J. J. Van Hellemond

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 extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei.

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

Leishmania infantum promastigotes have a poor capacity for anaerobic functioning and depend mainly on respiration for their energy generation

In earlier studies on the supposedly anaerobic metabolism of Leishmania promastigotes it was suggested that the reduction of fumarate to succinate functions as the main electron sink during anoxia. Interestingly, however, our preliminary results demonstrated that rhodoquinone, an essential component for efficient fumarate reduction in eukaryotes, was absent in L. infantum promastigotes. Therefore, we re-investigated the energy metabolism and succinate production of these promastigotes. Our studies demonstrated that L. infantum promastigotes could, to a certain extent, survive periods without respiration but had a low capacity for anaerobic metabolism. When oxygen could not be used as terminal electron acceptor, the degradation of glucose was severely inhibited, forcing the parasite to reduce its energy expenditure, which resulted in inhibited motility and proliferation. In addition, we studied the mechanism of succinate production under aerobic conditions and showed that in L. infantum promastigotes this succinate was mainly produced via an oxidative pathway, the Krebs cycle, and not significantly via fumarate reduction, which correlated with the absence of rhodoquinone. Taken collectively our studies show that L. infantum promastigotes depend mainly on respiratory chain activity for energy generation, have a poor capacity for anaerobic functioning, and go into metabolic arrest during anoxic conditions.

A Gene Encoding the Plant‐Like Alternative Oxidase is Present in Phytomonas but Absent in Leishmania spp.

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome‐containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron‐transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant‐like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant‐like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant‐like alternative oxidase.

Differential platelet adhesion to distinct life-cycle stages of the parasitic helminth Schistosoma mansoni.

Y . P . W U,* P . J . L ENT ING ,* A . G . M. T I ELENS , P . G . DE GROOT* and J . J . VAN H ELLEMOND *Department of Haematology, University Medical Centre Utrecht, Utrecht; Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht; and Department of Medical Microbiology and Infectious Diseases, Erasmus MC Medical Centre and Harbour Hospital Institute for Tropical Medicine, Rotterdam, the Netherlands

Rhodoquinone is synthesized de novo by Fasciola hepatica

Most adult parasitic helminths have an anaerobic energy metabolism in which fumarate is reduced to succinate by fumarate reductase. Rhodoquinone (RQ) is an essential component of the electron transport associated with this fumarate reduction, whereas ubiquinone (UQ) is used in the aerobic energy metabolism of parasites. Not known yet, however, is the RQ and UQ composition during the entire life cycle nor the origin of RQ in parasitic helminths. This report demonstrates the essential function of RQ in anaerobic energy metabolism during the entire life cycle of Fasciola hepatica, as the amount of RQ present reflected the importance of fumarate reduction in various stages. We also studied the origin of RQ, as earlier studies on the protozoan Euglena gracilis suggested that RQ is synthesized from UQ. Therefore, in parasitic helminths RQ might be synthesized by modification of UQ obtained from the host. However, we demonstrated that in F. hepatica adults RQ was not produced by modification of UQ obtained from the host but that RQ was synthesized de novo, as (i) the chain-length of the quinones of F. hepatica adults was not related to the chain length of the quinone of the host, (ii) despite many attempts we could never detect any in vitro conversion of UQ9 into RQ9 or into UQ10, neither by intact adult flukes nor by homogenates of F. hepatica adults and (iii) F. hepatica adults used mevalonate as precursor for the synthesis of RQ. We also showed that the rate of quinone synthesis in F. hepatica adults was comparable to that in the free-living nematode Caenorhabditis elegans. These results prompted the suggestion that RQ is synthesized via a pathway nearly identical to that of UQ biosynthesis: possibly only the last reaction differs.

Fasciola hepatica miracidia are dependent on respiration and endogenous glycogen degradation for their energy generation.

It is generally accepted that free-living stages of parasitic helminths are dependent on aerobic degradation of endogenous energy sources for their energy generation. This concept, however, is not the result of extensive experimental evidence, but originated mainly intuitively as oxygen is widely available in their habitat and these stages generally have a small size. Schistosoma mansoni, the sole parasitic helminth whose energy metabolism has been studied throughout its life-cycle indeed has aerobically functioning free-living stages. However, large differences exist in energy metabolism between adult stages of distinct parasitic helminths, and caution should be taken in predicting that all free-living stages of all parasitic helminths have the same, aerobic energy metabolism. Hence, this report studied the energy metabolism of Fasciola hepatica miracidia and demonstrated that F. hepatica miracidia are also dependent on aerobic degradation of their endogenous glycogen stores by glycolysis and on Krebs cycle activity for energy generation. However, in contrast to S. mansoni, F. hepatica miracidia cannot function anaerobically, as inhibition of the respiratory chain blocked motility and carbohydrate degradation, and finally resulted in death of the miracidia. Therefore, this report demonstrated that differences exist between miracidia of distinct species, in pre-adaptation of their energy metabolism to the occasional hypoxic conditions within their next host.

Schistosoma mansoni sporocysts contain rhodoquinone and produce succinate by fumarate reduction.

Although schistosomes were thought to be one of the few parasitic helminths that do not produce succinate via fumarate reduction, it was recently demonstrated that sporocysts of Schistosoma mansoni produce, under certain conditions, succinate in addition to lactate. This succinate production was only observed when the respiratory chain activity of the sporocysts was inhibited, which suggested that succinate is produced by fumarate reduction. In this report the presence of essential components for fumarate reduction was investigated in various stages of S. mansoni and it was shown that, in contrast to adults, sporocysts contained a substantial amount of rhodoquinone which is essential for efficient fumarate reduction in eukaryotes. This rhodoquinone was not made by modification of ubiquinone obtained from the host, but was synthesized de novo. Furthermore, it was shown that complex II of the electron-transport chain in schistosomes has the kinetic properties of a dedicated fumarate reductase instead of those of a succinate dehydrogenase. The presence of such an enzyme, together with the substantial amounts of rhodoquinone, shows that in S. mansoni sporocysts succinate is produced via fumarate reduction. Therefore, the energy metabolism of schistosomes does not differ in principle from most other parasitic helminths, which are known to rely heavily on fumarate reduction.

Immunologic activity of schistosomal and bacterial TLR2 ligands in Gabonese children

Schistosomes carry lipid moieties that interact with the immune system. To understand the consequence of interactions in terms of polarizing the cytokine profiles, the effect of two Toll‐like receptor‐2 (TLR2) activating schistosomal lipid fractions was studied on whole blood from Gabonese children living in a schistosomiasis endemic area. One fraction contained lysophosphatidylserine [monoacylglycerophosphoserine (lysoGPSer)] plus diacylphosphatidylserine [diacylglycerophosphoserine (GPSer)] while the other contained lysoGPSer and only a trace of GPSer. The effect of these schistosomal lipid fractions was compared with the known bacterial TLR2 ligands PAM3CSK4 and MALP‐2. PAM3CSK4 and MALP‐2 had preferential IL‐10‐activating capacities, while the fraction containing lysoGPSer plus GPSer had a strong TNF‐α‐inducing capacity. The fraction containing lysoGPSer was neutral with respect to pro‐ vs. anti‐inflammatory effects. When Th1 and Th2 cytokines were analysed, the schistosomal lipid fraction containing lysoGPSer plus GPSer showed a stronger Th2 response compared to PAM3CSK4, MALP‐2 and lysoGPSer alone. Therefore, the study indicates that not only TLR2 ligands derived from bacteria or from parasites can generate distinct cytokine profiles but also that the composition of lipid entities reaching the immune system can be important in leading to different immune outcomes. This information may be important for exploitation of immune modulatory molecules.