No description available.

The protozoan parasites Trypanosoma cruzi and Trypanosoma brucei are the causative agents of Chagas disease and Human African sleeping sickness respectively. Existing therapies are toxic and ineffective against the later stages of the two diseases, consequently safer, improved therapies are urgently required. Here, two areas of trypanosome biology are explored. In the first section, the process of cell division is approached from a fundamental biology perspective. Centromeres are the region of DNA where kinetochore structures form, allowing the attachment of microtubules to facilitate chromosome segregation. In T. brucei we have characterized the nature and location of centromeres by exploiting the localized activity of topoisomerase-II, a cancer chemotherapy target, at the centromere. Etoposide mediated DNA cleavage mapping revealed the presence of signature AT-rich repeat regions coupled with adjacent retrotransposons at the centromere. Further experiments demonstrate that of the two nuclear T. brucei topoisomerase-II isoforms, only topoisomerase-IIα is essential and active at the centromere. The second section centres on pro-drug development against a trypanosome type I nitroreductase. This enzyme has previously been implicated in activation of nifurtimox and benznidazole, the two therapies in clinical use against Chagas disease. Initially we have developed a luciferase based drug assay system in the clinically relevant intracellular T. cruzi stage and rapidly screened a range of nitroaromatic based compounds for trypanocidal activity. A series of derived nitrofuryl compounds previously developed against Chagas disease were also screened against T. brucei, where most demonstrate trypanocidal activities of less than 1 μM. Further we show that these compounds are active substrates of nitroreductase, and act as pro-drugs within the parasite by specific activation of nitroreductase to generate cytotoxic moieties.

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.

Trypanosomatid protozoan parasites are the causative agents of a number of diseases responsible for the death of thousands of people in developing countries. There is currently little hope for the development of vaccines and existing treatment regimens are associated with high toxicity. Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of these parasites are intimately linked to their shape and form which are in turn derived from a highly ordered microtubule-based cytoskeleton. Here I have investigated some of the critical structures of the cytoskeleton in terms of their molecular composition with a view toward interrogating their functions. I have used a combined reverse genetics/comparative proteomics approach to identify over 20 novel components of the paraflagellar rod, an essential structure for the mammalian infective form of the parasite. I have iterated this approach to define interdependent sub-groups within the cohort which provide clues to the function of the paraflagellar rod. I next applied the same comparative proteomics techniques to investigate the differences between the protein composition of two life-cycle stages of the parasite. I have identified novel components of a unique mobile transmembrane junction called the flagella connector, and of the flagellum attachment zone, a structure that is essential for cell division. In addition I have defined a pair of paralogous cytoskeletal proteins that show life-cycle stage specificity. Finally, I have used electron tomography, reverse genetics and in situ protein tagging to define the morphology of the flagellar pocket collar, a critical structure required for parasite viability, and provide new insights into its molecular composition, function and biogenesis.

The aim of this work was to explore an alternative to existing methods of detection for Human African Trypanosomiasis (also known as sleeping sickness). A new approach to diagnostics for sleeping sickness is needed, since the existing methods of detection employed in the field have significant shortcomings in terms of sensitivity, cost or ease of operation. In this work, the enrichment of trypanosomes from blood using travelling electric fields and the selective lysis of cells using optoelectronic tweezers will be presented. Both techniques allow for the enrichment of trypanosomes from blood samples but the first is more suited for an application as a point-of-care device, while the latter is also applicable to other cell types and offers greater flexibility. Besides demonstrating and quantifying the experimental results the work includes simulations to further explain the phenomena and investigate the underlying mechanisms. The results presented here offer a new method to enrich trypanosomes, a central step in any potential diagnostic tool. They open up the possibility to develop a new solution to the challenges posed by sleeping sickness diagnostics and allow for miniaturisation and automation of the process.

Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host, allowing a plethora of novel drug targets to be discovered and validated. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate produced by the enzyme CDP-DAG synthase (CDS), but nothing was known about CDS in T. brucei. Only one gene encodes CDS in Trypanosoma brucei (Tb927.7.220) and this was shown to encode a functional CDS by overexpression in E. coli and complementation of a yeast CDS null, which was created during this study. Expression and activity of TbCDS was confirmed in T. brucei, and was shown to be essential in both life cycle stages. Disruption of TbCDS altered the lipid profile of T. brucei, confirming a central role for CDP-DAG in phospholipid synthesis. Biochemical and morphological characterisation of mutants in TbCDS expression elucidated at least two separately localised and regulated pools of CDP-DAG and phosphatidylinositol in T. brucei. In bloodstream form these pools are localised to the Golgi and the ER, however in procyclics it is possible that both of these pools are localised to the Golgi, since no phosphatidylinositol synthase protein was detected in the ER of procyclics. Reduction in TbCDS was shown to affect cell cycle regulation and Golgi segregation possibly due to a depletion of phosphorylated phosphatidylinositols (PIPs). These studies also indicate that phosphatidylglycerol may be synthesised by the phosphatidylglycerol-phosphate synthase which may be capable of using phosphatidylserine as a substrate in a headgroup swapping reaction. TbCDS has now been genetically validated as a drug target, and has highlighted novel aspects of lipid biosynthesis in T. brucei. Collectively, these findings highlight the central role played by TbCDS and the new knowledge gained here may lead to the discovery and validation of other novel drug targets against African sleeping sickness.

Grâce à un modèle expérimental de la trypanosomose humaine Africaine (THA ou maladie du sommeil), le rat infecté par Trypanosoma brucei brucei, nous avons examiné l’implication du monoxyde d’azote (NO) dans le développement de cette pathologie. Des variations opposées de la concentration de ce composé ont été observées chez les animaux infectés, au niveau des compartiments périphérique et central : le NO diminue au niveau du sang et augmente au niveau cérébral. Ces changements sont dépendants de la NO-synthase inductible (iNOS). Au niveau périphérique, la diminution du NO qui survient favorise l’installation du parasite car la pression trypanocide de ce composé est diminuée. Dans cette situation, la L-arginine, le substrat à la base de la synthèse du NO, est utilisée pour la synthèse de polyamines, des composés nécessaires à la croissance du parasite. Ces mécanismes sont très probablement déclenchés par le trypanosome via ses facteurs solubles. Au niveau cérébral, la synthèse du NO est aussi soumise à des régulations qui impliquent l’arginase et la NG, NG-diméthylarginine diméthylaminohydrolase (DDAH). Tandis que l’activité de l’arginase demeure constante, celle de la DDAH augmente au cours de l’infection en accord avec les données des western-blot et des amino-acides. Cette augmentation, qui dépend essentiellement de l’isoforme DDAH-2, conduit à une augmentation du NO cérébral dont les propriétés sont trypanocides. Ces changements, contraires à ceux observés en périphérie, sont défavorables à la survie du trypanosome au niveau du cerveau. Ils pourraient constituer une protection supplémentaire contre l’entrée des trypanosomes dans cet organe / By way of an experimental model of human African trypanosomiasis (HAT or sleeping sickness), the rat infected by Trypanosoma brucei brucei, we examined the involvement of nitric oxide (NO) in the development of this pathology. In the infected animals, opposite variations in NO concentration were observedeither at peripheral or brain compartments: NO decreases in blood but increases in brain. These changes are dependent on the activity of the inducible NO-synthase (iNOS). In periphery, the decrease observed in NO concentration favors the parasite entrance because the trypanocidal pressure exerted by NO is decreased. In such a situation, L-arginine, the substrate conducing to the synthesis of NO, is employed for the synthesis of polyamines, a category of compounds necessary for the parasite growth. It is likely that above mechanisms might be triggered by parasites. In brain, NO synthesis is submitted to additive complex regulatory processes implying arginase and NG, NG-dimethylarginine dimethylaminohydrolase (DDAH). While the arginase activity remains constant, that of DDAH increases throughout the infection process in keeping with western-blot and amino acids data. This increase, depending mainly on DDAH-2 isoform, lasts in a brain NO increase which enhance the trypanocidal pressure. Above changes, opposite to those observed in periphery, are not favorable to the parasite survival in brain. They might constitute an additive protection against the parasite entry in this organ