No description available.

Active lipid metabolism is a key process required for the intra-erythrocytic development of the malaria parasite, Plasmodium falciparum. Enzymes that hydrolyze host-derived lipids play key roles in parasite growth, virulence, differentiation, cell-signaling and hemozoin formation. Therefore, investigating enzymes involved in lipid degradation could uncover novel drug targets. We have identified in P. falciparum, a glycerophosphodiester phosphodiesterase (PfGDPD), involved in the downstream pathway of phosphatidylcholine degradation. PfGDPD hydrolyzes deacylated phospholipids, glycerophosphodiesters to glycerol-3-phosphate and choline. In this study, we have characterized PfGDPD using bioinformatics, biochemical and genetic approaches. Knockout experiments showed a requirement for PfGDPD for parasite survival. Sequence analysis revealed PfGDPD possesses the unique GDPD insertion domain sharing a cluster of conserved residues present in other GDPD homologues. We generated yellow fluorescent fusion proteins that revealed a complex distribution of PfGDPD within the parasite cytosol, parasitophorous vacuole and food vacuole. To gain insight into the role of PfGDPD, sub-cellular localization was modulated and resulted in a shift in protein distribution, which elicited no growth phenotype. Kinetic analyses suggest PfGDPD activity is Mg₂⁺ dependent and catalytically efficient at the neutral pH environment of the parasitophorous vacuole. Next, our aim was to determine the upstream pathway that provides deacylated glycerophosphodiesters as substrate for PfGDPD. We identified via bioinformatics, a P. falciparum lysophospholipase (PfLPL1) that directly generates the substrate. Knockout clones were generated and genotyped by Southern and PCR analysis. The effects of PfLPL1 knockouts on parasite fitness were studied, and the results showed that PfLPL1was not required for parasite survival and proliferation. / Ph. D.

Malaria continues to be a global health burden that affects millions of people worldwide each year. Increasing demand for malaria control and eradication has led research to focus on sexual development of the malaria parasite. Sexual development is initiated when pre-destined intraerythrocytic ring stage parasites leave asexual reproduction and develop into gametocytes. A mosquito vector will ingest mature gametocytes during a blood meal. Sexual reproduction will occur in the midgut, leading to the production of sporozoites that will migrate to the salivary gland. The sporozoites will be injected to another human host during the next blood meal consequently, transmitting malaria. Due to decreased drug susceptibility of mature gametocytes, more investigation of the biology and metabolic requirements of malaria parasites during gametocytogenesis, as well as during the mosquito stages, are urgently needed to reveal novel targets for development of transmission-blocking agents. Furthermore, increasing drug resistance of the parasites to current antimalarials, including slowed clearance rates to artemisinin, requires the discovery of innovative drugs against asexual intraerythrocytic stages with novel mechanisms of action. Here, we have investigated the role of the apicoplast during Plasmodium falciparum gametocytogenesis. In addition, we describe drug-screening studies that have elucidated a novel mode of action of one compound from the Malaria Box, as well as identified new natural product compounds that may be serve as starting molecules for antimalarial development. / Ph. D.

Malaria is a parasitic infectious disease that results in millions of clinical cases per year and accounts for approximately 1 million deaths annually. Because the parasite has developed resistance to all current antimalarials, new therapies are urgently needed. Purine and pyrimidine biosynthesis for DNA and RNA synthesis has been recognized as a source of therapeutic targets. Targeted metabolite profiling has aided in the understanding of several biological processes in the parasite besides drug discovery. Therefore, having a robust analytical platform to quantify the purines and pyrimidines is of a great value. For this purpose an ion pair reversed phase ultra-performance liquid chromatography in tandem with mass spectrometry method was developed and validated. In addition, the apicoplast is an organelle present in the malaria parasite and other apicomplexan parasites. It was demonstrated that the apicoplast is essential for parasite's survival. The supply of isopentenyl diphosphate and dimethylallyl diphosphate for isoprenoid biosynthesis is the sole function of this organelle in the asexual intraerythrocytic stages. Isoprenoid precursors are synthesized through the methylerythritol phosphate (MEP) pathway in the malaria parasite while humans utilize the mevalonate pathway. Therefore, the MEP pathway is a source of drug targets for drug development. Our group has identified MMV008138 as anti-apicoplast inhibitor through phenotypic screening. Preliminary data suggest that the molecular target of MMV008138 may be within the MEP pathway. We used proteomic and metabolomic approaches to identify the molecular target of MMV008138 to aid future medicinal chemistry to improve the efficacy of this inhibitor. / Master of Science

No description available.

As part of an International Cooperative Biodiversity Group (ICBG) program and a collaborative research project with the Natural Products Discovery Institute, four plant extracts were investigated for their antiproliferative and antiplasmodial activities. With the guidance of bioassay guided fractionation, two known antiproliferative terpenoids (2.1 and 2.2) were isolated from Hypoestes sp. (Acanthaceae), four known antiplasmodial liminoids (3.1-3.4) were isolated from Carapa guianensis (Meliaceae), one inactive terpenoid (4.1) was isolated from Erica maesta (Ericaceae), and four cerebrosides (4.2-4.5) were obtained from Hohenbergia antillana (Bromeliaceae). The structures of these compounds were elucidated by using 1D (1H and 13C), 2D (HMBC, HSQC, COSY, NOESY) NMR spectroscopy and mass spectrometry. The structures of the compounds were also confirmed by comparing them with reported values from the literature. Compounds 2.1 and 2.2 showed moderate antiproliferative activity against the A2780 human ovarian cancer cell line with IC50 values of 6.9 uM and 3.4 uM, respectively. They also exhibited moderate antiplasmodial activity against chloroquine-resistant Plasmodium falciparum strain Dd2 with IC50 values of 9.9 ± 1.4 uM and 2.8 ± 0.7 uM, respectively. Compounds 3.1 to 3.4 had moderate antiplasmodial activity against Plasmodium falciparum Dd2 strain with IC50 values of 2.0 ± 0.3 uM, 2.1 ± 0.1 uM, 2.1 ± 0.2 uM and 2.8 ± 0.2 uM, respectively. Compounds 4.1 and 4.2 showed very weak antiplasmodial activity against Plasmodium falciparum Dd2 strain, with IC50 values between 5 and 10 ug/mL. / Master of Science / Cancer has a major impact all over the world and is one of the leading causes of death. Malaria remains as one of the most severe tropical diseases in the world. It is a common and often fatal disease caused by a parasitic infection. The treatment of cancer and malaria is a significant challenge, and has become a top priority in drug discovery field. The natural products from plants have been used for medicinal purpose for a long time, and a lot of well-known plant based natural product drugs have been discovered, including anticancer drug paclitaxel, and antimalarial drug chloroquine and artemisinin. However, the resistances for these drugs have developed, and it is urgent to find new drug that can take their place. This research is trying to find promising anticancer and antimalarial natural products from plant extracts. From four plant extracts, two antiproliferative compounds and four antiplasmodial compounds were discovered. In this thesis, the isolation and structure elucidation of these compounds will be discussed.

Replication-deficient adenovirus (Ad) and modified vaccinia virus Ankara (MVA) vectors expressing single Plasmodium falciparum antigens can induce potent T cell and antibody responses and have entered clinical testing using a heterologous prime-boost immunisation approach (Ad_MVA). This thesis describes a number of pre-clinical approaches aimed at enhancing the efficacy of these viral vectored vaccines targeting the blood-stage of malaria. First, the development of a highly efficacious malaria vaccine is likely to require a multi-antigen and/or multi-stage subunit vaccine. The utility of an Ad_MVA immunisation regime combining vaccines expressing the 42kDa C-terminus of the blood- stage antigen merozoite surface protein 1 (MSP142) and the pre-erythrocytic antigen circumsporozoite protein (CSP) in the P. yoelii mouse model was investigated. It was found that vaccine co- administration leads to maintained antibody responses and efficacy against blood-stage infection, but reduced secondary CD8+ T cell responses and efficacy against liver-stage infection. CD8+ T cell interference can be minimised by co-administering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy. The mechanisms of CD8+ T cell interference were explored. Second, Ad_MVA regimes expressing blood-stage antigens that can protect against P. chabaudi and P. yoelii blood-stage infection were tested against P. berghei, but did not confer protection. Similarly, IgG from rabbits immunised against P. falciparum MSP1 (PfMSP1) could not protect mice from a chimeric P. berghei parasite expressing PfMSP1. Third, two molecular adjuvants, the C4bp α-chain oligomerisation domain (IMX108/313) and the Fc fragment of murine IgG2a, were tested for their ability to enhance immunogenicity of recombinant adenoviruses when fused at the C-terminus of a blood-stage antigen. IMX108/313 was found to adjuvant T cell responses of small (< 80kDa) antigens and this was associated with antigen oligomerisation. However, the Fc fragment did not adjuvant responses. Finally, it was found that using a strong early promoter to drive antigen expression enhances the immunogenicity of single administration MVA vaccines, but that this did not enhance post-boost immunogenicity in an Ad_MVA regime.

Malaria caused by the protozan parasite Plasmodium is one of the most serious infectious diseases in the developing world. It is estimated that malaria causes an annual mortality rate of ~627,000. New drugs are urgently required, as the incidence of resistance is spreading rapidly. Glycosylphosphatidylinositol (GPI) anchored proteins decorate the merozoite surface and several of which, including merozoite surface proteins - 1 and -2 have previously been shown to be essential for erythrocyte invasion and parasite survival. Plasmodium GPI-anchors contain a glycan core consisting of four mannose residues. Therefore, the enzymes involved in the synthesis of activated mannose, guanidine diphosphomannose pyrophosphorylase (GDP-Man PP) and dolichol phosphate mannose synthase (DPMS), are thought to be crucial for GPI-anchor biosynthesis and as such potential drug targets. Double homologous recombination has been exploited to test whether PfGDP-Man PP and PfDPMS are essential during the erythrocytic portion of the parasite's life cycle. Additionally, overexpression parasite lines for both enzymes have been generated and have shown that the regulation of the two enzymes are intricately linked. Focused metabolomics by multi-reaction monitoring of the overexpression lines suggests that the fucosylation pathway may have a novel function within the parasite, possibly as a dynamic store for activated fucose/mannose. In order to determine the cellular concentration of key metabolites within the parasite, the volumes of the intra-erythrocytic stages have been determined and show that the concentration of metabolites in the ring stage parasites is substantially higher than previously thought. Furthermore, the sub-cellular localisation of GDP-Man PP and DPMS has been determined by immunofluorescence assay. The recombinant expression of DPMS in E. coli allowed its active site residues to be probed as well as establishing a platform for inhibitors to be screened against the enzyme. Finally, inhibitors of the T. brucei DPMS enzyme have been screened against the P. falciparum parasites in culture.

Le paludisme demeure une des maladies infectieuses les plus meurtrières au monde. Propagé par la piqûre d’un moustique femelle du genre Anopheles, le parasite du paludisme (Plasmodium) migre dans la circulation sanguine et infecte les cellules hépatiques de la victime. Dans le foie, le parasite se différencie et se reproduit par schizogonie à l’intérieur d’une vacuole parasitophore pour compléter la production de plusieurs milliers de mérozoïtes par cellule hépatique infectée. Ces mérozoïtes sont par la suite libérés dans la circulation sanguine où ils infectent les érythrocytes circulants dans lesquels le parasite subit des cycles d’infection, réplication et libération (processus de sortie actif provoqué par le parasite). Ces cycles répétitifs dans le sang sont à l’origine des symptômes cliniques de la maladie.Des études sur Plasmodium falciparum ont montré que P. falciparum SUB1 (PfSUB1), une protéase à sérine de la famille des subtilisines, est relâchée à l’intérieur de la vacuole parasitophore peu avant la libération des mérozoïtes des hématies. A l’intérieur de cette vacuole, la protéase intervient dans la maturation de protéines membres de la famille des SERA (famille de protéines du type papaïne) ainsi qu’un certain nombre de protéines de surface du mérozoïte (famille des MSP). Un grand intérêt a été porté sur cette protéase, car l’inhibition pharmacologique de l’activité de PfSUB1 bloque le processus de sortie et d’invasion des mérozoïtes dans le stade érythrocytaire du parasite in vitro.Le stade hépatique du parasite est une cible idéale pour le développement de traitements prophylactiques antipaludiques, car il précède la phase symptomatique de la maladie. En conséquence, il est important de mieux comprendre les mécanismes de fonctionnement du parasite à ce stade. Le présent projet avait pour but principal d’effectuer une étude du rôle de SUB1 dans le stade hépatique de Plasmodium. Pour ce faire, ce travail s’est effectué sur l’orthologue de PfSUB1 chez le parasite murin P. berghei. Dans un premier temps, l’expression de PbSUB1 dans les stades hépatiques du parasite a été confirmée en utilisant des anticorps spécifiques et en générant une lignée mutante de P. berghei exprimant la protéine endogène fusionnée à un marqueur hémagglutinine. Par la suite, l’enzyme a été exprimée sous forme recombinante et sa fonction et spécificité ont été partiellement caractérisées. Ce travail confirma que la protéase est capable de cliver des peptides basés sur les séquences de PbMSP1 et PbSERA3, substrats potentiels exprimés dans les stades hépatiques du parasite. Finalement, afin de mieux caractériser la fonction de PbSUB1, deux approches récentes permettant d’effectuer un knock-out conditionnel chez P. berghei ont été testées: le système Tet et la mutagenèse conditionnelle Flp/FRT. Afin d’utiliser cette dernière approche, une nouvelle méthode pour insérer des sites de reconnaissances Flp (sites FRT) dans les régions intergéniques de clones (dans ce cas, un clone comprenant pbsub1 et gènes voisins) provenant d’une bibliothèque génomique de P. berghei a été développée. Pour ce faire, plusieurs techniques d’ingénierie moléculaire ont été utilisées. Ces techniques, basées sur les systèmes de recombinaison de la levure (recombinase Flp) et de phages (recombineering) similaire à ceux utilisés par le projet PlasmoGEM (Pfander et al., 2012), surmontent les problèmes rencontrés par les méthodes conventionnelles pour le placement des sites FRT et sont aussi applicables aux longues séquences codantes. Avec ces nouveaux outils, un knock-out conditionnel de pbsub1 a été généré avec succès in vivo où la délétion du gène est accompagnée de l’expression d’un gène rapporteur (GFP) afin de faciliter l’identification des parasites ayant perdu le gène d’intérêt. A la fin de ce travail, une analyse préliminaire de ces parasites déficients en PbSUB1 suggère un rôle essentiel de cette protéase dans le développement de la phase hépatique du parasite. / Malaria remains one of the deadliest infectious diseases in the world. Propagated by the bite of an infected female Anopheles mosquito, the malaria parasite (Plasmodium) enters the bloodstream and infects hepatocytes. In the liver, the parasite differentiates and reproduces by schizogony within a membrane-bound parasitophorous vacuole (PV) resulting in the production of several thousands of merozoites per infected hepatic cell. These parasites are subsequently released into the blood stream where they infect circulating red blood cells and undergo repetitive cycles of infection, replication, and egress (active release of parasites) which are responsible for the clinical symptoms of the disease.Work on P. falciparum, has shown that P. falciparum SUB1 (PfSUB1), a serine protease of the subtilisin-like family, is discharged into the PV just prior to egress from the erythrocyte and mediates the proteolytic maturation of members of the SERA family (a family of papain-like proteins) as well as a number of merozoite surface proteins (MSPs). Pharmacological inhibition of PfSUB1 activity inhibits both egress and invasion of released merozoites in blood stages in vitro.The liver stage of the parasite is an ideal target for development of prophylactic anti-malarial drugs, as it is clinically silent. It is thus of importance to gain more detailed knowledge about parasite development in this stage. The main aim of this project was to study the role of SUB1 in the liver stage of the parasite life cycle. The work was performed on the orthologue of PfSUB1 in the murine malaria species P. berghei. Initially, expression of PbSUB1 in liver stages was confirmed using specific antibodies and by generating a transgenic P. berghei clone expressing epitope-tagged PbSUB1. Next, recombinant enzymatically-active PbSUB1 was expressed in insect cells and partially characterised with respect to its function and substrate specificity. This confirmed that the protease is able to process substrates based on PbMSP1 and PbSERA3, two putative substrates expressed in late hepatic stages of the parasite.Finally, to further study PbSUB1 function, two conditional gene knock-out approaches were applied to study the phenotypic consequences of loss of PbSUB1 expression. Working from a ~10 kb genomic DNA library clone comprising pbsub1 and flanking genes, a method to insert Flp recombinase recognition (FRT) sites into intergenic regions was developed. This was achieved by combining inducible Flp activation in E. coli with recombinase mediated engineering techniques similar to those that underlie the PlasmoGEM project (Pfander et al., 2012). This strategy overcomes challenges of existing techniques and is also suitable for flanking large genes with FRT sites. With these newly generated tools, an inducible knock-out of pbsub1 was successfully generated in vivo, in which stage-specific excision of the gene was accompanied by concomitant induction of GFP expression, facilitating identification of the knock-out parasites. A preliminary analysis of these PbSUB1-deficient parasites suggests an essential role for the protease in the development of liver stage schizonts.

L'objectif général de cette thèse était d'étudier le rôle des anticorps dans la susceptibilité ou la résistance au paludisme chez de jeunes enfants béninois exposés naturellement aux infections palustres durant leurs deux premières années de vie. Deux projets complémentaires (PALNOUGENENV et TOLIMMUNPAL) ont été mises en place au Bénin et consistaient à suivre des mères et leurs enfants. Dans le cadre du projet PALNOUGENENV, l'étude incluait 600 mères à l'accouchement et a suivi leurs enfants pendant les dix-huit premiers mois de vie dans le but d’étudier les conséquences de l’infection placentaire palustre chez les mères sur la survenue des premières infections palustres chez les nouveau-nés. Suite au projet PALNOUGENENV, il semblait important de connaitre le statut palustre de la mère pendant la grossesse et pas seulement à l’accouchement. Le projet TOLIMMUNPAL a donc été mis en place et incluait 400 mères et leurs enfants. Les mères ont été inclues à la première consultation prénatale et suivies au niveau parasitologique et clinique jusqu'à l'accouchement et leurs enfants ont été suivis de la naissance jusqu'à 24 mois dans le but d'étudier des déterminants environnementaux, biologiques et génétiques impliqués dans le développement de la tolérance immunitaire associée au paludisme et ses conséquences sur la protection de la femme enceinte et du jeune enfant. Plus spécifiquement, nous avons étudié chez ces enfants les relations entre l'infection palustre et les niveaux d'anticorps spécifiquement dirigés contre les antigènes candidats vaccins les plus avancés du stade érythrocytaire d'une part et la capacité des anticorps à inhiber le développement in vitro de P. falciparum d'autre part. Nos résultats mettent en évidence une association dans la cohorte PALNOUGENENV entre les niveaux élevés des sous-classes cytophiles IgG1 dirigées contre les antigènes candidats vaccins MSP1 (p<0,001, OR=0,90) et IgG3 anti-MSP2 (p<0,001, OR=0,89) et la protection contre l'infection palustre. Dans la cohorte TOLIMMUNPAL, les hauts niveaux d'IgG2 anti-GLURP R2 (p=0.05, OR=2.10) ont plutôt été associés au risque d'infection palustre. L'analyse fonctionnelle des IgG en utilisant le test GIA a révélé que l'infection par P. falciparum au moment du prélèvement affectait la capacité des IgG à inhiber la croissance parasitaire in vitro. Les IgG purifiées à partir d'échantillons collectés chez des individus infectés par P. falciparum avaient une capacité moyenne d'inhibition de la croissance parasitaire inférieure (p=0.003, Wilcoxon matched pairs test) de 19 % à celles purifiées à partir de plasmas d'enfants non infectés. Une corrélation inverse a été observée entre l'âge et la capacité des anticorps à inhiber l'invasion des globules rouges par le parasite. Aucune association entre les niveaux d'anticorps et leur capacité à inhiber le développement du parasite in vitro n'a été mis en évidence. Dans le cadre du développement du test Antibody-Dependent Respiratory Burst (ADRB), les cellules promyélocytaires de la lignée PLB 985W ont été utilisées. Ces cellules ont la capacité de se différencier en neutrophiles sous l'action du DMSO. Nos résultats ont montré que cette lignée a une faible capacité à produire les espèces réactives d'oxygène (ROS) comparés aux neutrophiles humains et les niveaux de ROS produits par cette lignée cellulaire sont insuffisants pour être utilisés dans le test ADRB. Nos résultats confortent le rôle important des antigènes candidats vaccins MSP1 et MSP2 dans la protection contre le paludisme. Cette relation est essentiellement établie au regard des quantités d’anticorps spécifiques produits, l’étude de fonctionnalité des anticorps employant le test GIA n’ayant pas permis de mettre en évidence de relation claire à la protection. (...) / The aim of this thesis was to study the role of antibodies in susceptibility or resistance to malaria in young Beninese children naturally exposed to malaria infections during their first two years of life. Two complementary projects (PALNOUGENENV and TOLIMMUNPAL) were implemented in Benin to identify individual factors of malaria susceptibility. The PALNOUGENENV cohort included 600 mothers at delivery and their children from birth to 18 months of age in order to study the effects of placental malaria infection in mothers on first occurrence of malaria infections in newborns. In the TOLIMMUNPAL cohort 400 mothers were included at the first antenatal visit and followed until delivery while their infants were followed from birth to 24 months in order to study environmental determinants, biological and genetic involved in the development of immune tolerance associated with malaria and its impact on the protection of pregnant women and infants. Specifically, the association between malaria infection and the level of antibodies specifically directed against the most advanced vaccine candidate antigens of the erythrocyte stage on one hand and the ability of antibodies to inhibit in vitro the development of P. falciparum on the other hand were investigated. Our results show an association in the cohort PALNOUGENENV between high levels of IgG1 to MSP1 19 vaccine candidate antigens (p <0.001, OR = 0.90) and IgG3 to MSP2 3D7 (p <0.001, OR = 0.89) and protection against malaria infection. In the TOLIMMUNPAL cohort, high levels of IgG2 to GLURP R2 (p = 0.05, OR = 2.10) were instead associated with an increased risk of malaria infection. Functional analysis of IgG using the GIA test revealed that the infection with P. falciparum at the sampling time affected the ability of IgG to inhibit parasite growth in vitro. The purified IgG from individuals infected with P. falciparum when the sample was collected had an average 19% lower capacity of inhibition of parasite growth (p = 0.003, Wilcoxon matched pairs test) than those that were uninfected at the time of sampling. An inverse correlation was found between age and the ability of antibodies to inhibit in vitro invasion of red blood cells by the parasite. There was no association between antibody levels and ability to inhibit the in vitro parasite development. In the development of the Antibody-Dependent Respiratory Burst (ADRB) assay, the promyelocytic cell line PLB 985W was used. These cells have the capacity to differentiate into neutrophils after exposure to DMSO. Our results showed a low ability of this cell line to produce reactive oxygen species (ROS) compared to human neutrophils and ROS levels produced by this cell line are insufficient to be used in the test ADRB. Our results confirm the important role of MSP1 19 and MSP2 3D7 vaccine candidate antigens in malaria protection. Although the levels of antibodies to MSP1 19 and MSP2 3D7 were associated with decrease risk of P. falciparum infection, the functional study of antibody using the GIA assay did not allow demonstrating the relationship to protection. Investigation on the functional role of antibodies is complex as IgG could act through multiple direct or indirect mechanisms. We will continue to investigate the functional role of antibody, particularly in plasma samples from our two birth cohorts by using the ADRB assay. Results will aid in providing new information to the existing knowledge gap and will help in malaria vaccine development.