Trypanosoma brucei est un parasite protozoaire responsable de la trypanosomiase humaine africaine. Il présente un cycle de vie complexe alternant entre des hôtes mammifères et un vecteur insecte, la mouche tsé-tsé. Au cours de ce cycle, il rencontre des environnements radicalement distincts auxquels il s’adapte en régulant son métabolisme. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique évoluant dans le tractus digestif de l’insecte vecteur. Dans cet environnement dépourvu de glucose, la néoglucogenèse est cruciale pour la croissance et la survie des parasites car elle permet la synthèse d’hexoses phosphates et en particulier du glucose 6-phosphate qui alimente plusieurs voies de biosynthèse essentielles. Nos travaux confirment ce flux néoglucogénique alimenté par la proline mais aussi par le glycérol. Nous montrons que le glycérol est une source de carbone efficacement métabolisée et préférentiellement utilisée par la forme procyclique à défaut de la proline et même du glucose pour alimenter son métabolisme intermédiaire. Cette situation qu in’a jamais été décrite auparavant met en évidence la répression du glycérol sur le métabolisme du glucose. Nous montrons également que l’enzyme fructose 1,6-biphosphatase(FBPase), spécifique de la néoglucogenèse, n’est pas essentielle à la survie du parasite en conditions dépourvues de glucose indiquant qu’il existe une alternative à cette enzyme.Toutefois, FBPase joue un rôle important dans la virulence de T. brucei dans l’insecte.De plus, nous avons mis en évidence une autre stratégie d’adaptation de T. brucei basée sur des réarrangements génomiques qui peuvent mener à la synthèse de gènes chimères. / Trypanosoma brucei is a protozoan parasite responsible for human African trypanosomiasis. His complex life cycle alternates between mammalian hosts and the insect vector, the tsetsefly. During this cycle, the parasite encounters dissimilar environments and adapts to the sechanging conditions by regulating his metabolism. We have studied intermediate and energetic metabolism of the procyclic form living in the midgut of the insect vector. In this glucose-depleted environment, gluconeogenesis is crucial for growth and viability of the parasites. Indeed, it allows the synthesis of hexoses phosphates and in particular glucose 6-phosphate which feeds several essential biosynthetic pathways. Our work has confirmed the existence of a gluconeogenic flux fed by proline and glycerol. We have shown that glycerol is an efficiently metabolized carbon source and is preferentially used by the procyclic form rather than proline or even glucose. This situation never described before highlights glycerol repression on glucose metabolism. We have also showed that the enzyme fructose 1,6-biphosphatase (FBPase), specific of the gluconeogenesis, is not essential for the viability ofthe parasite in glucose-depleted conditions, suggesting that there is an alternative to this enzyme. However, FBPase plays an important role for virulence of T. brucei in the insect. Moreover, we have showed another adaptation strategy developed by T. brucei which is basedo n genomic rearrangements leading to the synthesis of chimeric genes.

Les progrès récents dans la modélisation informatique des réseaux biologiques permettent maintenant aux chercheurs d'étudier le métabolisme cellulaire des organismes. Dans ce projet, ces approches ont été utilisées pour analyser le métabolisme de Trypanosoma brucei. Ce parasite protozoaire est responsable de la trypanosomiase africaine, une maladie mortelle chez l'homme et qui entraine des dégâts importants dans les élevages. Ce parasite est principalement retrouvé dans les régions d'Afrique sub-sahariennes. Durant cette thèse, des informations sur le métabolisme de T. brucei ont été recueillies à partir d'études publiées, bases de données et de communication personnelle avec des experts qui étudient les différents aspects du métabolisme des trypanosomatides. Cette information a été mise à disposition de la communauté à travers la base de données TrypanoCyc. La base de données a été publiée en Novembre 2014 et a eu plus de 4200 visiteurs provenant de plus de cent pays depuis Novembre 2015. Un modèle métabolique à l'échelle du génome de T. brucei a également été reconstruit sur la base des informations recueillies. Ce modèle a permis de faciliter l'étude du métabolisme de T. brucei en utilisant une approche de biologie des systèmes. Des algorithmes basés sur l'analyse de balance des flux ont été conçus pour optimiser la visualisation et l'étude des propriétés métaboliques du parasite. En utilisant l'algorithme iMat, des modèles spécifiques de la forme sanguine de T. brucei ont été générés à partir des informations fournies par les études publiées et les annotations présentent dans. Enfin, un algorithme a été conçu pour optimiser encore ces modèles spécifiques afin d'améliorer la cohérence de leurs prédictions avec les résultats publiés. Les modèles ainsi créés, spécifiques à la forme sanguine, ont montré une meilleure puissance prédictive que le modèle initial à l'échelle du génome, en particulier pour prédire le comportement métabolique spécifique de différents mutants de T. brucei. / Recent advances in computational modelling of biological networks have helped researchers study the cellular metabolism of organisms. In this project, these approaches were used to analyze Trypanosoma brucei metabolism. This protozoan parasite is the causative agent of African trypanosomiasis, a lethal disease which has been responsible for huge loss of lives and livestock in Sub- Saharan Africa since ancient times. Information on T. brucei metabolism was gathered from published studies, databases and from personal communication with experts studying different areas of Trypanosomatid research. This information has been presented to the public through the TrypanoCyc Database, a community annotated T. brucei database. The database was published in November 2014 and has had over 4200 visitors from more than 100 countries as of November 2015. A manually curated genome-scale metabolic model for T. brucei was also built based on the gathered information to facilitate the study of T. brucei metabolism using systems biology approaches. Flux balance analysis based algorithms were designed to optimize visualization and study interesting metabolic properties. Blood-stream form specific metabolic models were generated using information available from published studies and the TrypanoCyc annotations with the help of the iMAT algorithm. Finally, an algorithm was designed to further optimize these stage specific models to improve the consistency of their predictions with results published in previous studies. These stage-specific models were observed to have a clear advantage over the genome-scale model when predicting stage-specific behaviour of T. brucei, particularly when predicting mutant behaviour.

Le parasite Trypanosoma brucei est retrouvé en Afrique sub-Saharienne et est responsable de la maladie du sommeil chez l’homme et de la Nagana chez les animaux. Il cause de graves problèmes sanitaires et économiques car il affecte le bétail. La vaccination est impossible à cause de la variation antigénique. Les traitements actuels sont difficiles à mettre en place avec des effets secondaires importants. Il est donc urgent de trouver de nouvelles cibles thérapeutiques afin de développer de nouveaux médicaments. T. brucei possède un flagelle unique qui émerge de la cellule par une structure appelée la poche flagellaire (FP). Cette FP est une invagination de la membrane plasmique. Elle est nécessaire à la survie du parasite car c’est le seul site d’endo- et d’exocytose. Au cou de la FP on trouve le collier de la poche flagellaire (FPC) en forme d’anneau. Le FPC est composé de nombreuses protéines dont BILBO1 qui est nécessaire à la biogenèse de la FP et du FPC. De nombreux partenaires de BILBO1 ont été identifiés. Dans cette thèse, je caractérise deux d’entre eux : FPC5, une kinésine putative et FPC9, une synaptotagmine putative. J’ai pu montrer que FPC5 est localisée aux corps basaux mais aussi au FPC. Cette protéine n’est pas essentielle à la survie des parasites bien que des phénotypes de croissance et de ségrégation de la FP apparaissent après induction de l’ARNi. Nous ne sommes pas parvenus à prouver sa fonctionnalité, cependant j’ai pu montrer que son domaine moteur est capable de lier les microtubules. FPC9 est trouvée au niveau de la zone de transition du flagelle. L’ARNi contre cette protéine n’étant pas effectif, nous ne pouvons pas conclure quant à sa fonction dans la cellule. / Trypanosoma brucei is a parasite found in sub-Saharan Africa and is responsible for sleeping sickness in humans and Nagana in animals. It is the source of serious health and economic problems because it kills livestock. Vaccination is not possible because of antigenic variation and current treatments are difficult to implement or have toxic side effects. For these reasons it is urgent to find new therapeutic targets in order to develop effective treatments. T. brucei has a single copy flagellum that emerges from the cytoplasm through a unique structure called the Flagellar Pocket (FP). This pocket is an invagination of the pellicular membrane and because it is the sole site of endo- and exocytosis, it is essential for parasite survival. At the neck of the FP there is a cytoskeletal structure: the Flagellar Pocket Collar (FPC) that forms a “ring” around the flagellum. The FPC consists of numerous proteins, including the first to be identified - BILBO1, which is necessary for FP and FPC biogenesis. A number of potential BILBO1 partners were identified. In this thesis I characterize two of these proteins: FPC5, a putative kinesin and FPC9, a putative synaptotagmin. I show that FPC5 localizes mainly in the basal body area, but also at the FPC. This protein is not essential for parasite survival although reduced FP segregation and growth phenotypes appear after RNAi induction. We are not able to prove its functionality, however I could show its motor domain is able to bind microtubules. FPC9 is found in the transition zone of the flagellum. However RNAi knockdown against this protein was not efficient, so we are currently unable to define a function for this protein.

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

African trypanosomes are a major plague in sub-Saharan Africa. They cause sleeping sickness in humans and nagana in cattle. These parasites are transmitted between their mammalian hosts by tsetse flies. They are adapting to their different environments through differentiation processes. These processes involve, amongst other things, the expression of different surface coats. These coats are made of procyclin protein at the insect midgut procyclic stage and of variant surface glycoprotein (VSG) at the mammalian bloodstream stage. At a given time, one VSG is expressed from a single VSG gene out of a repertoire of more than 1500 VSG genes present in the trypanosomes genome. The expressed VSG gene is always located at one of fifteen telomeric polycistronic transcription units called expression sites (ES). The VSG coat is changed regularly in a process called antigenic variation allowing trypanosomes to escape the immune response. The exact mechanism controlling the selection of the active ES is not yet known and controversies have been raised concerning the ES transcription control. Although several molecular factors involved in the ES monoallelic-expression have been identified, none of them seems to be a critical regulator.<p><p>Thus during my thesis we decided to explore two aspects of ES expression: (A) deciphering the level at which this expression is controlled and (B) fishing for new protein factors controlling this expression.<p>A) It is not even clear at which level the ES transcription control takes place. In particular, there has been debate on whether it is taking place at the transcription initiation or elongation level. Previous experiments generated contradictory conclusions and gave rise to two different models. The first model suggested that transcription initiation takes place in all ESs simultaneously. The second model suggested that transcription is initiated in only two ESs, one being fully active and a second being pre-active. These two models were equally able to account for the finding of transcripts from different ES within a trypanosome population provided the pre-active ES differs between individual cells. In order to decide if a single or multiple ES promoters can initiate transcription in a given cell, single cell RT-PCR targeting the beginning of the ES was required. Thus single cell RT-PCR was performed and an analysis of the obtained transcripts showed that transcription initiation is taking place on many ES while only one VSG is transcribed. This permitted the unambiguous conclusion that the monoallelic expression of VSG is exerted by controls operating downstream from transcription initiation, suggesting transcription elongation or RNA processing as critical control steps. <p>B) We have characterized a new nuclear protein, Tb alba3, involved in the repression of silent VSGs. Its invalidation lead to chromatin opening in the silent expression sites and to a raise in their expression. As this protein is cytoplasmic and binding procyclin mRNAs at the procyclic stage, it could be a new versatile factor, shuttling between the cytoplasm and the nucleus and involved both in the inverse regulation of major surface antigens at different differentiation stages and the control of antigenic variation.<p><p>These results enhance our understanding of ES transcription control and of ES monoallelic expression. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Apolipoprotein L1 (APOL1) is a human-specific serum protein bound to high-density lipoprotein (HDL) particles. This protein allows human resistance to infection by African trypanosomes except for two subspecies, Trypanosoma brucei rhodesiense and T. b. gambiense, the causative agents of sleeping sickness or African trypanosomiasis. This disease infects 20 000 people in sub-Saharan Africa and without treatment, infection is almost always fatal. T. b. rhodesiense resists APOL1 through direct protein neutralization by the Serum Resistance-Associated (SRA) protein. T. b. gambiense does not express SRA, and its mechanism of resistance to APOL1 is orchestrated upon a recently characterized multifactorial defense mechanism.<p><p>The mechanism by which the human serum sensitive parasites are killed following APOL1 uptake is described as the result of the lysosomal swelling induced by the generation of ionic pores within the lysosomal membrane.<p>We show here that preventing the osmotic lysosomal swelling in a hyperosmotic culture condition does not prevent the cell death. In addition, APOL1 appears to trigger some programmed cell death events in the cell such as a fast mitochondrial depolarization followed by a DNA laddering and fragmentation. Furthermore, we show an implication of the endonuclease G (TbEndoG), known to be a key actor in the regulation of cell death process and a kinesin (TbKIFC1), which might be the transporter of APOL1 for the endosomes to the mitochondrion.<p> <p>In addition, by producing different recombinant human APOL proteins in E. coli and test their activity on T. brucei, we were able to show that APOL3, an other member of the APOL family, also possesses a trypanolytic activity like APOL1 beneath the fact it is not a secreted protein. APOL3 does not only kill T. b. brucei but is also able to lyse APOL1-resistant subspecies such as rhodesiense and gambiense, in vitro and confirmed in vivo when the recombinant APOL3 were injected in infected mice. A beginning of an action mechanism is described herein showing a pH-independent activity for this protein oppositely to APOL1, conferring its specificity.<p>It is thus conceivable to use this recombinant protein as a first step of a potent curative agent against gambiense or rhodesiense since the few currently available drugs for treatment of African trypanosomiasis, that are outdated, show problems with toxicity and resistance. <p><p>/ <p><p>L’ Apolipoprotéine L1 (APOL1) est une protéine sérique humaine associée aux lipoprotéines de haute densité (HDL). Cette protéine confère la résistance à l'infection des trypanosomes africains à l'exception des deux sous-espèces, Trypanosoma brucei rhodesiense et T. b. gambiense, les agents responsables de la maladie du sommeil ou trypanosomiase africaine. Cette maladie infecte 20 000 personnes en Afrique sub-saharienne et en l'absence de traitement, l'infection est presque toujours mortelle. T. b. rhodesiense résiste à l’APOL1 grâce à une neutralisation directe d’APOL1 par une protéine appelé SRA (Serum Resistant-Associated). T. b. gambiense n'exprime pas SRA, et sa résistance à l’APOL1 est orchestrée par un mécanisme de défense multifactorielle récemment caractérisé 1.<p>Le mécanisme par lequel les parasites sensibles au sérum humain sont tués suivant l’entrée de l’APOL1 est décrit comme le résultat d’un gonflement du lysosome induit par la génération de pores ioniques à l'intérieur de la membrane lysosomiale2. Nous montrons ici que le gonflement osmotique du lysosome peut être empêché en condition de culture hyper osmotique, sans néanmoins empêcher la mort de la cellule. En outre, l’APOL1 semble déclencher des événements de mort cellulaire programmée dans la cellule, tels qu’une dépolarisation mitochondriale rapide suivie d'une fragmentation de l’ADN. De plus, nous montrons une implication de l'endonucléase G (TbEndoG), connu pour être un acteur clé dans la régulation du processus de mort cellulaire et d’une kinésine (TbKIFC1) qui pourrait avoir le rôle de transporter l’APOL1 des endosomes vers la mitochondrie.<p>Nous avons également pu montrer que l’APOL3, un autre membre de la famille des APOLs humaines, possède tout comme l’APOL1, une activité trypanolytique bien que cette protéine ne soit pas sécrétée en condition physiologique. De manière intéressante, l’APOL3 ne tue pas seulement T. b. brucei, mais est également capable de tuer les sous-espèces résistantes à l’APOL1 tels que rhodesiense et gambiense, in vitro et in vivo lorsque de l’APOL3 recombinante est injectée dans des souris infectées. La spécificité d’action de l’APOL3 pourrait être liée à une indépendance au pH, au contraire de l’APOL1. Il pourrait être envisagé d'utiliser cette protéine recombinante comme agent curatif contre gambiense ou rhodesiense du fait que les médicaments actuellement disponibles pour le traitement de la trypanosomiase africaine montrent des problèmes de toxicité et de résistance.<p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The bloodstream form of Trypanosoma brucei differentiates into the stumpy form in the mammalian bloodstream, completing differentiation into the procyclic form on uptake by the tsetse fly. The underlying genetic events occurring during this differentiation process in pleomorphic cell lines were investigated through whole-genome microarray studies of key time points during differentiation from stumpy form cells to the procyclic form found in the insect midgut. The microarray was extensively validated and bioinformatic experiments conducted to detect motifs over represented in stumpy form or slender form cells. A positional-dependent motif was identified that was over represented in stumpy form cells, possibly representing a regulatory domain. The transcripts found to be enriched in stumpy form cells included a chloride channel, although RNAi directed against this gene showed no phenotype, suggesting the protein is redundant, as three other homologous proteins exist in the genome and showed similar mRNA profiles on the microarray. Stumpy form cells are G0 arrested and two proteins implicated in G0/G1 regulation in other organisms, Target of Rapamycin (Tor) and Cdh1, were investigated in T. brucei to determine whether these proteins were involved in differentiation. The result of depletion of either protein was rapid cell death in bloodstream form cells, although treatment with the drug rapamycin did not have any effect on the cells in contrast to other eukaryotes where this drug causes G1 arrest. A method for synchronisation of bloodstream form cells was also designed using a supravital dye and flow cytometry to allow investigation into cell cycle-dependent processes. This method was particularly suitable for harvesting populations enriched in G0/G1 stage cells, however differentiation of the isolated G0/G1 and G2/M populations did not show significantly different differentiation kinetics.

During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists in two different states: the proliferative slender form and the non-proliferative, transmissible, stumpy form. The transition from the slender to stumpy form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. The slender to stumpy transition and the contribution of stumpy formation to within-host dynamics have been difficult to analyse, however, because cell-type specific markers have been restricted to imprecise morphological criteria. PAD1 is a recently identified stumpy-specific protein which acts as a molecular marker for stumpy formation and a functional marker for transmission. Here, the control of stumpy-specific gene expression via the 3’UTR has been analysed, identifying that there are repressive elements in the 3’UTR preventing inappropriate expression during the slender life stage. Further, both pleomorphic and monomorphic transgenic reporter cell lines utilising the PAD1 3’UTR have been created that report on stumpy formation in vitro and these have been used for the analysis of stumpyinducing chemical compounds. Finally, a sensitive and accurate qRT-PCR assay has been developed and optimised that faithfully reports both parasitaemia and stumpy formation throughout host infection. Using a chronic infection rodent model, stumpy levels have been monitored on the basis of conventional morphological and cell cycle assays, as well as by qRT-PCR for PAD1 expression. The results define the temporal order of events that result in the generation of stumpy forms early in a parasite infection and thereafter describe the dynamics of slender and stumpy forms in chronic infections extending over several weeks. This quantitative data has allowed the mathematical modelling of transmission competence in trypanosome infections, suggesting dominance of transmission stages throughout infection.

The unicellular eukaryote Trypanosoma brucei causes African Sleeping sickness and multiplies extracellularly in the bloodstream of the infected host. The parasite evades antibody-mediated lysis by switching its Variant Surface Glycoprotein (VSG) coat. Blocking VSG synthesis results in an abrupt growth inhibition and a precise pre-cytokinesis cell cycle arrest, with an accumulation of cells with two nuclei and two kinetoplasts. Additionally, induction of VSG RNAi triggers a global block in translation, which is not due to a general decrease in transcript levels. The mechanism behind this translation arrest was investigated. It was observed that it correlated with a decrease in polysomes, indicating that translation was blocked at the level of initiation. It was also shown that the VSG RNAi-triggered growth inhibition was reversible, which suggests that this is not a lethal phenotype. The VSG221 RNAi-induced growth arrest could be alleviated if a second different VSG (VSG117), which was not recognised by the VSG221 RNAi, was expressed immediately downstream of the promoter of the active VSG221 Expression site. Further, it was possible to delete the telomeric VSG221 in these VSG double-expressors, leaving the cells completely reliant on the second complementing VSG117 gene. VSG117 expressed from a promoter-adjacent position in the active Expression site was shown to form a functional surface coat that protected the parasites from complement-mediated lysis in vitro. Transiently transfecting cells with anti-VSG221 morpholino oligonucleotides allowed us to specifically block translation of VSG221 mRNA without degrading it. This resulted in a pre-cytokinesis cell cycle arrest similar to that induced by VSG221 RNAi. This indicates that the VSG RNAi-triggered growth inhibition was due to a lack of VSG protein or its synthesis rather than the ablation of the abundant VSG mRNA. In addition, it was shown that blocking VSG synthesis reduced the rate of surface VSG internalisation in cells that were stalled precytokinesis, but had no effect on other endocytic markers. These experiments give us further insight into the importance of the protective VSG coat for pathogenicity in T. brucei.