Le Trypanosome africain, dont Trypanosoma brucei est le prototype, est un parasite sévissant en Afrique sub-tropicale. Il est responsable de la maladie du sommeil chez l’Homme et de diverses affections chez les animaux tant sauvages que domestiques.<p><p>T. brucei est un parasite extracellulaire qui se développe dans le sang de son hôte mammifère. Il est donc confronté en permanence au système immunitaire de l’hôte et a en conséquence, afin de générer un environnement plus favorable à sa croissance, établit différents mécanismes d’échappement tels que la variation antigénique ou l’immunomodulation. <p><p>Dans ce contexte, il a été montré que T.brucei libère des facteurs capables d’induire la voie arginase des macrophages. Cette induction peut favoriser la croissance des trypanosomes dans le sang de leur hôte de diverses manières. Premièrement, l’arginase participe à la synthèse de composés tels que les polyamines ou la trypanothione, facteurs de croissance des cellules. Deuxièmement, l’arginase partage le même substrat que la NO synthase inductible (iNOS), ces deux enzymes sont donc en compétition et l’activation de l’arginase pourrait contribuer à diminuer la quantité de NO, composé cytostatique et cytotoxique, produit par les macrophages en limitant le substrat disponible pour l’iNOS. Troisièmement, la déplétion du milieu en arginine suite à l’activation de l’arginase inhibe la prolifération de cellules du système immunitaire dont les lymphocytes T.<p><p>Nous avons identifié une chaîne lourde de kinésine chez T.brucei, TbKHC1 (Trypanosoma brucei Kinesin Heavy Chain 1), appartenant à la superfamille des kinésines, comme un candidat potentiellement capable d’induire la voie arginase des macrophages. TbKHC1 est principalement exprimée au stade sanguicole du parasite et est localisée au niveau de la région endo-exocytaire. Dans un modèle d’infection murin, une invalidation de l’expression de TbKHC1 (par ARN interférence ou par knock-out) conduit à une diminution du premier pic de parasitémie et à une prolongation de la survie des souris infectées. Nous avons montré que TbKHC1 joue un rôle dans l’interaction hôte/parasite à deux niveaux indépendants :premièrement, l’induction de la voie arginase des macrophages par TbKHC1 en début d’infection favorise la croissance du parasite et son établissement au sein de son hôte. Deuxièmement, elle joue un rôle dans l’induction de la pathologie liée à l’infection. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le trypanosome est le parasite responsable de la maladie du sommeil chez l’homme et de la Nagana chez le bétail. Afin d’échapper au système immunitaire de son hôte mammifère, il remplace périodiquement la protéine VSG (Variant Surface Glycoprotein) présente en 10 millions d’exemplaires à sa surface. Ce mécanisme a pour nom la variation antigénique. <p>Pour être exprimé, le gène de VSG (VSG) doit se trouver en fin d’un site d’expression (ES) particulier. Cet ES est polycistronique, télomérique et transcrit par une ARN polymérase de type ribosomique (Pol I). 20 à 40 ESs similaires et un millier de VSGs sont recensés dans le génome du trypanosome. Cependant, un seul ES est totalement transcrit (actif) et un seul VSG est exprimé. La variation antigénique est donc possible par deux mécanismes: soit l’activation d’un autre ES, soit le remplacement du VSG dans l’ES actif. La base de ce système est l’activation d’un seul ES à la fois (contrôle monoallélique).<p>Au laboratoire, un modèle a été proposé où la transcription s’initie au niveau de tous les ESs mais n’aboutit au VSG que dans le cas de l’ES actif (Vanhamme et al. 2000). Dans ce cas uniquement, le transcrit primaire subit une maturation correcte (épissage et polyadénylation) et est exporté dans le cytoplasme. Etant donné que des transcrits Pol I subissent une maturation identique à des transcrits Pol II, la régulation s’effectuerait par recrutement d’une machinerie d’élongation/maturation de l’ARN de type Pol II (Pol II « RNA factory »). Cette dernière serait uniquement localisée au niveau de l’ES actif dans le compartiment nucléaire appelé ES body (Navarro and Gull, 2001).<p>Durant cette thèse, diverses stratégies ont été élaborées pour tester la validité du modèle. La première visait à comparer l’état de maturation d’un ES en fonction de son activité. Nos résultats ont appuyé l’idée que les transcrits d’ESs ayant subi une maturation correcte provenaient préférentiellement de l’ES actif mais le(s) facteur(s) en quantité limitante ne permettant cette maturation qu’au niveau de l’ES actif doivent encore être identifiés. Le seconde stratégie analysait l’acétylation des histones ainsi qu’un éventuel attachement différentiel à la matrice nucléaire de l’ES suivant son activité. Le niveau d’acétylation d’un ES lorsqu’il est actif n’a pu être étudié. Des résultats préliminaires en faveur d’une association préférentielle de l’ES à la matrice nucléaire lorsqu’il est actif ont été obtenus. Enfin, nous avons cloné deux homologues d’un facteur général de la transcription appelé TFIIS. Ce dernier permet à la Pol de redémarrer lorsqu’elle est bloquée par un site de pause. Individuellement chacun de ces facteurs ne semble pas être essentiel au trypanosome. Cependant, un retard de croissance a été observé lorsque les deux facteurs sont invalidés dans la même lignée cellulaire. Ce phénotype particulier doit être caractérisé. En parallèle, nous avons envisagé de caractériser le complexe de la Pol I du trypanosome. Cette stratégie constituait la manière la plus simple de mettre en évidence un éventuel contact physique et/ou fonctionnel entre la Pol I transcrivant l’ES et la machinerie d’élongation/maturation de l’ARN de type Pol II « RNA factory ». 5 sous-unités du complexe ont été identifiées, associées à une protéine de fonction inconnue ainsi qu’à des histones. L’identification d’autres protéines associées au complexe constitue notre perspective principale. La phosphorylation de la plus grande sous-unité du complexe a été démontrée mais son rôle doit encore être élucidé.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Superóxido dismutases (SODs) são metaloenzimas que convertem o ânion superóxido em oxigênio molecular (O2) e peróxido de hidrogênio (H2O2). A presença de metal nessas enzimas está diretamente relacionada com seus mecanismos de catálise e com suas estruturas tridimensionais. Evolucionariamente, FeSOD e MnSOD podem ter evoluído de um gene ancestral comum, porque possuem sequências homólogas e estruturas cristalográficas sobreponíveis. Entretanto, a nível catalítico, ambas as proteínas divergiram o suficiente para que seus metais não possam ser intercambiáveis, produzindo uma enzima funcional, indicando que essas proteínas possuem alta especificidade por metal. O objetivo deste projeto de pesquisa é Identificar os determinantes estruturais do ajuste fino da especificidade por metal de MnSOD e FeSOD. Inicialmente, pretendese selecionar resíduos para mutagênese sítio-dirigida em TrMnSOD e TbFeSODB2, a partir de análise de acoplamento estatístico (SCA). Em seguida, mutantes serão construídos, expressos, purificados e cristalizados. A estrutura tridimensional dos mutantes será resolvida por cristalografia e sua atividade enzimática determinada, bem como a acomodação estrutural dos metais por Resonância Paramagnética Eletrônica. Nossa hipótese de trabalho é que através de SCA é possível elencar resíduos de aminoácidos candidatos para mutagênese sítio-dirigida para desenhar novas SODs, com características intermediárias de ligação por Fe/Mn, como possibilidade de interconversão de especificidade, caminhando na história evolutiva dessas moléculas. / Superoxide dismutases (SODs) are metalloenzymes that convert the superoxide anion in molecular oxygen (O2) and hydrogen peroxide (H2O2). The metal in the catalytic center of such enzymes is directly related to their catalysis mechanisms and tridimensional structures. Evolutionarily, FeSOD and MnSOD may have evolved from a common ancestor, because both proteins have homologous primary sequences and superposable crystallographic structures. However, at the catalytic level, both proteins diverged sufficiently to prevent interchange of their metallic centers, which would generate non-functional enzymes, indicating that these proteins have high metal specificity. The objective of this research project is to identify structural determinants of Fe/MnSODs necessary to metal specificity. We intend to use statistical coupling analysis (SCA) to select amino acid residues for site-directed mutagenesis in TrMnSOD e TbFeSODB2. Mutant genes will be constructed and their proteins expressed, purified and crystallized. The tridimensional structure of such mutants will be solved by X-ray crystallography and their enzymatic activities determined, as well as their electron paramagnetic resonance spectra. We hypothesize that SCA is useful to identify amino acid candidates for site-directed mutagenesis to design new SODs with intermediated Fe/Mn specificity, and even metal specificity interconversion, by studying the evolutionary history of these proteins.

Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results: The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion: Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the bloodbrain- barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.

This work allowed the first characterization of the three members of the chloride channel CLC family in T.brucei. The TbCLCs are expressed in the two proliferative stages of the parasite and two of their members appear non-essential. The three TbCLCs act as chloride transporters in X.laevis oocytes and some of their biophysical properties were determined. Furthermore, TbCLC-b appeared to be involved in lysis by the human innate immunity protein apoL-1<p>A novel function of T.brucei adenylate cyclases was discovered in their ability to suppress expression of the innate immunity protein TNF-α. The suppression of the innate response occurs before the first peak of parasitemia and reduces the host ability to control the parasite.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

In October 2006 an intervention was initiated to arrest the northerly advance through Uganda of the zoonotic parasite Trypanosoma brucei rhodesiense. This is a protozoal infection that is vectored by the tsetse fly. It is the aim of this thesis to review the impact of this large scale treatment programme in terms of animal health and human disease. The Stamp Out Sleeping Sickness (SOS) campaign was designed to target the cattle reservoir of T. b. rhodesiense in these newly affected areas by block treating >180,000 head of cattle. This was achieved in collaboration with final year vet students from the University of Makerere, Uganda. Farmers were also encouraged to spray their animals with deltamethrin in order to suppress the tsetse population. In order to monitor the impact of this intervention a base line survey was carried out. Evaluation of the logistics and implementation of the SOS campaign was assessed through interviews with personnel involved. Analysis by PCR revealed the prevalence of T. brucei s.l. as 15.57% (T. b. rhodesiense as 0.81%) within the cattle reservoir prior to SOS treatment. Follow up sampling was carried out at 23 locations at three, nine and 18 months. The prevalence of T. brucei s.l. was reduced post treatment, but in the absence of sustained vector control infections amongst the animals returned by nine months and subsequently exceeded the base line findings (P=<0.0001). It was observed that across most of the SOS area, T. b. rhodesiense did not re-establish following treatment. However, a significant cluster was identified where cases of both human and animal disease were continually reported. This cluster was noted to include the area immediately surrounding the Otuboi cattle market. This link between cattle movement and the spread of T. b. rhodesiense is an established one and is addressed by Ugandan governmental policy which states that ‘cattle traded at market must be treated with trypanocidal drugs prior to movement’. The findings presented here suggest that this policy may not be strictly enforced. The risk of spread is compounded at the northern districts of Uganda restock their domestic livestock following years of civil conflict. The majority of animals are traded in a northward direction – transporting infected animals from the endemic south. The scale of this trade is assessed through questionnaires, analysis of trade records and animal screening. Specific consideration is given to the implications of this cattle trade and impact this may have on the sustainability of the SOS campaign.

Trypanosoma brucei is a protozoan parasite that is the causative agent of sleeping sickness in sub-Saharan Africa. T. brucei has a complex life cycle involving passage between a mammalian host and the tsetse fly. The parasite evades the mammalian immune system via expression of Variant Surface Glycoprotein (VSG) on the cell surface. VSG genes are expressed at telomeric expression sites and at these sites are a number of Expression Site Associated Genes (ESAGs). One unusual ESAG, ESAG9, is developmentally regulated: RNA for these genes accumulates during the transition from slender to stumpy cells in the mammalian bloodstream and cellassociated protein is only detected transiently in stumpy and differentiating cells. Transgenic cell lines were generated which ectopically express one or more members of the ESAG9 gene family. Biochemical and cytological analyses using these cell lines indicated that some members of this family are glycosylated and GPI-anchored, and also that one gene, ESAG9-K69, is secreted. ESAG9-K69 is also secreted by wild-type stumpy parasites. In vivo experiments with tsetse flies did not conclusively show whether ESAG9 proteins play a role in the establishment of a tsetse fly mid-gut infection by transgenic trypanosomes. However, In vivo and ex vivo experiments using the mouse model of trypanosomiasis indicated that expression of ESAG9 proteins may alter parasitaemia in the mouse and results in a significant decrease in the proportion of CD4+ T cells in the mouse spleen.

Centriole and basal bodies are related nine-fold symmetric microtubule-based eukaryotic organelles central to the organisation of cilia/flagella and centrosomes. Mechanisms of eukaryotic centriole and basal body assembly are mainly based on studies in animal systems. To understand which centriolar proteins are the universally important ones in the assembly across eukaryotes, a bioinformatic survey presented here investigates the distribution of centriolar and cilia-associated proteins across a diverse range of eukaryotes. This analysis showed also that the basal body function is ancestral to eukaryotes, whereas centrosomal components are specific to Holozoa (which include animals). It also suggested that the ancestor of all eukaryotes possessed a cilium/cilia not only with motility function but also with a sensory role. The most frequently conserved proteins in extant ciliated eukaryotes found in this analysis included SAS-6, SAS-4 and WDR16. To test whether these proteins are also important for basal body assembly in distantly-related species to metazoan and other model organisms where the proteins have been studied to date, the proteins were investigated in Trypanosoma brucei. I used a combination of genetic tools and microscopy techniques to demonstrate that SAS-6 but not SAS-4 is essential for basal body assembly in T. brucei. I showed that WDR16 is a stably integrated component of the transition zone and axoneme but not the basal body. Furthermore, I identified a novel SAS-6 like protein which localises to a position consistent with the basal plate and has the capacity to form into filaments. This thesis provides new insights into the evolution of centrioles and basal bodies, and into the function of conserved centriole proteins in T. brucei, a distantly-related organism to animals.

In order to evaluate the suitability of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) as a potential drug target for an anti-parasite therapy, we are studying its role in the bloodstream form of Trypanosoma brucei brucei, the eukaryotic parasite that causes African Sleeping Sickness. Eukaryotic TDP1 removes covalently trapped topoisomerase IB and other adducts from the 3’ end of the DNA at DNA strand breaks. Covalent topoisomerase IB stalling is caused by endogenous DNA damage and by anti-cancer drugs such as camptothecin (CPT). A potential approach could be to use TDP1 inhibitors synergistically with CPT in a combined anti-parasite therapy. T. brucei TDP1 knock out cells are hypersensitive to CPT and accumulate in the late S phase of the cell cycle upon treatment with the drug. The CPT hypersensitivity of the TDP1-/- cells can be fully rescued through ectopic expression of wild type TDP1. The catalytic activity of TDP1 is required for complementation of the CPT sensitivity since overexpression of a catalytically inactive mutant form of TDP1 further sensitises TDP1-/- cells to CPT. In this context, expression of the mutant H358N, which shows reduced activity, also increases sensitivity of TDP1-/- cells to the drug. Surprisingly, expressing TDP1 carrying an analogous mutation to the one that causes SCAN1, a human neurodegenerative disease, does not sensitise TDP1-/- cells further. With this unique set of mutant TDP1 proteins in a TDP1-/- background we hope to answer questions concerning TDP1 function that have so far been elusive.

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.