The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the ‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis respectively. In their human infective form in the bloodstream all three trypanosomatid parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6- bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set of structural and regulatory differences compared to the mammalian host enzyme. These differences, coupled with the availability of trypanosomatid PFK crystal structures present an opportunity for the structure-based design of specific inhibitors against the enzyme. Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’) were designed against T. brucei PFK with the aim of developing novel treatments against Human African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to trypanosomatid PFKs. We show that the compounds are specific; neither competing with the natural substrates ATP or F6P nor inhibiting the human PFK enzyme. We describe the development and testing of highly potent and specific low molecular weight PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage I HAT in mice models. We describe the tight, 1:1 binding of these compounds with trypanosomatid PFKs, and the thermodynamic characteristics of binding through various biophysical assays. We also show the unprecedented characterisation of the reverse PFK reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and inhibition by the drug-like compounds against trypanosomatid PFKs.

Trypanosoma brucei is an extracellular kineotoplastid parasite that causes human African trypanosomiasis (HAT), also known as sleeping sickness. As trypanosomes undergo vector to host transition, heavy transcriptional adaptation such as metabolic shift to glycolysis and upregulated endocytosis occurs. Specifically, glycolysis in the infectious stage becomes the sole source of energy production; thus, the glucose transport mechanism in T. brucei provides one of the most promising therapeutic targets for development of new drugs to treat HAT. Despite an established trypanosome hexose transporter (THT) model for glucose transport across the plasma membrane, there remains gaps in the detailed mechanism of glucose transport especially as it relates to glucose transport across the glycosomal membrane. Using 2-NBDG, a fluorescent glucose analog, we measured glucose uptake rates in the presence of small molecule inhibitors and by using RNA interference (RNAi) to knockdown key proteins to investigate the mechanism of glucose transport in trypanosomes. We have confirmed a direct role of THT in glucose transport of BSF trypanosomes; however, in our investigations, we observed an unexpected ATP-dependence on glucose transport in live trypanosomes, which initiated further study where we focused on the role of endocytosis as an ATP-coupled bulk glucose transport mechanism. Experimental approaches that inhibited endocytosis reduced the observed glucose uptake rate confirming a role for endocytosis-coupled glucose transport in BSF trypanosomes. We provide evidence for an endocytosis-coupled glucose transport mechanism in BSF trypanosomes as an additional and important mechanism that functions in parallel with the established THT model.

No description available.

THE TRYPANOLYTIC FACTOR OF HUMAN SERUM: A TROJAN HORSE African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites of huge clinical, veterinary and economical importance. They develop in the body fluids of various mammals (including humans) where they face and manipulate many different aspects of the immune system. The extent of this interplay is pivotal to both host and parasite survival, and depending on parasite virulence and host susceptibility, infection duration ranges from some months to several years. At the end, host survival is invariably compromised. Humans and few other primates provide however a striking exception to this fatal outcome. They are indeed fully protected against most trypanosome infections through the presence in their blood of a so-called trypanosome lytic factor (TLF). The TLF is known to circulate mainly in the form of a high density lipoprotein particle characterized by the simultaneous presence of two primate-specific proteins: haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I). We have contributed to delineate the respective roles played by Hpr and apoL-I in the lysis process. ApoL-I was shown to be the exclusive toxin of the TLF. In its absence humans get fully susceptible to any trypanosome infection. The toxin was shown to kill the parasite after endocytosis through the generation of ionic pores in the lysosomal membrane. Those pores dissipate membrane potential and trigger the influx of chloride ions from the cytoplasm into the lysosomal compartment, leading to an eventually fatal uncontrolled osmotic phenomenon. ApoL-I efficient delivery to the parasite relies on Hpr. African trypanosomes indeed fulfil their heme nutritional requirements by receptor-mediated internalization of the complex formed by haptoglobin, an evolutionary conserved acute-phase protein, and hemoglobin, resulting from physiological intravascular hemolysis. This heme uptake by the auxotrophic parasites contributes to both growth rate and resistance against host oxidative burst. In human serum, the trypanosome receptor is unable to discriminate between Hp and the closely related TLF-bound Hpr, explaining TLF efficient endocytosis. As such, the TLF acts as a Trojan horse, killing the parasite from inside the cell after having deceived its vigilance through the high similarity between heme-delivering haptoglobin and toxin-associated Hpr.

African sleeping sickness is a parasitic infection spread by the protozoan parasite Trypanosoma brucei, and drugs used today are toxic and painful. Galactose metabolism is essential for the survival of T. brucei and without a functional UDP galactose 4’ epimerase (GalE) galactose starvation occurs and cell death will follow. In this Master thesis project two assays observing binding of small molecules to TbGalE has been investigated in attempt to establish an assay that in the future could be used for screening for drugs. TbGalE was biotinylated through the Pinpoint Xa vector and expressed in E. coli cells. The protein was successfully immobilized to a Streptavidin chip for Surface Plasmon Resonance experiments and the binding of the substrates UDP-galactose and UDP-glucose was observed. Unfortunately, the assay was not optimal for screening due to low signal response. However, the established protocol for expressing biotinylated proteins that bind to Streptavidin surfaces could be used in further experiments with TbGalE and other drug targets for African sleeping sickness. The fluorescent sugar nucleotide analogue UDPAmNS, which is a known inhibitor for E. coli GalE, was synthesised and purified and then used to establish a displacement assay. IC50 of UDPAmNS against TbGalE was determined and a synergic effect in fluorescence between the protein and the inhibitor was proven. Further, evidence for a reduction in fluorescence by displacing UDPAmNS with UDP was obtained. This reduction in fluorescence was also shown by a predicted cofactor inhibitor. The IC50 against TbGalE for this compound was determined before the displacement assay, which showed that the cofactor inhibitor, at least partly, binds to the active site of TbGalE. The UDPAmNS displacement assay could have the potential of becoming a robust screening assay for TbGalE, in the effort to find a better drug for African sleeping sickness.

L'ARN interférence (ARNi) est un phénomène découvert en 1998 par lequel la présence d'ARN double brin au sein d'une cellule entraîne la dégradation d'ARN de séquence homologue. L'ARNi est effectué par un complexe ribonucléoprotéique contenant des petits ARN double brin et au moins une protéine de la famille Argonaute. Cette thèse a été consacrée à l'étude de l'ARNi chez le protozoaire Trypanosoma brucei. Nous avons d'abord défini les conditions d'utilisation de l'ARNi au niveau de la spécificité et de l'efficacité, paramètres qui ont servi à l'élaboration d'un logiciel permettant la sélection de l'ARN double brin pour les études fonctionnelles. Ensuite, nous avons recherché plusieurs gènes candidats codant pour des protéines participant à l'ARNi. Le meilleur d'entre eux, TbAGO1, appartient à la famille Argonaute et se caractérise par la présence d'un domaine supplémentaire, capable de lier les ARN. Il est essentiel pour l'ARNi chez le trypanosome. Sa délétion produit des défauts significatifs lors de la mitose et nous avons établi que l'ARNi contribue à la formation du fuseau mitotique et à la ségrégation des chromosomes. Un second phénotype observé en l'absence d'ARNi est la surexpression des ARN de deux types de rétroposons (rétrotransposons sans LTR), sans toutefois augmentation de leur activité de rétroposition. Les deux phénotypes sont indépendants l'un de l'autre. Nous avons ensuite démontré que la présence d'ARN double brin entraîne la destruction d'ARN cible de séquence homologue dans le cytoplasme mais peut aussi conduire à une extinction de la transcription du gène correspondant. Ce type de mécanisme pourrait non seulement contrôler l'expression des ARN des rétroposons, mais aussi celle des gènes dans lesquels ils sont insérés.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves [120]-134).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 129-143).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-127).

Mitochondrial gene expression in trypanosomatid pathogens requires extensive post transcriptional modification called RNA editing. This unique molecular mechanism, catalyzed by a multiprotein complex (the editosome), generates translatable transcripts for essential components of parasite respiratory complex. How editosome proteins are assembled and perform RNA editing is not fully understood. Moreover, previous studies have shown that editosome proteins are essential for parasite survival, which makes editosome as a suitable target for drug discovery. Currently, researchers use radio-labeled based assays to monitor RNA editing process. However, these assays are not suitable for high throughput screening of editosome inhibitors, have low detection limits, and cannot monitor RNA editing in real time. / Therefore, to develop a sensitive high throughput RNA editing assay, we have designed a sensitive hammerhead ribozyme-based fluorescence assay. Ribozyme structure was remodeled by adding or removing uridylate in its conserved catalytic core to make an inactive ribozyme. In the presence of the editosome, inactive ribozyme is edited to an active ribozyme. Consequently, hammerhead ribozyme activity can be measured by cleaving its fluorescently labeled substrate. We have shown that higher sensitivity is achieved using fluorescent based assay than conventional radio-labeled assay. Moreover, we can use this assay for rapid identification and characterization of the editosome inhibitors against RNA editing activities in trypanosomatids.