Adaptations in the Energy Metabolism of Parasites

Adaptations in the energy metabolism of parasites Adaptaties in het energiemetabolisme van parasieten (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 4 juni 2009 des middags te 2.30 uur door Koen Willem Anton van Grinsven geboren op 28 november 1978, te Heesch Promotor: Prof. dr. A.G.M. Tielens Co-promotor: Dr. J.J. van Hellemond Üçtaneme…. ben ben değilim, ben dediğim biziz hep The printing of this thesis was financially supported by the J.E. Jurriaanse Stichting, and the Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University Layout by H. H. Otter Cover design by I. Agajev, M. Brouwer, T. di Cesare Printed by Ridderprint, Ridderkerk ISBN: 978-90-393-5056-0 Table of contents Chapter 1 General introduction 1 Chapter 2 Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis 19 Chapter 3 Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica 45 Chapter 4 Acetate formation in the energy metabolism of parasites 63 Chapter 5 Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolated from tsetse flies and during differentiation of bloodstream forms 85 Chapter 6 Rhodoquinone biosynthesis occurs partly via the ubiquinone biosynthetic pathway 101 Chapter 7 Summarizing discussion 121 Samenvatting 133 Curriculum vitae 139 List of publications 141 Dankbetuiging 143 Chapter 1 General introduction Chapter 1 Parasitism Parasites form a huge and diverse group of organisms, with the only common denominator that all parasites are for their own benefit associated with other organisms, for which this association is harmful. Eventhough this definition applies to a vast majority of all living creatures in biology, viruses, bacteria and fungi are not considered parasites, narrowing the group of parasites down to eukaryotic organisms, which are not fungi, to which the previous definition applies. Despite this exclusion of viruses, bacteria and fungi, parasites still form a vast and incredibly heterogeneous group of organisms, consisting of ectoparasites that live and feed on the outside of their hosts (e.g. fleas, ticks) as well as endoparasites, which live inside their hosts (e.g. intestinal worms, blood-dwelling parasites). Parasites can be unicellular (protozoan), or multicellular (metazoan), and where some parasites, like intestinal worms and Toxoplasma gondii, appear to live in relative harmony with their hosts, others like Plasmodium sp., Filaria nematodes and Trypanosoma brucei are known to cause severe or even lethal diseases (malaria, elephantiasis, and sleeping sickness, respectively). It is estimated that a majority of (eukaryotic) organisms display a parasitic lifestyle and in fact, parasites are so ubiquitous that all animals and plants can be (and usually are) infected by different types of parasites (1). Parasites generally have complex life cycles, which can comprise multiple host- species as well as free living stages. Examples of parasitic life cycles are depicted in Figures 1, 2 and 3. These complex life cycles include exposures to different environments, to which parasites adapt their metabolism. In some environments there might be a shortage of nutrients, forcing parasites to arrange their metabolism in such a way that metabolites are most efficiently used (2,3). This means that carbohydrates are degraded in the same way as occurs in mammals; glycolysis in the cytosol is followed by pyruvate decarboxylation and subsequent oxidation of acetyl-CoA by Krebs cycle activity in the mitochondria. Reoxidation of the thereby formed reduced cofactors, such as NADH, occurs by the oxygen-dependent mitochondrial electron-transport chain, which provides a proton gradient that is subsequently used for ATP synthesis. Other environments appear to be like the proverbial “land of milk and honey” turning parasites into real energy wasters, that ferment enormous amounts of glucose to lactate, pyruvate or ethanol (4-6). Another major factor to which many parasites have had to adapt their metabolism is the limited presence of oxygen in certain habitats, which prevents many parasites from using the classical mitochondrial electron transport chain all together, or only in certain lifestages (7,8). 2 General introduction This thesis describes studies performed on the adapted energy metabolism of three different parasites; Trichomonas vaginalis, Fasciola hepatica and Trypanosoma brucei. All these parasites adapted their energy metabolism to their specific habitats in their hosts. Trichomonas vaginalis The protozoan parasite Trichomonas vaginalis is a sexually transmittable parasite that has a relatively simple lifecycle, since it infects the genital tract of merely one host-species, namely humans (Fig. 1). Upon infection T. vaginalis causes trichomoniasis, a frequently asymptomatic infection, which can result in inflammation, preterm delivery and predisposition to Human Immunodeficiency Virus (HIV) and cervical cancer (9). Trichomoniasis is one of the most common sexually transmitted infections in industrialized countries (10), with a 3.1 % prevalence of infection in women in the United States (11). Fig. 1. Lifecycle of Trichomonas vaginalis, image adapted from the online public image library of the Center for Disease Control and Prevention (CDC), Division of Parasitic Diseases (DPD) 3 Chapter 1 Fasciola hepatica The liver fluke Fasciola hepatica is a parasitic flatworm that infests the bile ducts of various mammals, causing fascioliasis, a disease with common symptons such as fever, nausea, gastrointestinal disturbances, anaemia, ascites, hepatomegaly and splenomegaly. Fascioliasis does not only cause great economic losses in sheep and cattle, but it is also an important emerging, or re-emerging trematode infection in humans, with estimated worldwide human infections in the order of millions (12- 14). Fasciola hepatica has a complex life cycle, and development from egg to adult requires infection of both a snail and a mammalian host, as well as the passing of several free living stages (Fig. 2). Fig. 2. Lifecycle of Fasciola hepatica depicting the adult fluke residing in the bile ducts of mammals, and the various free living life stages as well as the intermediate snail host (image adapted from Petcare petalia.com). Trypanosoma brucei The mammalian life cycle stages of the protozoan parasite T. brucei reside in the bloodstream, lymph system and central nervous system of their host. T. brucei is endemic in sub-Saharan Africa, where it causes trypanosomiasis (also known as 4 General introduction sleeping sickness in people or nagana in cattle) a lethal disease with estimated numbers of over 300,000 human cases per year (15,16). Infection with T. brucei occurs after the bite of an infected tsetse fly. Metacyclic trypomastigotes are injected from the salivary glands of the fly into the circulatory system of the mammalian host. Here the metacyclics differentiate into long and slender bloodstream form (BSF) trypanosomes, which actively multiply in the blood, lymph or spinal and cerebral fluid. At the peak of parasitaemia, long and slender BSF trypanosomes transform into non-dividing short and stumpy BSF trypanosomes (Fig. 3), which are pre-adapted to the midgut of the tsetse fly (17,18). If short and stumpy BSF trypanosomes are ingested by tsetse flies during a bloodmeal on an infected mammal, they will differentiate into replicating procyclic form (PCF) trypanosomes in the midgut of the fly (Fig. 3). The lifecycle is completed when the procyclics transform into epimastigotes that multiply in the fly’s salivary gland and differentiate into metacyclic trypomastigotes, the stage that will infect mammals upon a bite from the infected tsetse fly (Fig. 3). Fig. 3. Lifecycle of Trypanosoma brucei depicting the morphological changes trypanosomes undergo during their life cycle (taken from Lee et al. (68)) 5 Chapter 1 Adaptations in the energy metabolism of Trichomonas vaginalis, Fasciola hepatica and Trypanosoma brucei These three parasites all adapted their energy metabolism to the environment in their hosts, resulting in strikingly different ways of glucose breakdown. One feature that these three metabolic pathways have in common however, is the formation of acetate as an end product of carbohydrate catabolism, a process that does not occur in their mammalian hosts. Whether this acetate formation occurs via homologous enzymes in these different parasites was unknown and is one of the questions that will be addressed in this thesis. T. vaginalis were long considered amitochondriate protozoa, which complied with their microaerobic habitat and the fact that they feature an anaerobic metabolism. Trichomonad cells do however contain alternative metabolic organelles called hydrogenosomes. Eventhough the evolutionary origin of these hydrogenosomes has been a subject of discussion for many years, genomic evidence demonstrated that hydrogenosomes are undoubtedly of mitochondrial origin (19). In the hydrogenosomes of T. vaginalis pyruvate is degraded to acetate. Acetate formation concomitantly results in carbondioxide formation and the hydrogen production giving this organelle its name (20,21) (and Figure 4). Pyruvate is decarboxylated by Pyruvate Ferredoxin Oxidoreductase (PFO) to acetyl-CoA, yielding carbondioxide. Protons act as

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