Aloysius G.M. Tielens

Biochemistry and evolution of anaerobic energy metabolism in eukaryotes.

SUMMARY Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.

Mitochondria as we don't know them

Biochemistry textbooks depict mitochondria as oxygen-dependent organelles, but many mitochondria can produce ATP without using any oxygen. In fact, several other types of mitochondria exist and they occur in highly diverse groups of eukaryotes - protists as well as metazoans - and possess an often overlooked diversity of pathways to deal with the electrons resulting from carbohydrate oxidation. These anaerobically functioning mitochondria produce ATP with the help of proton-pumping electron transport, but they do not need oxygen to do so. Recent advances in understanding of mitochondrial biochemistry provide many surprises and furthermore, give insights into the evolutionary history of ATP-producing organelles.

An anaerobic mitochondrion that produces hydrogen.

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product—biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

Energy generation in parasitic helminths

Although parasitic helminths are a very heterogeneous group of organisms, they share many interesting properties in their energy metabolism. In certain stages of their life cycle, they all have a large capacity for anaerobic functioning. In other stages, an aerobic energy metabolism prevails. Parasites have to adapt to different environments in which the availability of oxygen and food varies widely. These variations in their external conditions strongly influence their energy metabolism. Here, Louis Tielens presents an introduction to the current ideas on the bioenergetics of parasitic helminths, focusing on the differences in energy metabolism between various stages (free-living and parasitic), and paying special attention to the mechanisms involved in the transitions between the different methods of energy generation.

Differences in Energy Metabolism Between Trypanosomatidae

Although various members of the family Trypanosomatidae generate energy in a similar way, fundamental differences also exist and are not always recognized. In this review, Louis Tielens and Jaap Van Hellemond discuss the known differences in carbohydrate metabolism among trypanosomatids, and especially compare Leishmania with trypanosomatids such as Trypanosoma brucei and Phytomonas spp. Special attention will be paid to differences in end-products of carbohydrate degradation, to differences in anaerobic capacities between the various trypanosomatids and to the components of their respiratory chains, including the presence or absence of a plant-like alternative oxidase. Furthermore, evidence will be discussed which indicates that the succinate produced by trypanosomatids is formed mainly via an oxidative pathway and not via reduction of fumarate, a process known to occur in parasitic helminths.

The electron transport chain in anaerobically functioning eukaryotes

Many lower eukaryotes can survive anaerobic conditions via a fermentation pathway that involves the use of the reduction of endogenously produced fumarate as electron sink. This fumarate reduction is linked to electron transport in an especially adapted, anaerobically functioning electron-transport chain. An aerobic energy metabolism with Krebs cycle activity is accompanied by electron transfer from succinate to ubiquinone via complex II of the respiratory chain. On the other hand, in an anaerobic metabolism, where fumarate functions as terminal electron acceptor, electrons are transferred from rhodoquinone to fumarate, which is the reversed direction. Ubiquinone cannot replace rhodoquinone in the process of fumarate reduction in vivo, as ubiquinone can only accept electrons from complex II and cannot donate them to fumarate. Rhodoquinone, with its lower redox potential than ubiquinone, is capable of donating electrons to fumarate. Eukaryotic fumarate reductases were shown to interact with rhodoquinone (a benzoquinone), whereas most prokaryotic fumarate reductases interact with the naphtoquinones menaquinone and demethylmenaquinone. Fumarate reductase, the enzyme essential for the anaerobic functioning of many eukaryotes, is structurally very similar to succinate dehydrogenase, the Krebs cycle enzyme catalysing the reverse reaction. In prokaryotes these enzymes are differentially expressed depending on the external conditions. Evidence is now emerging that also in eukaryotes two different enzymes exist for succinate oxidation and fumarate reduction that are differentially expressed.

New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle.

We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of α-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

Phospholipids in parasitic protozoa.

Parasitic protozoa are surrounded by membrane structures that have a different lipid and protein composition relative to membranes of the host. The parasite membranes are essential structurally and also for parasite specific processes, like host cell invasion, nutrient acquisition or protection against the host immune system. Furthermore, intracellular parasites can modulate membranes of their host, and trafficking of membrane components occurs between host membranes and those of the intracellular parasite. Phospholipids are major membrane components and, although many parasites scavenge these phospholipids from their host, most parasites also synthesise phospholipids de novo, or modify a large part of the scavenged phospholipids. It was recently shown that some parasites like Plasmodium have unique phospholipid metabolic pathways. This review will focus on new developments in research on phospholipid metabolism of parasitic protozoa in relation to parasite-specific membrane structures and function, as well as on several targets for interference with the parasite phospholipid metabolism with a view to developing new anti-parasitic drugs.

Triggering of Innate Immune Responses by Schistosome Egg Glycolipids and Their Carbohydrate Epitope GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc

To investigate the interactions of glycoconjugates with the innate immune system, peripheral blood mononuclear cells were stimulated with glycolipids derived from Schistosoma mansoni eggs and worms and with biochemically synthesized neoglycoconjugates. Egg glycolipids stimulated the production of interleukin (IL)--10, IL-6, and tumor necrosis factor--alpha in monocytes, whereas worm glycolipids failed to do so. When monoclonal antibodies that specifically recognize defined carbohydrate epitopes were used, the binding of a GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc (LDN-DF) reactive antibody was pronounced on egg glycolipids but was absent on worm glycolipids. The binding of antibodies that recognize Gal beta 1-4(Fuc alpha 1-3)GlcNAc (LewisX), GalNAc beta 1-4GlcNAc (LDN), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (LDN-F) was comparable for both preparations. Cytokine production in response to neoglycoconjugates containing enzymatically synthesized glycans also was measured. The LDN-DF neoglycoconjugate was the most potent cytokine inducer, which indicates that this difucosylated glycan can act at the host-parasite interface and can trigger innate immune responses.

Surprising variety in energy metabolism within Trypanosomatidae

The metabolism of Trypanosomatidae differs significantly between distinct species and can even be completely different between various life-cycle stages of the same species. It has been proposed that differences in energy metabolism are related to differences in nutrient supply in the environments of the various trypanosomatids. However, the literature shows that availability of substrates does not dictate the type of energy metabolism of trypanosomatids, as Trypanosoma theileri, Trypanosoma lewisi and African trypanosomes all live in the bloodstream of their mammalian host, but have surprisingly large differences in metabolism. Furthermore, in trypanosomatids no obvious relationship exists between energy metabolism and phylogeny or mode of transmission. We provide an overview of the metabolic capacities in the energy metabolism of distinct trypanosomatids, and suggest that these can be divided into four different metabolic categories of increasing complexity.