenase passes the electrons on to the ubiquinone/ubiquinol pool. From reduced ubiquinol the electrons are donated to oxygen by a plant-like alternative oxidase that is present in the inner membrane of the trypanosome's single mitochondrion.
All of the glycolytic enzymes necessary to convert glucose into 3-phosphoglycerate are localized in membrane-bounded microbody-like organelles, called glycosomes (Fig. 1). These organelles, that are related to the peroxisomes in other eukaryotes, are unique to the trypanosomatid flagellates, such as Trypanosoma, Leishmania, Phytomonas and Crithidia. Glycosomes are bounded by a single membrane, extremely homogeneous in size, measure about 0.3 μm in diameter and represent more than 4 % of the total cell volume. In T. brucei, more than 200 glycosomes are present while in other genera these organelles are not as abundant.
The possible functional advantage of the extreme subcellular compartmentation and enzyme association within the trypanosome cell is still debated. About 90% of the glycosomal protein content consists of glycolytic enzymes which appear to be in close association. Originally it was suggested that compartmentalization of glycolysis in the glycosome enables the parasite to sustain its high rate of glycolysis because of the high concentration of substrates and enzymes which provided some kind of channeling in the pathway. There is, however, no experimental evidence for metabolite channeling in trypanosomes. Furthermore, recent calculations performed during modeling of glycolysis in blood stream trypanosomes indicated that compartmentation of the enzymes in glycosomes is not necessary to obtain the observed glycolytic flux. Even if the glycolytic enzymes were not sequestered in a single compartment, diffusion should not be controlling the glycolytic flux in T. brucei. In T. brucei, the glycolytic flux seems to be limited by the catalytic activity of the enzymes rather than by the diffusion of metabolites from one enzyme to the next. In addition, T. brucei indeed has a fairly high glycolytic flux when compared to mammalian tissues, but similar and even higher fluxes are also observed in other micro-organisms, which do not possess glycosomes. Furthermore, all kinetoplastids possess glycosomes, including those that are not solely dependent on glycolysis for energy generation. Glycosomal metabolism is also involved in CO2 fixation and in the biosynthesis of pyrimidine nucleotides and ether-lipids.
Under anaerobic conditions, bloodstream trypomastigotes convert glucose to equimolar amounts of pyruvate and glycerol (Fig. 1). Under these circumstances, high concentrations of glycerol 3-phosphate and ADP are expected to accumulate inside the glycosomes. This would allow the glycerol kinase (which is present in extremely high specific activities in trypanosomes capable of producing glycerol under anaerobiosis) to proceed in the direction of ATP formation. As shown in Fig. 1, this results in an overall net synthesis of one mol of ATP per mol of glucose catabolized, indicating that a functional glycerol 3-phosphate shuttle and alternative oxidase are not essential for the survival of the organism. It is still unclear, however, if this halved efficiency of ATP production is sufficient for the long-term survival and proliferation of the parasite.
When bloodstream trypomastigotes of T. brucei differentiate into procyclic vector forms, they switch from a predominantly glycosomal to a more mitochondrial type of energy metabolism (Fig. 1). In procyclics, pyruvate, the end-product of glucose metabolism in the bloodstream form, is not excreted but is further metabolized inside the mitochondrion. This compound is first decarboxylated by pyruvate dehydrogenase to acetyl-CoA, which can then be further degraded to carbon dioxide via the TCA cycle. However, a large part of the acetyl-CoA is not degraded via the TCA cycle, but is converted into acetate via an acetate:succinate CoA-transferase. In this pathway, a succinate/succinyl-CoA cycle is involved, thereby generating extra ATP (Fig. 1).
Transformation of the long slender bloodstream form of T. brucei into the procyclic insect stage is associated with a remarkable alteration in the concentration and distribution of various enzymes and is accompanied by an increase in the relative mitochondrial volume from 5% to 25%. While in the acristate promitochondrion of the bloodstream stages typical oxidative pathways, such as the TCA cycle and a cytochrome-linked respiratory chain, are missing, these complex enzymatic systems become established within the fully developed, cristate mitochondrion of procyclic stages. The midgut forms of T. brucei, which correspond quite closely in morphology and ultrastructure to the procyclic culture forms, may rely on the oxidation of amino acids and TCA cycle intermediates rather than on carbohydrate for their energy requirements. Of particular importance in this respect is proline which is metabolized to CO2, alanine and aspartate following the routes shown in Fig. 2. Utilization of amino acids would correlate well with the change in the trypanosome's environment. Under resting conditions, proline is present in tsetse hemolymph in excessively high concentrations, and the midgut of the fly is deficient in carbohydrates but rich in amino acids and peptides.
The reducing equivalents (NADH), generated by substrate oxidation, are in all trypanosomatids delivered to a respiratory chain that uses oxygen as the final electron acceptor, but essential differences in respiratory chains exist among the various species and developmental stages. The long-slender bloodstream stage of T. brucei lacks cytochromes and a classical respiratory chain, but contains instead a plant-like alternative oxidase. Reducing equivalents produced in the glycosomes are transferred to the mitochondrion via the classical mammalian-type glycerol 3-phosphate/dihydroxyacetone phosphate shuttle (Fig. 1). The mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase donates electrons to the ubiquinone/ubiquinol pool, and the reduced ubiquinol is then the electron donor for the alternative oxidase. This process of electron transfer via the alternative oxidase is not linked to ATP production, does not involve cytochromes, is insensitive to cyanide and antiycin A, but is susceptible to inhibition by aromatic hydroxamic acids like salicylhydroxamate (SHAM) and the antifungal agents ascofuranone and miconazole.
The procyclic insect stages of trypanosomes, in contrast, possess not only the alternative oxidase system, but also a classical, cyanide-sensitive cytochrome-containing respiratory chain, coupled to oxidative phosphorylation (Fig. 1). Apart from complex I, this part of the respiratory chain of trypanosomatids probably strongly resembles the mammalian type respiratory chain, as no dissimilarities have been found yet. It is unknown yet, what physiological advantage the multiple oxidase assembly may offer to trypanosomes. Under the conditions of the midgut of the tsetse fly where oxygen concentrations are low, expression of the alternative oxidase system would be down-regulated, but the cytochrome aa3-linked respiratory chain would be induced and become functionally operative. As the latter system is coupled to oxidative phosphorylation, the economic efficiency of the overall energy generating metabolism of procyclic trypanosome stages would by far exceed that of the bloodstream forms. This would well reflect the environmental transition from the vertebrate blood with its abundant supply of nutrients and oxygen to the insect midgut, where limited substrate and oxygen concentrations may have forced the parasite to develop a more efficiently functioning and versatile energy-generating system. The stimuli eliciting the biosynthesis of the complex enzymatic system accomplishing these energetic requirements within the procyclic stages remain largely to be elucidated. Intermediate and short stumpy bloodstream forms of T. brucei have better-developed mitochondria than the long-slender forms and are not dependent only on glycolysis for energy generation. Apparently, the transition in energy metabolism between the glycolysis-dependent long-slender form and the TCA cycle-dependent procyclics occurs already in the bloodstream. The transitional bloodstream forms are thus pre-adapted to functioning in the insect vector if ingested with a blood meal.
The substrates and metabolic pathways utilized for energy generation by other kinetoplastids are similar to those observed in the procyclic culture forms of T. brucei. The differences in energy metabolism between the two main morphological forms of Leishmania species are less pronounced than those of T. brucei. In general, the bioenergetics of these organisms resemble those functional in T. brucei and T. cruzi culture forms. The glycolytic enzymes are present in glycosomes, and the cristate mitochondria of Leishmania possess a functional TCA cycle and a cytochrome-oxidase-linked respiratory chain. The relative importance of carbohydrates and amino acids as an energy source is not completely clear, however, and may vary with the stages, species and phase of growth.
Leishmania promastigotes have an energy metabolism in which a small part of the carbohydrate is completely oxidized to carbon dioxide via the TCA cycle, but in which large amounts of partly oxidized products, like acetate, pyruvate and succinate, are also produced as end-products of glucose metabolism. A small part of the pyruvate is transaminated to alanine, which is then excreted. In Leishmania promastigotes, the partly oxidized end-products are the result of an aerobic metabolism involving an electron-transport chain, with oxygen as final electron acceptor, as in trypanosomes under aerobic conditions. The succinate produced during aerobic incubations of Leishmania promastigotes is mainly produced via an oxidative pathway involving part of the TCA cycle (from oxaloacetate via citrate to succinate) and NADH oxidation by the respiratory chain. Leishmania promastigotes, like the insect stage of T. brucei, have a classical respiratory chain but lack the alternative oxidase that is present in the other Trypanosomatidae. Leishmania promastigotes are strongly dependent on this classical respiratory chain for their energy generation, which is in agreement with the observation that Leishmania promastigotes possess an energy metabolism in which most of the carbohydrate is degraded to partially oxidized end-products, a process concomitantly producing NADH that is re-oxidized by the respiratory chain.
Leishmania amastigotes most likely have an energy metabolism very similar to that of the promastigotes, both stages being dependent on TCA-cycle activity and a mammalian-type respiratory chain, although fatty acids probably are a more important substrate in the insect stage, at least in L. mexicana. This would correlate with the nutritional conditions of their intracellular habitat, the macrophage, in which a sufficient supply of lipids is likely to be available.
The energy metabolism of T. cruzi strongly resembles that of Leishmania, i.e. all stages of this parasite possess TCA cycle activity and a mammalian-type respiratory chain linked to the generation of ATP. However, like Leishmania, T. cruzi catabolizes glucose only partially to carbon dioxide and produces, in addition, various organic compounds as metabolic end-products, including acetate and succinate.
Anaerobic Protozoa
Protozoan parasites which have been classified as anaerobes occur in the taxonomic groups of trichomonad flagellates, Entamoeba and Giardia species. These organisms differ from most other eukaryotes in that they lack morphologically recognizable mitochondria (amitochondriate) and such biochemical attributes as the tricarboxylic acid cycle, cytochromes and oxidative phosphorylation. They do not require oxygen for their survival and multiplication, but can tolerate low oxygen concentrations. As a consequence of this metabolic organization, energy generation in these organisms functions anaerobically and is exclusively associated with substrate level phosphorylations. An important source of energy for anaerobic protozoa are carbohydrates which are stored in large amounts in the form of glycogen and degraded via an extended glycolytic pathway to different organic end products and CO2. On the other hand, these organisms do consume O2 when it is available, but the mechanism of this oxygen utilization process is unclear (see below).
Trichomonad flagellates distinguish from Entamoeba and Giardia in their ability to eliminate substrate-derived reducing equivalents in the form of molecular hydrogen by a pathway resembling that observed in some anaerobic eubacteria. As illustrated in Fig. 3, catabolism of carbohydrate in these parasites proceeds by classical glycolysis up to the step of pyruvate which is further metabolized to acetate, CO2 and hydrogen. Trichomonas vaginalis and Tritrichomonas foetus show the same metabolic pattern, except that the former trichomonad produces lactate and the latter succinate as additional end products. The process of acetate formation in trichomonads is highly unusal for a eukaryote in that it is achieved by oxidative decarboxylation of pyruvate via a ferredoxin-linked step catalyzed by pyruvate:ferredoxin oxidoreductase. In a subsequent reaction, catalyzed by the iron-sulfur enzyme hydrogenase, the electrons are transferred from ferredoxin to protons to form molecular hydrogen. The ferredoxins involved in pyruvate oxidation of anaerobic protozoa are 12 kDa iron-sulfur proteins that differ in their structural properties among different species. The trichomond ferredoxin possesses only a [2Fe-2S] cluster similar to mitochondrial iron-sulfur proteins, whereas the ferredoxins of Entamoeba and probably Giardia contain two clusters of four iron atoms bound to four acid-labile sulfur atoms (2[4Fe-4S]). The characteristic trichomonad ferredoxin-dependent metabolic properties account for the selective toxicity of 5-nitroimidazole antiprotozoal drugs.
Another feature unique to the trichomonads is that the enzymes involved in pyruvate oxidation are carried in a separate organelle, the hydrogenosome, which is bounded by two closely apposed unit membranes and thus represents a metabolic compartment separate from the cytosol. The evolutionary position of the hydrogenosome is still an open question, but functional and morphological considerations led to the suggestion that hydrogenosomes and mitochondria are related organelles. According to a recent hypothesis hydrogenosomes may have evolved from an ancestral organelle with combined features of both mitochondria and hydrogenosomes. The presence of hydrogenosomes is not restricted to trichomonad flagellates since they have been discovered also in anaerobic rumen ciliates, in fungi and in free-living protozoans of anaerobic sediments. The establishment of these organelles appears to be one of the various evolutionary approaches to adapt to an anaerobic mode of life. The advantage achieved with this strategy over that evolved in other anaerobic systems is that substrate-derived reducing equivalents can be directly eliminated in the form of molecular hydrogen and that substrate oxidation beyond the pyruvate step increases the economic efficiency of the energy generating system over that observed in simple lactate or ethanol fermentation. The conversion of acetyl CoA, the primary product of anaerobic pyruvate oxidation in trichomonads, to free acetate can be coupled to substrate level ATP synthesis, a process catalyzed by the combined action of two enzymes involving a CoA transferase and succinyl CoA synthetase.
Like trichomonad flagellates, the energy metabolism of Giardia and Entamoeba is entirely fermentative (Fig. 3), irrespective of whether oxygen is present or not, which is in accordance with the lack of mitochondria and the functions associated with these organelles. The utilization of carbohydrate results in the formation of a mixture of acetate, ethanol and CO2. Anaerobically, more ethanol and less acetate are produced, whereas in the presence of oxygen the catabolism switches to produce more acetate. In this pathway, the major fate of glycolytically formed pyruvate is its oxidative decarboxylation to acetyl CoA, catalyzed by an enzyme analogue to the ferredoxin-linked oxidoreductase of trichomonad flagellates. However, unlike trichonomads, Giardia and Entamoeba, because of the lack of a ferredoxin-linked hydrogenase, are not able to form molecular hydrogen. As an alternative, in these anaerobes reduced ferredoxin is utilized in the formation of ethanol as catalyzed by the bifunctional enzyme, acetyl CoA reductase/alcohol dehydrogenase. Oxygen may also act as terminal acceptor for the reducing equivalents removed from pyruvate, which would explain the aerobic requirement for acetate formation, but the precise steps involved in this pathway of electron flow are still unclear. As in trichomonads, hydrolysis of the acetyl CoA in these parasites can also be linked to energy generation by substrate level phosphorylation, but this step is catalyzed by a single enzyme, a novel type of acetate thiokinase (Fig. 3), which is known to occur only in some prokaryotes.
A notable feature of glycolysis in anaerobic protozoa is its dependency on pyrophosphate (PPi.) as a phosphate donor rather than on adenine nucleotides (Fig. 3). In a first step, a phosphofructokinase (PPi-PFK) utilizes PPi to phosphorylate the glycolytic intermediate fructose 6-phosphate to form the corresponding l,6-bisphosphate. The second PPi -dependent enzyme, pyruvate phosphate dikinase (PPDK), is located in lower glycolysis, where it replaces the pyruvate kinase (PK). In T. vaginalis, only the phosphofructokinase is PPi-specific, whereas in Entamoeba and in Giardia both PPi-PFK and PPDK are present. Recently, the coexistence of PPDK with PK in Giardia and the occurrence of a glycosomal PPDK in addition to cytosolic PK have been reported for Giardia and T. brucei, respectively. Other parasitic protozoa, including some apicomplexans, were also found to contain PPi-PFK instead of the corresponding ATP-dependent enzyme, though these species are not considered to be anaerobic. A third PPi-linked enzyme, phosphoenolpyruvate (PEP) carboxyphosphotransferase, is a constituent of a three-enzyme system functioning in Entamoeba as an alternative route for PEP utilization (Fig. 3). In this step, the high-energy phosphate bond of PEP is transformed into a PPi bond with simultaneous fixation of CO2. Entamoeba appears to be the only eukaryotic cell in which this enzyme is present. The possible physiological roles of inorganic PPi. utilization may be to conserve the energy of the PPi generated during anabolic processes and, as the PPi-linked enzymatic reactions are reversible under physiological conditions, they may have functions not only in catabolic but also anabolic routes.
Anaerobic protozoa experience aerobic conditions within their natural environments, consume oxygen when provided at rates comparable to those observed for aerobic protozoa and can tolerate surprisingly high concentrations of this gas in vitro. T. vaginalis has even been shown to grow optimally in the presence of small amounts of oxygen, and therefore these organisms may be classified as microaerophiles rather than anaerobes. Although the trichomonads, Giardia and Entamoeba, are devoid of any heme-containing proteins, reducing equivalents can be transferred from substrates to molecular oxygen. This reaction is catalyzed by NAD(P)H oxidoreductases which are located in the cytosol and apparently produce water as the reaction product. As an alternative, electrons to oxygen may be also donated through a succession of electron carriers containing flavins, non-heme iron, and other high potential carriers of an unknown nature. The precise architecture and physiological role of these presumptive respiratory systems of anaerobic protozoa, which are not associated with energy generation, remain to be elucidated. The fact that, with the exception of the large intestine, neither of the tissues invaded by these parasites are notably deficient in oxygen suggests that aerobic processes may be operative in vivo, and it was suggested that the respiratory systems may have a role in protecting the cell against oxygen damage.
Apicomplexans
Detailed knowledge of the bioenergetics of apicomplexan protozoa is limited to the intraerythrocytic stages of Plasmodium and Eimeria, while this aspect in the case of other genera has received only little attention. Generally, carbohydrates serve as the main energy source throughout the life cycle of apicomplexans. The erythrocytic stages of the malarial parasite lack energy stores and, consequently, use blood glucose as the primary nutrient. Eimeria species and Toxoplasma gondii bradyzoites store carbohydrate in the form of amylopectin, a known reserve polysaccharide of plants and some free-living protozoa and rumen ciliates. Like most other endoparasites, apicomplexan protozoa have a limited capacity to oxidize substrates completely to CO2 and water but instead satisfy their energy requirements by fermentative mechanisms. The erythrocytic forms of mammalian malarial parasites convert glucose almost quantitatively to lactate, regardless of whether oxygen is present or not. Carbohydrate metabolism of the intraerythrocytic stages of Babesia seems to parallel that of human malarial parasites in that glucose is primarily or solely degraded to lactate. Eimeria, Toxoplasma and Cryptosporidium also ferment carbohydrate to lactate with minor formation of acetate and glycerol. In common with anaerobic protozoa, glycolysis of various apicomplexans is unusual in that it contains PPi-PFK instead of the conventional ATP-dependent enzyme. Another unique features of these protozoans is that their lactate dehydrogenases, including those of Plasmodium, Eimeria and Toxoplasma, contain a five-amino-acid insert around the active site, which may explain, at least in part, the unusual specificities of these enzymes in apicomplexans. A common feature of coccidian parasites is also the accumulation of mannitol, a carbohydrate previously known to be present only in fungi. The presence of mannitol in Eimeria, and probably also in Toxoplasma and Cryptosporidium, is associated with a cyclic pathway (mannitol cycle) that involves a synthetic and catabolic part and is linked to glycolysis via fructose 6-phosphate. The function of mannitol in coccidial parasites is still unclear, but suggested possibilities are its role as an energy reserve or osmoregulator, or it may act as a mechanism for dealing with oxygen radicals.
The lack of a full complement of the tricarboxylic acid cycle (TCA cycle) enzymes in the apicomplexan parasites investigated has left a classical mitochondrial function involving complete substrate oxidation doubtful. On the other hand, several apicomplexans, notably all life cycle stages of Eimeria and Toxoplasma, contain distinctive, cristate mitochondria. The presence of various cytochromes have been detected in these parasites and their respiration is sensitive to inhibition by cyanide and other mitochondrial electron transport inhibitors. For Eimeria it was suggested that during the processes of sporulation and excystation, which are associated with high respiratory activities, but also within those parasite stages preparing for an extracellular phase of life, a more aerobic type of metabolism may be established using the TCA cycle and cytochrome-linked substrate oxidation. As oxygen is required for optimal growth in vitro and inhibitors of respiration have significant effects on the survival of malarial and other apicomplexan parasites, a function of the mitochondrion in a capacity other than energy generation must be crucial. A possible functional significance of respiration in apicomplexans could be its coupling to the pyrimidine biosynthetic pathway as appears to be the case for blood stage P. falciparum. The observation that the anticoccidial quinolones and pyridinols inhibit respiration in Eimeria sporozoites and oocysts rather selectively suggests major differences between the coccidial and mammalian type of mitochondrial electron transport systems. Avian malarial parasites seem to have rather different bioenergetic capacities. Their cristate mitochondria appear to possess a functional TCA cycle, and in accordance with these properties, free erythrocytic stages of these species are able to oxidize a significant portion of the carbohydrate utilized to CO2. Cryptosporidium parvum seems to lack mitochondria suggesting that oxygen is unimportant and glycolysis is the major strategy of energy generation in this apicomplexan.
Helminths
Adult Helminths
As substrate for energy conservation the adult stages of most helminths use primarily carbohydrate, of which glycogen is the main storage polysaccharide, consisting in many cases of as much as l0 % of the worm's wet weight. The catabolism of amino acids does not seem to be of particular importance for energy generation with the exception of glutamine, whose co-utilization with carbohydrate was shown to be energetically advantageous for some helminths species. Generally, the bioenergetic pathways found in adult helminths function primarily anaerobically. Effective terminal oxidative processes, such as the TCA cycle and a conventional type of respiratory chain, are very often absent or of limited activity, which precludes the utilization of fatty acids as an energy source. For the same reason, the degradation of carbohydrates and amino acids beyond the acetyl-CoA stage is only hardly feasible. As a consequence, most adult helminths are not capable of oxidizing organic compounds to a significant extent to CO2 and water. More pronounced oxygen dependent routes, however, appear to exist in small helminth species and in the outermost tissue layers of larger worms.
Although the pattern of end products varies greatly between different species of adult helminths, none of them degrades carbohydrates completely to carbon dioxide, as the free-living stages do. As helminths in general do not use oxygen as final electron acceptor, they must have a fermentative metabolism instead and excrete organic substances as metabolic end products. When oxygen cannot function as terminal electron acceptor, the degradation of substrates has to be in redox balance. Two approaches are pursued by helminths to fulfil this requirement. Some species, including adult schistosomes and filarial nematodes, use the classical adaptation to anaerobic metabolism by degrading carbohydrates to lactate or ethanol. This so-called anaerobic glycolysis yields two mol of ATP per mol of glucose degraded. Although most adult parasites use this strategy to some extent, the majority uses a different approach (malate dismutation), in which carbohydrates are degraded to phosphoenolpyruvate (PEP) via the conventional Emden-Meyerhof pathway. PEP is then carboxylated by phosphoenolpyruvate carboxykinase (PEPCK) to form oxaloacetate, which is subsequently reduced to malate (Fig. 4). The fate of PEP at the pyruvate kinase/PEPCK branch point will depend on the activity ratios and kinetics of the two enzymes involved, but is also determined by substrate concentrations, the rate of subsequent reactions and other factors. This part of the pathway occurs in the cytosol and is comparable to the formation of lactate or ethanol in being redox balanced and yielding two mol of ATP per mol of glucose degraded. However, the malate produced in the cytosol is, unlike lactate, not excreted but transported into the mitochondria for further degradation. In a branched pathway, a portion of malate is oxidized to acetate and another portion of it is reduced to succinate. The oxidation occurs via two consecutive steps of oxidative decarboxylation, first to pyruvate via malic enzyme and subsequently to acetyl CoA via pyruvate dehydrogenase . The pyruvate dehydrogenase complex of Ascaris suum was shown to be specifically adapted to anaerobic functioning. The acetyl CoA formed is converted to acetate via a succinate/succinyl-CoA cycle. In the other part of the pathway, reduction of malate occurs in two reactions which reverse part of the TCA cycle. Many helminths metabolize succinate further to propionate, which is then excreted. This so-called malate dismutation is in redox balance when twice as much propionate is produced as compared to acetate.
Apart from the electron-transport-associated ATP formation in the reduction of fumarate (see below), malate dismutation is also accompanied by substrate-level phosphorylations (Fig. 4). Formation of acetate from acetyl-CoA occurs via two consecutive enzymatic steps with the concomittant production of ATP. The presence of the first enzyme of this reaction, acetate:succinate CoA-transferase, has now been unequivocally demonstrated in F. hepatica. The formation of propionate from succinate occurs through a sequence of metabolic reactions required for propionate utilization in animal tissues but working in reverse (Fig. 4). This cyclic pathway involves a set of enzymatic reactions which require deoxyadenosylcobalamin and biotin and accomplish the loss of a carboxyl group as CO2. Each pass of the sequence promotes the synthesis of one molecule of ATP through coupling of ADP phosphorylation with the decarboxylation of methylmalonyl CoA. In total, the anaerobic production of propionate and acetate yields approximately 5 mol of ATP per mol of glucose degraded.
In the anaerobically functioning mitochondria of helminths capable of malate-dismutation, the electron-transport chain is different from the one present in mammals in that endogenously produced fumarate functions as the terminal electron acceptor instead of oxygen. In this case, electrons are transferred from NADH to fumarate via complex I and fumarate reductase (Fig. 5). Free-living stages of helminths, however, possess an aerobic energy metabolism with a classical mammalian type respiratory chain (see below). This implies that during the development of free-living into the parasitic stages, a transition occurs from succinate oxidation via succinate dehydrogenase in the TCA cycle to the reverse reaction, reduction of fumarate to succinate. Bacteria contain two homologous but distinct enzyme complexes to catalyse these reactions, one to oxidize succinate (succinate dehydrogenase) and one to reduce fumarate (fumarate reductase). Different enzymes are needed for these two reactions because the electron flow through the two complexes is in opposite direction, which implies differences in the affinity for electrons (standard electron potential) of the electron-binding domains of these enzyme complexes. Distinct enzyme complexes have now also been described in the parasitic nematodes Haemonchus contortus and Ascaris suum. These complexes were shown to be differentially expressed during the life cycle of the parasites and are suggested to function either as a succinate dehydrogenase or as a fumarate reductase. In addition to customary enzyme complexes for succinate oxidation and fumarate reduction, also distinct quinones are involved in these processes in helminths.
In many helminths, the primary products of anaerobic malate metabolism, acetate, succinate and propionate, accumulate as major excretory products. Some other helminths metabolize these compounds further to branched-chain fatty acids. In A. suum, the branched-chain fatty acids, 2-methylbutyrate and 2-methylvalerate, are the predominant end products of anaerobic carbohydrate metabolism. As demonstrated in Fig. 6, these acids arise from the condensation of acetyl CoA with propionyl CoA or of two propionyl CoA units, with subsequent reduction of the condensation products to the unsaturated acids. This pathway is also located inside the anaerobically functioning mitochondrion and is similar to the reversal of b-oxidation of fatty acids, although some of the enzymes involved in branched-chain fatty acid production of helminths differ in their kinetics and regulatory properties from the corresponding mammalian β-oxidation enzymes. There is evidence to suggest that two sites of ATP generation may be operative in this reductive process (Fig. 5). One is associated with the penultimate step in which the dehydroacyl CoA compound is reduced to the saturated CoA ester and involves phosphorylation linked to electron transport, in a way similar to that occuring during fumarate reduction. NADH is reoxidized and the electrons are used for enoyl CoA reduction. A second site of energy generation is thermodynamically feasible in the final step of free branched-chain fatty acid formation which occurs with a large drop in free energy upon hydrolysis of the CoA thioester bond. Probably ATP formation proceeds either by a thiokinase or by the combined action of two enzymes, analogous to acetate formation from acetyl CoA. The production of these branched-chain fatty acids does not generate more energy than the production of acetate and propionate but functions as an alternative electron sink. More rarely occuring pathways of glucose utilization, such as those leading to propanol in H. contortus, have not yet been elucidated and thus their relevance to energy conservation is unknown.
The anaerobic energy generation in helminths can apparently be associated with several sites of ATP formation. At the substrate level, these are coupled to glycolysis, PEP carboxylation, methylmalonyl-CoA decarboxylation (in the formation of propionate) and succinyl CoA synthetase (in the formation of acetate), while NADH-coupled reductions of fumarate and enoyl CoA compounds serve to produce ATP via electron-transport-associated phosphorylation. Energy generation in helminths mediated by mixed fermentations and anaerobic electron transport would thus display a clear advantage over simple lactate or ethanol fermentation. The latter pathways yield only 2 ATP per mol of glucose catabolized, whereas for an organism like F. hepatica, which degrades glucose almost solely to acetate and propionate in a ratio of 1:2, approximately 5 mol ATP is produced per mol of glucose catabolized. In spite of the obvious advantage of multiple anaerobic catabolic systems over simple lactate fermentation, the economic efficiencies of these pathways are still very low compared to that obtained from complete substrate oxidation to CO2 and water, which generates approximately 30 mol of ATP per mol glucose degraded.
Free-Living and Larval Stages of Helminths
Most free-living stages of helminths are self-supporting, i.e., they do not obtain food or substrates other than oxygen from their environment. They are completely dependent on the endogenous food stores that they have obtained in their previous host. Glycogen is present in many free-living stages and is used to span the gap in food supply until the next host is entered. The free-living and parasitic developmental stages of helminths have, unlike the adult parasites, usually an aerobic energy metabolism. Many of them, such as the free-living infective eggs of A. suum, eggs and developing larvae of Nippostrongylus brasiliensis and H. contortus, juvenile liver flukes and young schistosomula, and various free-living cercariae, have a high oxygen requirement, mainly for energy generation, with high TCA-cycle and mitochondrial respiratory chain activities. These organisms thus are capable of completely oxidizing substrate carbon to CO2 and water. In addition, smaller amounts of oxygen are needed for synthetic purposes, such as the formation of collagen and fatty acid desaturation. Another feature of the developmental stages of helminths is that the diversity of energy sources that can be utilized is, in many cases, greater compared to that of the respective adult forms. For instance, the free-living stages of many parasitic nematodes and some cercariae rely heavily on lipids for their energy generation, implying that a functional β-oxidation sequence is present in these stages. Generally, there is, however, great variation in the oxygen requirements and the capabilities for substrate utilization in the developmental stages of helminths, and the situation in one species may not be necessarily relevant for another species.
If the metabolism of helminths differs between larval and adult stages, then the developmental cycle of these organisms must involve, at some stage, a transition from one metabolic strategy to another, triggered by appropriate environmental and physiological conditions. In the development of A. suum, the transition from aerobic to anaerobic energy metabolism is likely to occur during the third molt when larvae develop in the small intestine from the third to the fourth stage. In contrast to the adult, which lacks a functional tricarboxylate cycle and cyanide-sensitive respiration, the earlier larval stages of this parasite rely on aerobic strategies for energy conservation resembling those functional in mammalian cells. A similar adaptation is exhibited in the life cycle of F. hepatica (Fig. 7). While the early liver-parenchymal stages of this parasite possess a predominantly aerobic metabolism and are capable of complete substrate oxidation, these oxidative capacities decline gradually during development. In flukes between two and eight weeks old, TCA-cycle activity is already largely suppressed, but aerobic reactions still remain functional as characterized by the oxygen-linked formation of acetate. In this pathway, most of the chemical energy may still be conserved by mitochondrial oxidative phosphorylation, but the reducing power necessary to drive respiration is not derived from the TCA cycle but from the formation of acetate. Compared to complete substrate oxidation, the relative efficiency of this energy-generating pathway is much lower, resulting in approximately 14 mol of ATP per mol of glucose catabolized to acetate and CO2. After arrival of the flukes in their definitive environment, the bile ducts, a further drop in oxidative capacities and relative efficiency for energy generation occurs. From this stage on, anaerobic redox-processes, resulting in the formation of acetate and propionate, remain functional throughout adult life. In the development of the liver fluke, therefore, two pronounced shifts in energy metabolism seem to occur: one being characterized by the transition from complete substrate oxidation to aerobic acetate formation, and the other by a change from aerobic to anaerobic metabolism as is observed during the development of the late immature worms into the adult stage.
Also in schistosomes such a switch occurs from an aerobic metabolism in the free-living stages to an anaerobic one in the parasitic stages. The free-living cercariae and miracidia of Schistosoma mansoni possess an aerobic energy metabolism in which their endogenous glycogen reserves are degraded, mainly to carbon dioxide. Adult schistosomes, on the other hand, live in the blood stream of their host and despite their small size and life in an aerobic environment, they have a fermentative metabolism and degrade glucose, mainly to lactate. When cercariae penetrate the skin of the final host and transform into schistosomula, they switch rapidly from TCA-cycle activity to lactate production via glycolysis. This metabolic switch was shown to be initiated by the sudden presence of external glucose when the free-living stages penetrate the new host, and is not linked to a decreased availability of oxygen, as in F. hepatica. The mere presence of external glucose results in an increased glycolytic flux, probably caused by the rapid uptake of glucose that occurs upon expression at the surface of a specific schistosomal glucose transporter protein, SGTP4. This increased glycolysis is maintained as a result of the specific kinetic properties of schistosomal hexokinase, the first enzyme in glucose catabolism. The observed rapid switch to lactate production occurs only in cercarial heads, the region of the larvae that develops into the mature parasite. The tail of the cercaria, which only propels the organism through the water, is fully dependent on the degradation of endogenous glycogen reserves as it contains little or no SGTP4 and hexokinase, and it degenerates following the separation from the penetrating schistosomulum. In contrast to Ascaris, F. hepatica and several other helminth species, no significant changes in the energy metabolism occur during the further development of schistosomes. Lactate remains the main end product, although TCA-cycle activity and oxidative phosphorylation also contribute significantly to ATP production, even in adults.
The reason for the more pronounced oxidative capacities in developmental stages of helminths than in adults is not completely understood, but may be due to their smaller body size, which causes less oxygen diffusion problems than in adults, in conjunction with a sufficient availability of oxygen in the habitats of free-living and many parasitic larval stages. Both circumstances are likely to be essential for the establishment and function of the processes responsible for complete substrate degradation, i.e. the tricarboxylate cycle, β-oxidation route and a conventional type of phosphorylating respiratory chain. Upon increasing in body size which, for a migrating parasite, is often accompanied by a decreasing access to oxygen and changes in other environmental conditions, a marked drop in oxidative capacities often occurs in the adult, where anaerobic strategies are becoming the major ATP-generating sites. While in the latter stage these pathways cannot be reversibly replaced by aerobic processes, most developmental aerobic stages can survive anaerobically by utilizing anaerobic energy-conserving strategies which often coexist with aerobic routes. An example is F. hepatica, in which the ability of the adult to catabolize glucose anaerobically to acetate and propionate is present immediately after excystment and persists in all stages until the mature parasite. The interesting question of how the metabolic transitions occurring during the developmental cycle of helminths are regulated remains largely to be determined.
