14Life and Death of Trypanosoma brucei: New Perspectivesfor Drug DevelopmentTorsten Barth, Jasmin Stein, Stefan Mogk, Caroline Schonfeld,Bruno K. Kubata, and Michael Duszenko
AbstractCell death is a life-long companion for any cell and molecular processes evolved inorder to deal with life-threatening situations like starvation or poisoning. It is alsoknown that in metazoa different pathways exist by which a cell undergoes suicidefor the benet of the whole organism, collectively known as apoptosis. Althoughprotozoa generally lack caspases, a formerly thought indispensable prerequisitefor apoptosis, an inducible caspase-independent form of apoptosis as a mecha-nism of cell density regulation has been described in African trypanosomes. Here,we review the current status of cell death mechanisms in Trypanosoma bruceiincluding necrosis, autophagy, and apoptosis. We will discuss which of theseevents are necessary to cope with stressful situations, and which are probablyneeded to ensure continuance of infection and transmittance to the insect vector.Since autophagy and apoptosis are essential for the parasites survival, we will alsodiscuss possible pathway junctions suitable for drug development.
Any cell will undergo necrosis if changes of the environmental conditions arecontradictory to cell integrity. This might be extensive temperature shifts, highpressure, non-isotonic conditions, or the appearance of substances that eitherdissolve the membrane integrity or lead to a breakdown of the energy metabolism.In any case, this is an accidental cell death and mostly a rather rapid process.Detection of necrosis is usually performed by light or electron microscopy, orby uptake of specic dyes that are unable to cross intact membranes. Propidiumiodide is often used in this respect, because it is excluded from viable cells, butpenetrates damaged membranes, intercalates into DNA (one molecule of dyebinds to about 5 bp), and is then readily detectable by uorescence-activated cellsorting measurements (Figure 14.1). However, if conditional changes appeargradually, a cell would react accordingly to cope with the respective stress
Trypanosomatid Diseases: Molecular Routes to Drug Discovery, First edition. Edited by T. Jger, O. Koch, and L. Floh.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
condition. In this case, signs of autophagic cell death or apoptosis may also bedetected as a consequence of the cellular response .Necrosis in bloodstream-form trypanosomes is easily detected by incubating the
parasites in, for example, glucose-free media or using sufcient concentrations oftrypanocidal drugs (Figure 14.1). Although induction of necrosis in trypanosomesmight appear as a perfect alternative to remove parasites from the host, severe side-effects may appear during this process that could threaten the hosts life. First, drugsintervening with the parasites metabolism in a way to cause necrosis will probablyalso interact with at least some of the hosts cells. For example, depletion of glucosewould kill the trypanosomes, but would also lead to anemia, to coma, and nally todeath caused by a hypoglycemic shock. The reason is that most, if not all, of thecentral metabolic pathways are evolutionarily highly conserved. Here again, glycoly-sis is a very good example: trypanosomes as well as the other agellates of the orderKinetoplastida possess an organelle called a glycosome that contains most of theglycolytic enzymes [2,3]. Thus, although the organization of this pathway as well as
Figure 14.1 Necrosis in African trypanosomesafter H2O2 treatment. (a) Scanning electronmicrograph of trypanosomes incubated for 3 hin culture medium containing 40 mMH2O2.(b) Flow cytometry double staining withpropidium iodide (PE-A) and Annexin-V-FLUOS(FITC-A) to detect necrosis and apoptosis. Leftpanel: control trypanosomes incubated in
culture medium; right panel: trypanosomesincubated for 3 h in culture medium containing40mMH2O2. Quadrants: dots in the lower-leftquadrant represent viable cells; dots in thelower-right quadrant represent apoptotic cells(Annexin positive); dots in the upper-rightquadrant represent necrotic cells (Annexin andpropidium iodide positive).
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the structure of several glycolytic enzymes show differences and peculiarities ascompared with their mammalian counterpart, so far no specic inhibition ofglycolysis in trypanosomes could be established, although crystal structures ofmany glycolytic enzymes exist of both mammalian and trypanosomal origin .Secondly, mass destruction of parasites would lead to a massive inammationreaction that, especially in local areas like the meningeal compartment, may havedeleterious effects.
Autophagic Cell Death
Autophagy in general is a survival mechanism of a cell to cope with different stresssituations. The most plausible and best-analyzed scenario is starvation. In this casepart of the cytosol including organelles is engulfed by a double membrane that isformed elsewhere within the cell and delivered to the lysosome for degradation.Following fusion of the outer membrane of the autophagosome with the lysosome,the inner autophagosomal membrane as well as its contents are degraded bylysosomal hydrolases and the remaining substrates (e.g., nucleosides, amino acids,carbohydrates, and lipids) are released into the cytosol for biosynthesis of essentialbiopolymers (nucleic acids, proteins, etc.). In this way, a cell can survive for sometime by eating its less important constituents to form urgently needed molecules. Ifnutrition does not return to a normal supply, the cell will enter autophagic cell death,most obviously characterized by a massive increase of lysosomes and vacuolization.In contrast to the constitutively occurring engulfment of parts of the cytosol by thelysosome itself (microautophagy), the process has been called macroautophagy.Interestingly, this is not the only cellular event wheremacroautophagy is involved. Infact, it became increasingly clear during the last decade that autophagy is alsoinvolved during cell differentiation and generally during cellular remodeling as anadaptation to environmental changes . In addition, it plays signicant roles indisease  and infection . We will briey describe the general molecularmachinery involved in the autophagic process, before we concentrate on trypano-somes. Macroautophagy (further referred to as autophagy) starts with the formationof a phagophore (also called an isolation membrane) at a certain place within the cellthat has been called the phagophore assembly site or pre-autophagosomalstructure (PAS). It is a long-standing question where the PAS originates. Datahave been presented that, on the one hand, it is formed as an elongation of theendoplasmic reticulum  or, on the other hand, that the outer mitochondrialmembrane contributes to PAS formation . Although autophagy occurs consti-tutively at a low basal level, the PAS is massively formed in response to a denedsignal (e.g., in the absence of amino acids). The central regulator is the serine/-threonine kinase TOR, an enzyme that was named due to its inhibition byrapamycin, hence its name target of rapamycin. Rapamycin, a macrolide, wasoriginally isolated from Streptomyces hygroscopicus and used as an anti-fungal drug,before it was discovered that it has a pronounced immunosuppressive effect byinhibiting the intracellular response of B- and T-cells to interleukin-2 . As we
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know now, rapamycin associates with the soluble protein FK-binding protein 12(FKBP12) before it directly binds to the TOR complex 1 (TORC1) to inhibit its kinaseactivity. TORC1 and TORC2 are two distinct cytosolic complexes consisting ofseveral proteins with TOR as the catalytic center of both complexes . TORC1senses many different cellular signals like the amino acid concentration, theavailability of growth factors, the energy status of the cell, and stress. Sufcientsupply of these signals and the absence of stress lead the cell to an anabolic ratherthan catabolic metabolism, inducing cell growth, cell cycle progression, and cellproliferation. On the contrary, deciency of these signals, an increase in stress, orbinding of rapamycinFKBP12 leads to the opposite effects and induces autophagy(Figure 14.2a) . Usually, TORC2 does not bind rapamycinFKBP12 and does notinduce autophagy, but is involved in cytoskeleton organization and cell volumecontrol . Inhibition of TORC1 blocks phosphorylation of the downstream signal
Figure 14.2 Schematic description ofautophagy. (a) Regulation of the induction ofautophagy; (b) vesicle nucleation andgeneration of membranes; (c) autophagosome
formation by the Atg8 and Atg12 ubiquitin-likepathways; and (d) fusion of a lysosome andautophagosome to an autophagolysosome andvesicle breakdown. (See text for more details.).
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molecules and induces autophagy. Key players in autophagy induction are autoph-agy-related proteins (Atg) which are encoded in autophagy-related genes (ATG).TORC1 phosphorylates Atg13 to inhibit autophagy. Under starvation conditions,TORC1 becomes inactive and Atg13 is readily dephosphorylated, forming a complexwith Atg1 and Atg17. The activated complex induces formation of the PAS byactivating a protein complex consisting beside others (e.g., Atg6, Atg14, Vps15) ofclass III phosphatidylinositol-3 kinase (Vps34). This Vps34 (Vps vacuolar proteinsorting) leads to formation of phosphatidylinositol-3-phosphate that is enriched inthe inner bilayer of the autophagosome membrane thereby attracting additional Atgproteins like the Atg18Atg2 complex to increase the membranes size(Figure 14.2b). The next step (i.e., formation of a vesicular structure) includesbending of the double membrane, which is induced by binding of Atg8 tophosphatidylethanolamine (PE). Atg8, a ubiquitin-like protein, reacts with thecysteine protease Atg4, which removes amino acids until a glycine residue isexposed at the C-terminus. It is then conjugated to PE by the E1-like activatingenzyme Atg7 and the E2-like conjugating enzyme Atg3. Conjugation of Atg8 issupported by Atg12, Atg5, and Atg16 a protein complex that works as an E3-likeenzyme and needs activity of Atg7 and Atg10 for its own formation. Finally, Atg8 isreleased from PE by Atg4 (Figure 14.2c) . The newly formed autophagosomescarry a cargo of engulfed cytoplasmic materials including cytosol with all solutes,macromolecules like ribosomes, and organelles. The outer membrane of theautophagosome fuses with a lysosome, thereby forming an autophagolysosome.Now the lysosomal hydrolases will degrade the inner autophagosomal membraneand all constituents of the cargo to release the degradation products (amino acids,nucleosides, sugars, lipids) into the cytosol (Figure 14.2d).Wewill now describe differences in the autophagymechanisms in trypanosomes
in order to discuss possible drug targets. There is no doubt that trypanosomesundergo autophagy, which is clearly visible in electron micrographs (Figure 14.3).Obviously, autophagy is already visible under control conditions (Figure 14.3a), butis especially induced during starvation or incubation with nanoparticles(Figure 14.3b and c). Since a bioinformatics survey had revealed that a considerablylower number ofATG genes seem to be present in Kinetoplastida, it was speculatedthat autophagy in these parasites may be more simply organized than in highereukaryotes . However, as we know now, the major principles are very muchcomparable, although some peculiarities exist that could indeed serve as targets fordrug development. It already starts with the TORC complexes. As described above,TORC1 is usually the target of rapamycin and its inhibition leads to the induction ofautophagy. In African trypanosomes, both TORC1 and TORC2 are present andbuild from two different TOR kinases (TbTOR1 and TbTOR2) and different adapterproteins, including TbTOR-like 1 and TbTOR-like 2 . As in the mammaliansystem, both complexes are involved in cell cycle progression and cell division, butin contrast, TORC1 is not affected by rapamycin in the nanomolar range intrypanosomes. Instead, rapamycin inhibits TORC2, leading to a disruption ofcytokinesis and subsequently cell death . Since TOR and TOR-like proteins areconsiderably different to their mammalian counterparts and rapamycin inhibits
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specically TORC2 instead of TORC1 as in the hosts system, the parasitic TOR andTOR-like proteins could indeed be valuable targets for drug development, espe-cially as they are involved in cell cycle control and progression. This is furthersupported by the nding that the TOR-like 1 kinase seems specically involved information of acidocalcisomes unique and essential organelles in Kinetoplastida. The next step, namely formation of the autophagosome bilayer, dependsprimarily on Vps34. The respective ortholog of this protein has been investigated intrypanosomes, and seems especially involved in Golgi segregation as well as inreceptor-mediated endocytosis and in exocytosis . Its involvement in autophagywas not explored in this study, but RNA interference-induced knockdown mutantsshowed severe growth defects, indicating that TbVps34 is needed for a correctcytokinesis. It might thus be advisable to investigate more about PAS membraneformation in trypanosomes in order to gainmore information about this step and toanalyze its suitability for drug development. Finally, autophagosome formation andprogress of autophagy has been investigated in several laboratories .
Figure 14.3 Electron micrographs showing thedevelopment of autophagy in T. brucei. (a)Untreated control cells showing a very low butvisible rate of autophagy; (b) autophagyinduced by nanoparticles labeled with bovineserum albumin (BSA) for uptake bytrypanosomes; and (c) final stage of autophagyclose to autophagic cell death induced by amino
acid starvation for 24 h. AP, autophagosome;APL, autophagolysosome; APP,autophagophore; fp, flagellar pocket; G,glycosome; Gol, Golgi apparatus; kDNA,kinetoplast DNA; L, lysosome; M,mitochondrion; PAS, phagophore assemblingsite; N, nucleus.
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Trypanosomes contain all orthologs for the Atg8 pathway and possess threepossible ATG8 genes, named ATG8.1, ATG8.2, and ATG8.3 . The respectiveprotein Atg8.3 contains an insertion of 16 amino acids and seems not to be involvedin autophagosome formation. The function of Atg8.1 is confusing. Since it is oneamino acid shorter and ends with a glycine residue on its C-terminal end, it will notbe processed by Atg4. It is, however, expressed (as judged from our unpublishedreverse transcription-polymerase chain reactio...