Dissertations / Theses on the topic 'Plasmodium falciparum'

Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is responsible for 300 to 600 million clinical cases annually (Snow et al., 2005), resulting in the deaths of up to three million people every year (Breman, 2001, Breman et al., 2004). There is a clear need for further research aimed at identifying novel drug targets (Ridley, 2002). Reversible phosphorylation of proteins is a major regulatory mechanism in most cellular processes, and protein kinases are considered promising drug targets, comprising as much as 30% of all protein targets under investigation (Cohen, 2002). The divergences between human and plasmodial protein kinases suggest that specific inhibition of the latter is an achievable goal (Doerig, 2004, Doerig and Meijer, 2007). This study investigates protein kinase CK2 of Plasmodium falciparum, seeking to establish by reverse genetics and biochemical approaches whether it represents a possible antimalarial drug target. Protein-kinase CK2, formerly known as Casein Kinase II, is a dual-specificity (Serine/Threonine and Tyrosine) protein kinase ubiquitously expressed in eukaryotes. It has over 300 cellular substrates catalogued to date (Meggio and Pinna, 2003). Consistent with its multiple substrates, the enzyme plays a crucial role in many cellular processes, and is essential to viability in yeast and slime mould (Padmanabha et al., 1990, Kikkawa et al., 1992). The human CK2 holoenzyme consists of two catalytic a or a’ subunits and two regulatory b subunits, and recent evidence indicates that the latter interact with several protein kinases in addition to CK2a (reviewed in (Bibby and Litchfield, 2005)), pointing to a likely role in the integration of numerous signalling pathways. A putative CK2a orthologue and two predicted CK2b subunits were identified in the P. falciparum genome (Ward et al., 2004, Anamika et al., 2005). Here we present the biochemical characterisation of the PfCK2a orthologue and both PfCK2b orthologues, and demonstrate by using a reverse genetics approach that each of the three subunits is essential for completion of the erythrocytic asexual cycle of the parasite, thereby validating the enzyme as a possible drug target. Recombinant PfCK2a possesses protein kinase activity, exhibits similar substrate and co-substrate preferences to those of CK2a subunits from other organisms, and interacts with both of the PfCK2b subunits in vitro. PfCK2a is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2a enzymes to a small molecule inhibitor. Taken together, the data indicate that PfCK2a is an attractive, validated target for antimalarial chemotherapeutic intervention.

Apicomplexan parasites of the genus Plasmodium are the causative agent of malaria, one of the most prevalent infectious diseases worldwide. Five different Plasmodium species can cause malaria in humans, leading to a total of approximately 500 million cases each year and of these, P. falciparum causes the most deadly form of the disease and is responsible for more than 1 million deaths annually. A major problem in the global fight against malaria is the widespread resistance of the parasites against the currently available drugs. It is of great importance to identify new drug target as well as to understand the mechanisms that lead to drug resistance in the first instance in order to potentially reverse the resistant phenotypes and to avoid the development of resistance in the future. The tripeptide glutathione (GSH) or γ-glutamylcysteinyl-glycine is the most abundant low molecular weight thiol in most eukaryotic organisms and serves a number of important functions as sulfhydryl-buffer, cofactor for enzymes and for the detoxification of xenobiotics and drugs. GSH is an important component of the antioxidant machinery and because malaria parasites live in an environment rich in iron and oxygen and thus increased oxidative stress, they depend on functional antioxidant systems. The biosynthesis pathway for GSH, consisting of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GS) is present in malaria parasites as well as in their host cells. Previous studies have shown that depletion of GSH has an antimalarial effect, but it remained unclear whether parasites were killed directly or died because their host cell could not survive the depletion of GSH. To address this question, the knockout of both genes encoding the enzymes of the GSH biosynthesis pathway in P. falciparum was attempted. While both gene loci were targeted by control constructs, the knockout of either pfγgcs or pfgs was impossible, indicating both genes are essential for parasite survival in the erythrocytic stages. To analyse the localization of γGCS and GS, GFP-tagged recombinant fusion proteins were expressed in the parasites and showed that GSH biosynthesis is cytosolic. Apart form its other functions GSH has previously been suggested to be involved in resistance to the antimalarial drug chloroquine (CQ). CQ was for a long time the first line antimalarial drug due to its high efficiency, low cost and low toxicity, but is now widely inefficient in the treatment of the disease. CQ resistance is associated with mutations in the CQ resistance transporter (PfCRT), a membrane protein of the digestive vacuole that allows the efflux of the drug form its site of action. However, PfCRT mutations alone cannot explain the full array of phenotypes found in resistant parasites. GSH is able to degrade heme, the target of CQ, in vitro and it has been suggested that elevated GSH levels contribute to CQ resistance. However, analyses of isogenic parasite lines bearing different forms of PfCRT in this study revealed lower GSH levels and higher susceptibility to inhibition of GSH biosynthesis in the CQ resistant lines. These changes did not correlate with changes in the expression of enzymes involved in the de novo biosynthesis or consumption of GSH. However, the cellular accumulation ratio for CQ indicated a decrease of free heme in the resistant parasites. Mutant forms of PfCRT expressed in oocytes of Xenopus laevis were able to transport GSH, while the sensitive wild-type form did not transport the tripeptide. The findings of this study suggest that in parasites bearing mutant PfCRT, GSH is transported into the digestive vacuole where it is able to contribute to resistance by degrading heme, before the tripeptide itself is degraded by peptidases inside the vacuole, consistent with the overall reduction of GSH levels in CQ resistant parasites.

Thesis (Ph.D.) -- University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Metabolite transport pathways of the malaria parasite, Plasmodium falciparum, are an important area for study in order to further the understanding of the parasite's biology. Identification and characterisation of the transporters involved in these pathways may also provide potential novel drug targets or drug delivery mechanisms. This is especially valuable as chemotherapy remains one of the main management strategies in the fight against malaria and the usefulness 0 f the current range 0 f antimalarial drugs is seriously threatened by the emergence and spread of resistance . .' In this thesis the Xenopus laevis oocyte heterologous expression system was used to functionally characterise a gene-specific cDNA library of 48 putative membrane proteins and the previously annotated putative amino acid transporter PFF1430c for the uptake of several amino acids. This screening failed to identify any definite amino acid transport by the cDNA library or PFF1430c, however this could have been due to the fact that uptake of a relatively narrow range of amino acids was tested and these were used at concentrations lower than found physiologically. Inherent issues with the X laevis expression system may also have been an issue, including the expression of endogenous transporters for the substrates being investigated.

Plasmodium falciparum var genes encode the PfEMP1 family of variant antigens expressed on the surface of infected erythrocytes. PfEMP1 mediates the adhesion of the parasitized erythrocyte to the venular endothelium and to uninfected erythrocytes. PfEMP1 variants use a range of different host molecules as receptors in these adhesive interactions. The expression of different PfEMP1 variants may directly affect the clinical course of malaria infection by defining the distribution and intensity of parasite adhesion in the microvasculature of various organs within the host. PfEMP1 is a major focus of the host immune response, and the slow onset of clinical immunity in endemic areas may be explained by the gradual accumulation of effective responses to a wide range of PfEMP1 variants present in the local parasite population. It has been hypothesised that the immune response to PfEMP1 may act to stratify the parasite population into co-circulating 'strains' defined by discrete, non-overlapping repertoires of var genes. Here, the DBL1 region of 56 var gene variants from 6 genetically distinct co-circulating Sudanese parasites have been cloned and sequenced. Sequence comparisons suggest that recombination and gene duplication are important mechanisms in the generation of new var variants. A model of the basic structural framework of DBL1 sequences is described and "sequence subtypes" identified within variable regions of sequence. Phylogenetic analysis of the Sudanese and other var sequences from GenBank fail to support the 'strain' model for Sudanese P. falciparum and suggests that the global pool of var genes is finite. 40 Sudanese variants have been expressed as GST-fusion proteins with yields of varying quantity and degree of degradation. 10 of the recombinant proteins have been tested against a cohort of sera in a pilot ELISA, and the role of anti-PfEMP1 immune responses in the development of clinical immunity is discussed.

Persistence of drug resistant Plasmodium falciparum is a major problem to management and control malaria in endemic areas. The focus of this thesis was to study the dynamics of resistant P. falciparum parasites. The study was performed in two African countries: 1) Sudan: Asar village in eastern Sudan, here we examined the persistence of drug sensitive and resistant P. falciparum genotypes among individuals with single-clone and multiple clones infection during the dry season. We genotyped microsatellite loci in the vicinity of the dihydrofolate reductase gene (dhfr) and the dihydropteroate synthase gene (dhps). Microsatellite investigation showed that asymptomatic parasitemia persisted in some patients for several months throughout the dry season and into the next transmission season. In some samples mixed infections were detected, and we noted several cases where the microsatellite haplotype varied from month to month, suggesting turnover of different parasite populations in the blood. This demonstrates that even during asymptomatic infections there can be dynamics within the parasite population in an individual. In addition, we calculated the parasite density throughout the dry season to the next transmission season by using allele-specific quantitative PCR. Parasite density during the dry season fluctuated, but was generally lower than in the first transmission season. A significant difference (P<0.05) between dry and first transmission season was found in regard to the parasite density, whereas no significant difference was observed when dry and second transmission season were compared (P>0.05). 2) Ethiopia: West Arsi zone, one of the malaria endemic zones of the Oromia region. In the first study we determined the prevalence of asymptomatic malaria carriages from November-December 2012. According to PCR the prevalence of sub-microscopic P. falciparum carriage was 19.2%, microscopy-based prevalence was 3.7% while the prevalence was 6.9% using RDT. Based on this, PCR was considered a better tool for measuring Plasmodium prevalence than microscopy and RDT. A second study addressed the genetic diversity of chloroquine resistance (CQR) in P. falciparum by analysing four microsatellite markers in and around the pfcrt gene. Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the Pfcrt K76T mutation. Only the CVIET haplotype was identified. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Both intronic and MS flanking the pfcrt gene showed low levels of diversity.

Plasmodium falciparum var genes encode the PfEMPI family of variant antigens expressed on the surface of infected erythrocytes. PfEMPlmediates the adhesion of the parasitized erythrocyte to the venular endothelium and to uninfected erythrocytes. PfEMP 1 variants use a range of different host molecules as receptors in these adhesive interactions. The expression of different PfEMPI variants may directly affect the clinical course of malaria infection by defining the distribution and intensity of parasite adhesion in the microvasculature of various organs within the host. PfEMPI is a major focus of the host immune response, and the slow onset of clinical immunity in endemic areas may be explained by the gradual accumulation of effective responses to a wide range of PfEMPI variants present in the local parasite population. It has been hypothesised that the immune response to PfEMPI may act to stratify the parasite population into co-circulating 'strains' defined by discrete, non-overlapping repertoires of var genes. Here, the DBL1 region of 56 var gene variants from 6 genetically distinct cocirculating Sudanese parasites have been cloned and sequenced. Sequence comparisons suggest that recombination and gene duplication are important mechanisms in the generation of new var variants. A model of the basic structural framework of DBL1 sequences is described and "sequence subtypes" identified within variable regions of sequence. Phylogenetic analysis of the Sudanese and other var sequences from GenBank fail to support the 'strain' model for Sudanese P. falciparum and suggests that the global pool of var genes is finite. 40 Sudanese variants have been expressed as GST-fusion proteins with yields of varying quantity and degree of degradation. 10 of the recombinant proteins have been tested against a cohort of sera in a pilot ELISA, and the role of anti-PfEMPI immune responses in the development of clinical immunity is discussed.

Le parasite Plasmodium falciparum, l’agent responsable du paludisme, a un cycle de vie complexe et emploie des stratégies de multiplication atypiques, adaptées à l'expansion rapide de la population pendant la colonisation de l'hôte et la transmission, qui ne sont pas entièrement comprises. Afin de comprendre l'initiation de la réplication de l'ADN de ce pathogène humain, mon projet s'est concentré sur deux aspects principaux de ce processus : l'identification et la caractérisation des origines de la réplication et de la machinerie de réplication.Dans la première partie de ce projet, j'ai combiné trois approches différentes pour cartographier les origines de réplication chez P. falciparum : ChIP-seq, le séquençage des brins naissants d'ADN (SNS-Seq) et la méthode NanoForkSpeed (NFS). J'ai d'abord réalisé les expériences ChIP-seq sur deux sous-unités du complexe de reconnaissance des origines de réplication (PfORC1 et PfORC2) au début de la schizogonie afin d'obtenir une cartographie de tous les sites d'initiation potentiels. Ensuite, j'ai identifié les sites de réplication actifs en utilisant deux stratégies : SNS-seq et la cartographie de l'incorporation de l'analogue de la thymidine BrdU dans l'ADN en réplication, en utilisant le séquençage nanopore et l'algorithme NFS. En combinant les données issues de ces différentes méthodes, j'ai obtenu un ensemble robuste d'origines de réplication qui présentent des caractéristiques similaires à celles des origines des mammifères, comme la distribution non aléatoire dans des zones d'initiation ou ‘clusters’ et l'association avec des séquences formant des G-quadruplex. Les origines de réplication montrent également des caractéristiques uniques. Elles sont associées à des gènes fortement transcrits, mais dépourvus de sites de début de transcription (TSS). En outre, les résultats montrent que la vitesse de la fourche de réplication est uniforme sur l'ensemble du génome, à l’exception d’une diminution significative au niveau des centromères et des télomères. Des informations obtenues sur molécules uniques, utilisant des lectures contenant de multiples événements d'initiation qui n'auraient pu provenir que de cellules individuelles, ont révélé une relation entre la vitesse à laquelle les fourches de réplication se déplacent et la distance par rapport à l'origine la plus proche. Cette approche à multiples facettes a fourni la première analyse complète du paysage génétique des origines de réplication chez P. falciparum.La deuxième partie de mon projet s'est concentrée sur la caractérisation du complexe réplicatif de P. falciparum en isolant des protéines sur l'ADN naissant, qui font potentiellement partie de la machinerie du réplisome. Dans l'ensemble, ce travail contribue aux études en plein essor, sur la réplication de l’ADN des parasites Plasmodium falciparum, dans l'hôte humain, et fournira une base solide pour les futures recherches dans ce domaine
Plasmodium falciparum parasites have a complex life cycle and employ atypical multiplication strategies, tailored for fast population expansion during host colonisation and transmission, that are not fully understood. In an effort to unravel the initiation of DNA replication of these human pathogens my project focused on two main aspects of this process: (i) identifying and characterising the origins of replication and (ii) identifying and characterising the replication machinery.Regarding the first part, I combined three different approaches to map the origins of replication in P. falciparum. These were ChIP-seq, SNS-Seq and NFS. I first performed ChIP-seq on two subunits of the origin recognition complex (of PfORC1 and PfORC2) at the beginning of schizogony to obtain a cartography of all potential replication initiation sites. Next, I mapped sites of active replication using two strategies: sequencing DNA nascent strands (SNS-seq); and mapping the incorporation of the thymidine analogue BrdU into replicating DNA, using nanopore sequencing combined with NanoForkSpeed (NFS). By combining data from these different methods, I have obtained a robust set of origins of replication that display some characteristics similar to those of mammalian origins, such as the non-random distribution in initiation zones or clusters and the association with G-quadruplex forming sequences. Strikingly, they also display unique characteristics, since they are associated with highly transcribed genes but depleted from transcription start sites (TSS). Additionally, the results showed a uniform fork speed across the genome with a significant decrease in centromeres and telomeres. Single molecule information, using reads containing multiple initiation events which could have only come from individual cells, revealed a relationship between the pace at which replication forks travel and the distance to the nearest origin. This multifaceted approach provided the first comprehensive analysis of the genetic landscape of the origins of replication in P. falciparum.The second part of my project focused on the characterization of the replicative complex of P. falciparum by isolating proteins bound to nascent DNA at active replication forks.Overall, this work contributes to the growing field of studying how Plasmodium falciparum parasites replicate their genome within the human host, and I am confident that it will serve as a solid foundation for further investigations

La pompe ATPase Ca2+ du réticulum sarco-endoplasmique Plasmodium falciparum (PfATP6) est une protéine de dix transmembranes, impliqué dans la régulation de l'homéostasie du calcium dans le parasite. L'importance d'étudier cette protéine repose sur l'hypothèse d'être engagé dans le mécanisme d'action et de résistance des artémisinines. Des travaux précédents, fondé sur l'expression hétérologue dans des ovocytes de Xenopus laevis et Saccharomyces cerevisiae, ont montré des résultats opposés, générant de nombreux corollaires vérifiables. Par conséquent, des travaux supplémentaires sont nécessaires pour mieux comprendre la nature des interactions entre les artémisinines et transporteurs de type SERCA.Afin d'évaluer le caractère essentiel du gène de Plasmodium spp., une approche de génétique inverse a été utilisée. Knockout du gène, soit P. falciparum et berghei, ne pouvant pas être obtenu. La complémentation de sauvetage épisomique a été jugée impossible. Marquage à la fin 3' de PfATP6 et PbATP6 a été, également, tenté pour étudier la localisation et l'expression de la protéine chez les parasites. La manipulation des gènes à cette place n'a pas permis la survie du parasite. Nos résultats, pris ensemble, montrent que ATP6 est essentiel dans Plasmodium spp..Au cours de nos études génétiques, un phénotype stable et particulier de parasites du genre Plasmodium falciparum 3D7 a été distingué. Les étranges parasites “monstres" contiennent une vacuole digestive inhabituelle gonflées à travers toutes les étapes du développement du parasite. Caractérisation de l'insolite Plasmodium a été réalisée, montrant une sensibilité accrue à la chloroquine, mais pas à l'artémisinine ou de la méfloquine. Tenant compte de la similitude du PfATP6 avec la pompe SERCA orthologue mammifère, de nouvelles molécules, connu et synthétisé pour cibler spécifiquement la protéine chez les mammifères, ont été testés sur P. falciparum. Quatre classes différentes de composés (sHA 14-1, BHQ, chalcone et des analogues de l'ACP) a montré le blocage de la croissance in vitro du P. falciparum 3D7 et Dd2 à des concentrations inférieure au range micromolaire. En outre, une nouvelle classe de molécules (thaperoxides), conçu comme un hybride entre l'artémisinine et thapsigargine, a été testé contre le type sauvage 7G8 et la ligne muté L263E. Ce dernier porte une mutation ponctuelle unique de nucléotides dans PfATP6, déjà connu d'être impliqué dans la résistance du l'artémisinine.Compte tenu de la difficulté à manipuler les gènes du parasite, et afin de mieux caractériser PfATP6, un gène synthétique a été optimisé pour l'expression hétérologue chez S. cerevisiae. De cette façon, la complémentation d'une ligne de levure mutée (K616) sans les pompes endogènes Ca2+ de type P a été permis avec succès, montrant le sauvetage de la croissance de la levure en présence de forte concentration de calcium libre. Différents inhibiteurs de SERCA, comme la thapsigargine et l'acide cyclopiazonique, ont été testés sur la levure complémenté K616 PfATP6, afin de vérifier l'inhibition de la croissance. Tous les composés ont bloqué la croissance de levure sélectivement ciblant le PfATP6. En outre, le test a été développé comme un criblage de haute performance, afin de tester de nouvelles molécules pour leur activité. La méthode s'est révélée être un outil rapide et très fiable et reproductible pour l'identification de nouveaux composés actifs
The Plasmodium falciparum sarco-endoplasmic reticulum ATPase Ca2+ pump (PfATP6) is a ten transmembrane protein involved in the regulation of the calcium homeostasis in the parasite. The importance of studying this protein relies on the fact that it has been hypothesized to be involved in the mechanism of action and resistance of artemisinins. Previous works, based on heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae, have shown contrasting results, generating many testable corollaries. Therefore, further work is needed to better understand the nature of interactions between artemisinins and SERCA-type transporters.In order to assess the essentiality of the gene in Plasmodium spp., a reverse genetics approach has been used. Knockout of the gene, in either P. falciparum and berghei, could not be achieved. Complementation for episomal rescue was found to be not possible. Tagging at the 3' end of PfATP6 and PbATP6 has been, also, attempted to study localization and expression of the protein in parasites. Manipulation of the gene at this position did not permit parasite survival. Our results, taken together, show that ATP6 is essential in Plasmodium spp..During our genetic studies, a stable and peculiar phenotype of Plasmodium falciparum 3D7 parasites has been noticed. The odd “monster” parasites contain an unusual swollen food vacuole throughout all stages of parasite development. Characterization of the unusual Plasmodium has been carried out, showing an increased sensitivity to chloroquine, but not to artemisinin or mefloquine. Taking into account the similarity of PfATP6 with the mammalian orthologue SERCA pump, new molecules, designed and synthesized to specifically target the mammalian protein, were tested on P. falciparum parasites. Four different classes of compounds (sHA 14-1, BHQ, chalcone and CPA analogues) showed to inhibit P. falciparum 3D7 and Dd2 growth in vitro at concentrations in the lower micromolar range. In addition, a novel class of molecules (thaperoxides), designed as an hybrid between artemisinin and thapsigargin, has been tested against 7G8 wild type and mutated L263E line. The latter carries a single nucleotide point mutation in PfATP6 that has been previously shown to be involved in artemisinin resistance. Considering the difficulty in manipulating the gene in the parasite and in order to better characterize PfATP6, a synthetic gene was optimized for heterologous expression in S. cerevisiae. This enabled successful complementation of a mutated yeast line (K616) lacking the endogenous P-type Ca2+ pumps, showing rescue of the yeast growth in presence of high concentration of free calcium. Different SERCA inhibitors, such as thapsigargin and cyclopiazonic acid, have been tested on K616 PfATP6 complemented yeast, in order to check for growth inhibition. All compounds showed to inhibit yeast growth selectively targeting PfATP6. In addition, the assay has been developed as a high throughput screening, in order to test new molecules for their activity. The method has proved to be a fast, highly reliable and reproducible tool for identification of new active compounds

Background: In P. falciparum a similarity between the transit peptides of apicoplast and mitochondrial proteins in the context of net positive charge has previously been observed in few proteins. Existing P. falciparum protein localization prediction tools were leveraged in this study to study this similarity in larger sets of these proteins.

Results: The online public-domain malarial repository PlasmoDB was utilized as the source of apicoplast and mitochondrial protein sequences for the similarity study of the two types of transit peptides. It was found that

many of the 551 apicoplast-targeted proteins (NEAT proteins) of PlasmoDB may have been wrongly annotated to localize to the apicoplast, as some of these proteins lacked annotations for signal peptides, while others also had annotations for localization to the mitochondrion (NEMT proteins). Also around 50 NEAT proteins could contain signal anchors instead of signal peptides in their N-termini, something that could have an impact on the current theory that explains localization to the apicoplast [1].

The P. falciparum localization prediction tools were then used to study the similarity in net positive charge between the transit peptides of NEAT and NEMT proteins. It was found that NEAT protein prediction tools like PlasmoAP and PATS could be made to recognize NEMT proteins as NEAT proteins, while the NEMT predicting tool PlasMit could be made to recognize a significant number of NEAT proteins as NEMT. Based on these results a conjecture was proposed that a single technique may be sufficient to predict both apicoplast and mitochondrial transit peptides. An implementation in PERL called TargetPf was implemented to test this conjecture (using PlasmoAP rules), and it reported a total of 408 NEAT

proteins and 1504 NEMT proteins. This number of predicted NEMT proteins (1504) was significantly higher than the annotated 258 NEMT proteins of plasmoDB, but more in line with the 1200 predictions of the tool PlasMit.

Conclusions: Some possible ambiguities in the PlasmoDB annotations related to NEAT protein localization were identified in this study. It was also found that existing P. falciparum localization prediction tools can be made to detect transit peptides for which they have not been trained or built for.

The stevor family is the third largest multigene family in the P. falciparum 3D7 genome (Gardner et al., 2002). With 30 copies estimated per genome, each encoding a predicted transmembrane spanning protein: a conserved N-terminal domain is followed by hydrophobic regions, a 100 amino acid 'hyper-variable' region (HVR), conserved transmembrane domain and short highly conserved cytoplasmic C-terminus. In 3D7, stevor is transcribed in trophozoites and STEVOR protein was found in cytoplasmic membranous structures known as Maurer's clefts (Kaviratne et al., 2002). The Maurer's clefts are implicated in trafficking of proteins to the infected red blood cell (iRBC) surface, and pre-assembly of proteins destined for knob structures on the surface (Craig and Scherf, 2001). Stevors have also been detected in gametocyte stages and a STEVOR peptide found in sporozoite extracts (Sutherland, 2002 and Florens et al, 2002). The HVR of STEVOR may play a role in immune evasion. To investigate this further, we have started to characterise the genomic repertoire and expression profile of stevor genes in laboratory lines and field parasite isolates from Kenya, East Africa. Using stevor specific primers, we have identified 152 stevors in P. falciparum laboratory lines and Kenyan isolates. In addition, 27 stevors were identified in the genome of a Ghanaian field isolate (www.plasmodb.org) and 17 in a second laboratory isolate IT. Western blots identified a protein of approximately 30 kDa expected STEVOR mean molecular mass is 36.75kDa. Immunofluorescence assays showed that in mature, segmented schizonts, STEVOR is redistributed from the Maurer's clefts to throughout the iRBC cytosol. Interestingly, STEVOR was also observed in the apex of merozoites. An extremely important observation for the application of in vitro cultured P. falciparum clones is the huge difference in STEVOR expression to that in the field parasite isolates. STEVOR protein is expressed in only 5% of P. falciparum 3D7 schizonts, whereas it is found in 50% of Kilifi field isolate schizonts. We investigated field isolates further to see whether STEVOR is on the iRBC surface together with other multigene families, such as the var encoded PfEMPl and RIFINS, where STEVOR could play a role in antigenic variation which enables the P. falciparum parasite to evade the host immune response. Alternatively, STEVOR may have ligand binding functions/ RBC adherence properties during final schizont stage, rupture and merozoite reinvasion.

Plasmodiumfalciparum malaria poses one of the most important disease problems in the world. Despite decades of effort to improve disease outcome, the emergence and rapid dissemination of multi-drug resistant parasites has led to a disturbing increase in malaria mortality and morbidity. A critical limitation in managing multi-drug resistant falciparum malaria has been the incomplete understanding of both the underlying molecular mechanisms of drug resistance and the mode of action of widely used drugs. This study aimed to characterise the molecular mechanisms underlying multi- drug resistant malaria by studying the role of gene amplification in the P. falciparum multi-drug resistance gene 1 (pfmdrl) in determining parasite response to a variety of antimalarials in vitro and in vivo. In addition, P. falciparum ATPase 6 (PfATP6), a putative drug target of the widely used artemisinins, was also examined for possible drug-modulating mutations. First a real-time peR technique to measure amplification of pfmdri was developed and validated. This technique was used to determine pfmdri copy number in a unique set of field sample set (n = 600) collected in Northern Thailand, an area harbouring the world's most drug-resistant parasites. This allowed a comprehensive analysis of the importance of pfmdri amplification in (1) in vitro resistance to drugs, (2) in vivo response to mefloquine or mefloquine- artesunate therapy, (3) evolution of amplification in pre- and post-treatment samples. Subsequent studies also investigated the prevalence of pfmdrt amplification in Gabon, a Sub-Saharan country with very little mefloquine resistance. In addition, P. falciparum field isolates were studied for possible polymorphisms in PfATP6 and plasmid constructs generated to study the role of single nucleotide point mutations in the putative active site of the enzyme.

A new era of malaria eradication programs relies on increased knowledge of the parasite through sequencing of the Plasmodium genome. Programs call for re-orientation at specific epidemiological markers as regions move from control towards pre- and total elimination. However, relatively little is known about the effects of intervention strategies on the parasite population or if the epidemiological cues correspond to effects on the parasite population. We hypothesized that genomic tools could be used to track population changes in Plasmodium falciparum to detect significant shifts as eradication programs apply interventions. Making use of new whole-genome sequencing data as well as GWAS and other studies, we used SNPs as biological markers for regions associated with drug resistance as well as a set of neutral SNPs to identify individual parasites. By utilizing tools developed as proxy for full genomic sequencing of the human pathogen Plasmodium falciparum, we characterized and tracked parasite populations to test for changes over time and between populations. When applied to markers under selection - those associated with reduced antimalarial drug sensitivity - we were able to track migration of resistance-associated mutations in the population and identify new mutations with potential implications for resistance. Using a population genetic analysis toolbox to study changes in neutral allele frequencies in samples from the field, we found significant population changes over time that included restricted effective population size, reduced complexity of infections, and evidence for both clonal and epidemic propagation of parasites.

The main thrust of this thesis was to characterize P.falciparum genetic diversity in northern Ghana. To do this, I used simple techniques to purify P. falciparum DNA from clinical samples across a rural setting for whole-genome sequencing. The goal was to provide a framework for exploring host-parasite genetic interactions. Utilizing Illumina deep sequencing data for 277 isolates I analyzed P. falciparum genetic diversity and described within-host diversity across this area. I observed random mating (ie no population structure) in the local parasite population, and a high genetic diversity indicative of high out-crossing. Moreover, when I aggregated my data with similar published data from Burkina Faso and Mali (sites ≈500km apart), no population structure was evident. In contrast, sites sampled in Cambodia and Thailand (≈ 800km apart) were found to have greater population structure and high potential for inbreeding. This may be driven by differences in transmission intensity between the sites sampled in West Africa and southeast Asia. To demonstrate the utility of deep sequencing data, I focused on the genomic regions of pfdhfr, pfdhps and pfcrt, known to be under antimalarial drug selection. I surveyed the full diversity of point mutations already characterized in these genes and discovered previously unknown variants. However, in order to provide a means to follow up on new variants or interesting candidate regions in large clinical samples with limited parasite DNA, I assessed the Sequenom iPLEX platform for high-throughput genotyping of P. falciparum polymorphisms. This necessitated developing a method appropriate for assigning genotypes in haploid genome mixtures common in natural infections. Finally, I used this method to type host and parasite markers in a case-control sample set from this region for exploring host-parasite genetic interactions. I found that children who have the sickle-cell trait and carry parasites that have pfdhfr resistant alleles lose their protection against severe malaria as compared to children who have normal haemoglobin and are infected with parasites with these resistant alleles.

Nous avons analyse par electrophorese d'isoenzymes (7 a 12 loci) le polymorphisme genetique de 5 populations (113 stocks) du plasmodium falciparum, l'agent de la forme la plus severe de paludisme. Les resultats ont ete interpretes en termes de genetique des populations, dans le but d'analyser la structure des populations naturelles de ce parasite. Un fort desequilibre de liaison apparait dans trois des populations etudiees, ainsi que dans l'echantillon global. Une quatrieme population montre egalement des indices de desequilibre, quoique dans une moindre mesure. Une seule serie n'a montre aucune deviation par rapport aux predictions de la panmixie. Ces resultats ne sont explicables, ni par la structuration geographique des populations, ni par la selection naturelle. L'hypothese la plus parcimonieuse pour rendre compte de nos donnees est l'existence d'une propagation uniparentale chez p. Falciparum dans certaines circonstances. De forts indices d'autofecondation peuvent expliquer ce resultat. La structure des populations naturelles de plasmodium falciparum et le mode de reproduction du parasite ont des consequences importantes sur l'epidemiologie de la maladie. Le modele subclonal ou partiellement clonal que nous proposons ici est radicalement different, quant a ses implications taxonomiques et epidemiologiques, du modele potentiellement panmictique qui avait ete propose pour p. Falciparum. En effet, si des lignees parasitaires subissent une propagation uniparentale, elles se comportent comme des clones, et gardent intactes leurs caracteristiques genetiques d'une generation a l'autre, tandis que le patrimoine genetique de chaque genotype fait l'objet d'un brassage energique a chaque generation dans le cas du modele potentiellement panmictique. Les problemes poses sont: (a) la nature du processus de reproduction uniparentale; (b) l'importance respective des cycles sexues et asexues; (c) la stabilite dans l'espace et dans le temps des lignees uniparentales

Includes bibliographical references (leaves 165-180).
Mefloquine has been in use for over twenty years and still very little is known about its interaction with Plasmodium falciparum. In 1979, Fitch er al carried out the only other published extensive investigation of mefloquine accumulation, but were not able to demonstrate energy dependent uptake. They later indicated that an energy requirement may be being masked by mefloquine’s ability to bind membrane phospholipids to a large extent (Chevli & Fitch, 1982).Until now no energy requirement for mefloquine accumulation has been uncovered. This thesis investigates the relationship between chloroquine and mefloquine resistance, and characterizes the mechanism of mefloquine accumulation in Plasmodium falciparum. Conditions were established that enabled the amplification of the parasites' contribution to overall mefloquine accumulation in the parasitised erythrocyte. It was found that mefloquine accumulation is stimulated by glucose and is inhibited by the glycolysis inhibitor, iodoacetate, and also by incubation at low temperature. Mefloquine accumulation was also found to be partly dependent on the pH gradient between the acidic food vacuole and the external medium. It has also been determined that mefloquine-resistant Plasmodium falciparum accumulate approximately half the amount of mefloquine than do mefloquine-sensitive parasites. It has been shown that the accumulation of both chloroquine and mefloquine have two components, a high affinity saturable component and a low affinity non-saturable component (Fitch et aI., 1979; Fitch et al., 1974; Bray et al., 1998). The saturable component has been well characterized, but until now the non-saturable component has not been identified. This thesis shows that chloroquine and mefloquine adsorption to synthetic β-haematin and pure isolated haemozoin is non-saturable. It is proposed that the malaria pigment is responsible for the low affinity, non-saturable component of chloroquine and mefloquine accumulation. The effect of chloroquine, mefloquine and artemisinin on haemoglobin levels in parasitised erythrocytes was also measured. Chloroquine caused a buildup in haemoglobin and mefloquine caused a decrease in haemoglobin levels. This adds weight to previously published work (Famin & Ginsburg, 2002) suggesting that chloroquine prevents the degradation of haemoglobin, while mefloquine inhibits the endocytosis of haemoglobin.

Malgré d'intenses recherches, le paludisme représente toujours à l'heure actuelle la première endémie parasitaire mondiale, et un problème majeur de santé publique. Au cours de notre thèse, les travaux ont porté essentiellement :- Sur l'activité antipaludique d'aminoquinolines ferrocéniques et de la ferroquine- Sur la mise en évidence d'une nouvelle famille de protéines (LRR) chez P. Falciparum et sur la caractérisation du gène PfLRR1L'étude de l'activité antipaludique d'aminoquinolines ferrocéniques (portant le ferrocène sur l'azote terminal de la chaîne latérale de l'aminoquinoline) et de ferroquines (où le ferrocène est intéfré dans la chaîne latérale de l'aminoquinoline) a permis de montrer que : 1) la position de la ferroquine dans la chaîne latérale de l'aminoquinoline est indispensable à l'activité antipaludique. 2) la présence d'un second ferrocène sur l'azote terminal des ferroquies annule l'activité antipaludique. 3) La présence du ferrocène sur l'azote terminal de l'aminoquinoline n'améliore pas son activité. Lors du développement industriel, la métabolisation de la ferroquine a été étudiée en détail et la structure des différents métabolites a été déterminée. Nous avons pu montrer que le premier et principal métabolite, la mono-N-diméthyl ferroquine (DMFQ), possédait une activité antipaludique presque aussi importante que celle de la ferroquine sur les clones de P. Falciparum résistants à la chloroquine. La DMFQ participe donc activement à l'activité antiparasitaire de la ferroquine, qui peut-être en conséquence considérée comme un antipaludique à action prolongée. Enfin, nous avons analysé, à partir de résultats collectés sur le terrain, la relation entre le polymorphisme et le niveau d'expression du gène pfcrt (à l'origine de la resistance à la chloroquine) et la sensibilité à la ferroquine. Aucune corrélation n'a été trouvée. En conséquence, nous avons exposé un clone de P. Falciparum à une pression de 100 nM de ferroquine afin d'essayer de sélectionner un mutant résistant dont aurait été possible de caractériser le génotype sur l plan moléculaire. Aucun clone résistant n'a pu être obtenu dans nos conditions d'experience. Le séquençage récemment achevé du génome de Plasmodium falciparum, agent du paludisme, a ouvert de nouvelles voies de recherche tant sur la biologie du parasite que sur le développement potentiel de stratégies thérapeutiques ou vaccinales adaptées. Dans cet esprit, sur des bases immunologiques et une analyse génomique comparative, nous avons identifié chez P. Falciparum, une nouvelle famille de protéines riches en Leucine ou LRR (les PfLRR) encore inconnue à ce jour. Grâce à leurs structures caractériqtiques, ces protéines sont connues pour être impliquées dans des interactions protéines-protéines. Elles participent à des processus biologiques essentiels et nombre d'entre-elles sont notamment impliquées dans les interactions hôte/pathogène. Afin d'élucider le rôle essentiel de ces protéines dans la biologie complexe de P. Falciparum, nous avons entrepris la caractérisation moléculaire et fonctionnelle de la PfLRR1, un orthologue d'une protéine appelée Sds22 impliquée dans la mitose, et ensuite de démontrer qu'elle est un partenaire de la protéine phosphatase de type 1. Le gène qui code pour une protéine de 30 kDa est situé sur le chromosome 10. Nous avons démontré que la PfLRR1 est régulée au niveau transcriptionnel et traductionnel, suggérant une expression différentielle de ce gène chez la souche 3D7 de Plasmodium falciparum. Des études fonctionnelles ont montré la capacité de la PfLRR1 à se lier et inhiber à la fois la protéine phosphatase de type 1 de Plasmodium falciparum et des ovocytes de xénope, provoquant ainsi la maturation des ovocytes et l'apparition du pôle animal (GVBD). L'ensemble de ces résultats laissent suggérer la participation de la PfLRR1 dans la régulation du cycle celulaire de P. Falciparum, et constitue une base pour entreprendre l'analyse fonctionnelle de cette nouvelle protéine LRR chez Plasmodium falciparum.

Le parasite du paludisme humain le plus virulent, Plasmodium falciparum, a un cycle de vie complexe entre son hôte humain et le moustique vecteur. Chaque étape est pilotée par un programme de transcription spécifique, mais avec un rapport relativement élevé de gènes à des facteurs de transcription spécifiques, on ne sait pas comment les gènes sont activés ou réduits au silence à des moments spécifiques. Le génome de P. falciparum est relativement euchromatique par rapport au génome des mammifères, à l'exception de gènes spécifiques qui sont uniquement hétérochromatinisés via HP1. Il semble y avoir une association entre l'activité des gènes et l'organisation spatiale ; cependant, les mécanismes moléculaires derrière l'organisation du génome ne sont pas clairs. Alors que P. falciparum manque de protéines organisatrices du génome clés trouvées dans les métazoaires, il possède tous les composants essentiels du complexe cohésine. Chez d'autres eucaryotes, la cohésine est impliquée dans la cohésion, la transcription et l'organisation du génome des chromatides soeurs. Pour étudier le rôle de la cohésine dans P. falciparum, nous avons combiné l'édition du génome, la spectrométrie de masse, l'immunoprécipitation et le séquençage de la chromatine (ChIP-seq) et le séquençage de l'ARN pour caractériser fonctionnellement la sous-unité cohésine Structural Maintenance of Chromosomes protein 3 (SMC3). L'inactivation de SMC3 aux premiers stades du cycle de développement intraérythrocytaire (IDC) a entraîné une régulation positive significative d'un sous-ensemble de gènes impliqués dans la sortie et l'invasion des érythrocytes, qui sont normalement exprimés à des stades ultérieurs. ChIP-seq de SMC3 a révélé que sur l'IDC, l'enrichissement au niveau des régions promotrices de ces gènes est inversement corrélé à leurs niveaux d'expression et d'accessibilité à la chromatine. Ces données suggèrent que la liaison de SMC3 aide à réprimer des gènes spécifiques jusqu'à leur moment d'expression approprié, révélant un nouveau mode de répression génique spécifique au stade et indépendant de HP1 chez P. falciparum
The most virulent human malaria parasite, Plasmodium falciparum, has a complex life cycle between its human host and mosquito vector. Each stage is driven by a specific transcriptional program, but with a relatively high ratio of genes to specific transcription factors, it is unclear how genes are activated or silenced at specific times. The P. falciparum genome is relatively euchromatic compared to the mammalian genome, except for specific genes that are uniquely heterochromatinized via HP1. There seems to be an association between gene activity and spatial organization; however, the molecular mechanisms behind genome organization are unclear. While P. falciparum lacks key genome-organizing proteins found in metazoans, it does have all core components of the cohesin complex. In other eukaryotes, cohesin is involved in sister chromatid cohesion, transcription, and genome organization. To investigate the role of cohesin in P. falciparum, we combined genome editing, mass spectrometry, chromatin immunoprecipitation and sequencing (ChIP-seq), and RNA sequencing to functionally characterize the cohesin subunit Structural Maintenance of Chromosomes protein 3 (SMC3). SMC3 knockdown in early stages of the intraerythrocytic developmental cycle (IDC) resulted in significant upregulation of a subset of genes involved in erythrocyte egress and invasion, which are normally expressed at later stages. ChIP-seq of SMC3 revealed that over the IDC, enrichment at the promoter regions of these genes inversely correlates with their expression and chromatin accessibility levels. These data suggest that SMC3 binding helps to repress specific genes until their appropriate time of expression, revealing a new mode of stage-specific, HP1-independent gene repression in P. falciparum

Cette etude constitue la premiere evaluation de la diversite genetique et de la structure de population de plasmodium falciparum, au venezuela, par analyse rapd et par pcr des sequences polymorphes des genes codant pour les proteines msp-1, msp-2, resa et csp. Par ailleurs, nous avons realise la caracterisation moleculaire des mutations dans les genes de la dihydrofolate reductase (dhfr) et dihydropteroate synthetase (dhps), supposees etre associees a l'echec des traitements a la pyrimethamine-sulfadoxine (ps) au venezuela. L'analyse rapd a montre une variabilite considerable dans les souches collectees pendant les mois de mai et juillet (epoque de haute transmission) par rapport a celles qui ont ete collectees en novembre (epoque de faible transmission). Un desequilibre de liaison tres significatif (p<104) a ete detecte dans les populations naturelles de p. Falciparum au venezuela. Ceci suggere que la propagation clonale des genotypes joue un role important dans la structure des populations de ce parasite au venezuela, ce qui peut etre explique par de forts taux d'autofecondation. Cependant, la structuration occasionnee par la propagation clonale est clairement moins accentuee que pour d'autres especes parasitaires telles que trypanosoma cruzi. L'analyse phylogenetique de la variabilite rapd montre clairement le caractere monophyletique de p. Falciparum, qui peut etre caracterise par plusieurs caracteres rapd specifiques (tags). Cette etude a trouve en haute proportion des mutations simultanees sur les genes dhfr et dhps. L'effet cumulatif de ces mutations pourrait etre un mecanisme important dans l'emergence d'une resistance elevee a la ps dans cette region. La haute prevalence et diffusion rapide de genotypes resistants pourrait etre une consequence directe de la clonalite et de l'autofecondation, lesquelles peuvent diffuser avec plus de rapidite le mecanisme de multi-resistance aux drogues antipaludiques, comme c'est le cas au venezuela.

Orientadores: Fabio Trindade Maranhão Costa, Stefanie Costa Pinto Lopes
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A malária é responsável por cerca de 300 milhões de casos de infecção e 1 milhão de mortes por ano. No Brasil, em 2012, foram registrados cerca de 1500 casos sendo a malária vivax responsável por 85% destes. Ainda, é frequentemente reportada falha terapêutica aos antimaláricos convencionais (como cloroquina e mefloquina) em infecções por P. falciparum, principalmente, mas também por P. vivax. Sendo assim, terapias combinadas com artemisinina e seus derivados (ACT) são atualmente recomendadas. No entanto, resistência aos derivados da artemisinina já é evidente e a busca por novos compostos com atividade antimalárica é urgente. A violaceína, pigmento violeta extraído de bactérias Gram negativas, demostrou apresentar elevada atividade antimalárica in vitro e in vivo em trabalho anterior de nosso grupo. Neste sentido, este projeto tem como objetivo aprofundar a investigação a respeito da atividade antimalárica da violaceína avaliando seu papel em isolados amazônicos de P. falciparum adaptados recentemente em cultura, e em P. vivax após imediata coleta de sangue de pacientes infectados. Além disso, investigamos o potencial da violaceína como terapia combinada junto ao artesunato no tratamento de parasitas murinos resistentes a este e a outros antimaláricos como a cloroquina. Inicialmente foram testadas dois diferentes tipos de violaceína in vitro contra P. falciparum 3D7: uma comercial extraída de Janthinobacterium lividum (vJl- IC50: 227 nM) e outra extraída de Chromobacterium violaceum (vCv- IC50: 390 nM). Apesar de não termos encontrado uma diferença na atividade antimalárica entre as duas violaceínas, a extraída de C. violaceum teve uma baixa toxicidade em eritrócitos (<400 nM) em células de hepatoma humano (<800 nM). Devido a esta baixa toxicidade, somente vCv foi avaliada quanto sua atividade antimalárica. Demonstramos que vCv apresentou IC50 similar ao encontrado para P. falciparum 3D7 (IC50 média= 419,8 nM) em 7 isolados de campo de P. falciparum. Ainda, em ensaios in vivo, utilizando cepas murinas de P. chabaudi, vCv conseguiu diminuir significativamente (P<0,05) a parasitemia no dia pico da infeção em cepas resistentes à cloroquina (30CQ) e a artesunato e mefloquina (ATNMF1). Adicionalmente, nos testes realizados em P. vivax a vCv parece evitar o amadurecimento parasitário nos quatro isolados testados. Coletivamente, podemos concluir que a vCv apresentou um efeito antimalárico em cepas de P. falciparum e pode ser especialmente útil quando usada em combinação com artesunato no tratamento de camundongos infectados com cepas resistentes. Finalmente, ensaios adicionais de amadurecimento com isolados de P. vivax necessitam ser conduzidos para a comprovação do efeito da vCv nesta espécie
Abstract: Malaria is responsible for about 300 million infections and one million deaths per year. In Brazil in 2012, about 1500 cases were reported and malaria vivax accounts for 85% of these. Still, it is frequently reported treatment failure with conventional antimalarials (as chloroquine and mefloquine) mainly in infections with P. falciparum but also by P. vivax parasite. Because of that combination therapies with artemisinin and its derivatives (ACT) are now currently recommended. In a previous work, our group demonstrated that violacein was able to inhibit the in vitro growth of laboratory strains of P. falciparum and also to strongly control the parasitemia of mice infected with P. chabaudi. This project aims to investigate further the antimalarial activity of violacein evaluating its activity in Amazonian isolates of P. falciparum recently adapted in culture, and in P. vivax isolates immediately after collecting blood from infected patients. Furthermore, we investigate the potential of violacein as combination therapy with artesunate in the treatment of murine strains which are resistant in different levels to artesunate, mefloquine and chloroquine. The antimalarial activity of violacein were initially investigated in vitro against P. falciparum 3D7 using two different types of violacein, one comercial, extracted from Janthinobacterium lividum (vJl-IC50: 227 nM), and another extracted from Chromobacterium violaceum (vCv-IC50: 390 nM) by our collaborators. In spite of no difference in the antimalarial activity between the two violaceins, the one extracted from C. violaceum had the lowest toxicity in erythrocytes (<400 nM) and in human hepatoma cells (<800 nM). Because of this, the antimalarial activity only of vCv was evaluated against 7 field isolates of P. falciparum showing a similar IC50 to that found for P. falciparum 3D7 (IC50= 419.8 nM). The antimalarial activity was also evaluated in murine strains of P. chabaudi showing a significant (P < 0.05) parasitemia decrease in the peak day in the two clones of P. chabaudi tested, one resistant to chloroquine (30CQ) and another resistant to artesunate and mefloquine (ATNMF1). Additionally, in P. vivax vCv was capable to reduce the parasite maturation in the four isolates tested. Therefore we can conclude that the vCv has an antimalarial effect on field isolates of P. falciparum and can be especially useful when used in combination with artesunate in the treatment of mice infected with resistant strains. Moreover, more assays should be conducted using blood infected with P. vivax to corroborate its effect in inhibiting the maturation of trophozoites
Mestrado
Imunologia
Mestra em Genética e Biologia Molecular

El objetivo del presente trabajo fue identificar antígenos relevantes de valor diagnóstico de aislados de P. vivax y P. falciparum provenientes del departamento de Loreto, mediante la técnica de inmunoblot. Se seleccionaron pacientes entre 3 y 64 años con diagnóstico de malaria, gota gruesa positiva, procedentes de centros de salud en el departamento de Loreto. Fueron analizadas 4 mezclas de antígenos, una de P. falciparum (PF1) y tres de P. vivax (PV1, PV4 y PV5), preparadas a partir de 36 muestras de pacientes con alta parasitemia por P. vivax (2 700 – 69 000 parásitos/μL) y P. falciparum (2 750 – 10 000 parásitos/μL). Las mezclas de antígenos fueron enfrentadas a 39 sueros (12 de P. falciparum y 27 de P. vivax) mediante ensayos de inmunoblot.
Tesis

Les plastes sont des organites semi-autonomes qui dérivent d'un événement unique d'endosymbiose entre une cyanobactérie et une cellule eucaryote ancestrale. Le plaste le mieux caractérisé est le chloroplaste des cellules de plantes et d'algues vertes. À la différence des systèmes membranaires eucaryotes qui sont en général riches en phospholipides, les membranes des plastes végétaux se composent à plus de 70% de galactoglycérolipides (monogalactosyldiacylglycérol, MGDG ; digalactosyldiacylglycérol, DGDG). Leur synthèse est assurée par des galactosyltransférases, les MGDG synthases (EC 2. 4. 1. 46) et les DGDG synthases (EC 2. 4. 1. 241) localisées dans les membranes de l'enveloppe qui limitent l'organite. Chez les plantes, les galactolipides sont essentiels pour la biogenèse des plastes mais aussi à la synthèse de membranes extraplastidiales sous certaines conditions physiologiques telles qu'en carence de phosphate. La plupart des parasites Apicomplexes (protozoaires parasites à mode de vie intracellulaire) possèdent une structure plastidiale, non photosynthétique, contenant un ADN circulaire. Cet organite, baptisé apicoplaste, a pour origine phylogénétique une endosymbiose secondaire entre deux eucaryotes, avec ingestion d'une algue rouge unicellulaire par un protozoaire ancestral, suivie d'une disparition de la plupart des structures subcellulaires de l'algue. Des protéines codées par des gènes nucléaires sont importées dans l'apicoplaste et impliquées dans des voies métaboliques typiques des plantes telles que la biosynthèse d'acides gras par le système FASII. Des lipides aux propriétés chromatographiques proches de celles du MGDG et du DGDG ont pu être détectés dans des extraits de Plasmodium falciparum et Toxoplasma gondii, suggérant l'existence d'une voie de biosynthèse des galactolipides comparable à celle existant dans le chloroplaste. Au moyen d'une série d'anticorps dirigés contre le DGDG, un premier objectif de ce travail de thèse a été de caractériser la localisation d'un épitope de structure proche du digalactolipide chloroplastique (DGLE pour digalactolipid-like epitope) chez P. Falciparum et déterminer l'évolution de la distribution subcellulaire de cet épitope au cours du cycle cellulaire du parasite. Ces études suggèrent qu'un lipide de structure proche du DGDG est associé à des systèmes membranaires en périphérie de la cellule, en particulier le complexe membranaire interne. Des parasites transgéniques exprimant une MGDG synthase exogène de plante ont été générés. L'accumulation remarquable à la fois de MGDG et de DGDG chez ces transformants démontre d'une part que l'enzyme de plante est fonctionnelle, catalysant la synthèse de MGDG, et d'autre part qu'une glycosyltransférase de P. Falciparum est capable ensuite de catalyser de grandes quantités de DGDG. Le rôle possible de cette glycosyltransférase dans la synthèse du DGDG reste à établir. Un second objectif de cette thèse était d'évaluer le potentiel dans P. Falciparum de composés inhibant la synthèse des galactolipides de plante dans une visée thérapeutique. Un criblage à haut débit robotisé d'une banque de 24. 000 molécules nous a permis d'identifier des inhibiteurs de la MGDG synthase 1 d'Arabidopsis thaliana, parmi lesquels deux composés présentent un effet inhibiteur mesuré par une CI50 de l'ordre 10 µM sur l'activité MGDG synthase. Nous avons pu caractériser l'effet de ces molécules comme compétiteurs de la fixation du diacylglycérol. Une activité inhibitrice de la prolifération de P. Falciparum a pu être mesurée in vitro bien qu'aucune enzyme homologue à la MGDG synthase n'ait pu être identifiée in silico sur les banques de données relatives aux Apicomplexes. Une diversification de la structure du châssis moléculaire a été conduite afin d'améliorer 1) l'effet herbicide et 2) l'inhibition de la croissance des parasites et de développer ainsi de nouvelles molécules antipaludiques, qui puissent être qualifiées de médicaments herbicides. L'étude de 250 analogues a permis de progresser de façon substantielle dans le sens d'une meilleure sélectivité avec des composés actifs à 200 nM. Il n'est pas exclu que les molécules sélectionnées pour leur capacité à inhiber une activité MGDG synthase exercent, chez P. Falciparum, un effet sur une autre cible. Nous avons conduit une expérience visant à isoler la(les) protéine(s) cible(s) des molécules bioactives par chromatographie d'affinité, puis identifié ces protéines après digestion trypsique par spectrométrie de masse. Une perspective de ce travail consiste à caractériser certaines de ces cibles candidates. Le développement de candidats-médicaments est un processus long selon le schéma classique, depuis la validation d'une cible jusqu'aux essais cliniques. La nouvelle classe de compétiteurs du diacylglycérol caractérisée dans ce travail de thèse présente des propriétés intéressantes dans une visée thérapeutique, qu'il sera important d'optimiser dans l'avenir
Plastids are semi-autonomous organelles that derive from a unique endosymbiotic event between an ancestral eukaryotic cell and a cyanobacteria. The best characterized plastid is the chloroplast from plant cells and green algae. Differing from eukaryotic membrane systems that are naturally phospholipid-rich, plant chloroplasts membranes are composed of >70% galactoglycerolipids (monogalactosyldiacylglycerol, MGDG ; digalactosyldiacylglycerol, DGDG). Their synthesis is catalyzed by galactosyltransferases, namely MGDG synthases (EC 2. 4. 1. 46) and DGDG synthases (EC 2. 4. 1. 241), localized in the organelle limiting envelope membranes. In plant cells, galactolipids are essential to plastid biogenesis as well as for the composition of non-plastidial membranes under specific physiological conditions, such as phosphate deprivation. Most of the apicomplexan parasites (obligate intracellular protozoan parasites) harbour a non-photosynthetic plastid, containing a circular DNA. This organelle, called the apicoplast, is considered as a relic of an algal plastid, resulting from a secondary endosymbiosis between two eukaryotic cells, with the ingestion of a unicellular red algae by an ancestral protozoan, followed by the disappearance of most of the algal subcellular structures. Interestingly, some apicomplexan nuclear gene products are imported into the apicoplast and involved in typical plant biosynthetic pathways such as FASII fatty acids biosynthesis. Lipids with chromatographic properties similar to those of MGDG and DGDG, have been detected in Plasmodium falciparum and Toxoplasma gondii total extracts, suggesting the existence of a chloroplast-like galactolipid biosynthetic pathway. Using a series of antibodies directed against DGDG, a first aim of this thesis work was to investigate the localization of a chloroplastic digalactolipid-like epitope (DGLE) in P. Falciparum and to determine its evolution along the parasitic cell cycle. These studies suggest that a DGLE may be associated to endomembrane systems in the cell periphery, particularly the inner membrane system. Transgenic parasites expressing an exogenous plant MGDG synthase have been generated. The remarkable accumulation of both MGDG and DGDG demonstrate that the exogenous plant enzyme is functional, catalyzing the synthesis of MGDG, and that a P. Falciparum glycosyltransferase is subsequently capable of catalyzing large amounts of DGDG. The possible role of this DGDG-producing glycosyltransferase in the synthesis of the DGLE remains to be demonstrated. A second aim of this thesis consisted in the evaluation of compounds inhibiting the galactolipid synthesis for a therapeutic purpose. An automated high throughput screening of a 24000 molecule-chemolibrary allowed the identification of inhibitors of Arabidopsis thaliana MGDG synthase 1, among which two compounds harbour an inhibiting effect in the 10 µM range on MGDG synthase activity. We characterized the effect of these molecules as competitors of diacylglycerol binding. An antiparasitic activity was measured in vitro. In order to improve 1) the herbicidal effect of the lead compounds, 2) the parasite growth inhibition and to develop new antiplasmodial candidates that could be qualified as herbicidal-drugs, a structure-based diversification of the molecules was conducted. The characterization of 250 analogues allowed the optimization of the molecular selectivity with compounds being active in the 200 nM range. It cannot be excluded that molecules selected for their ability to inhibit a MGDG synthase activity could exert a distinct effect on a non-related Plasmodium falciparum target. We developed an experimental procedure to isolate the protein target(s) of the bioactive compounds by affinity chromatography against a biotinylated analogue, leading to the identification of several proteins by mass spectrometry after trypsic digestion. The development of drug candidates is a long process from the target validation to the clinical trials. The properties of the novel class of compounds characterized in this thesis work are promising. Future prospects include therefore sustained efforts to improve the antiparasitic properties of this novel class of diacylglycerol competitors