83 research outputs found
Anopheles/Plasmodium interactions at the ookinete-to-oocyst developmental transition
The ookinete to oocyst developmental transition of the Plasmodium parasite represents a
major population bottleneck in the malaria life cycle. This suggests that it could be a target for
intervention strategies, such as transmission blocking vaccines, provided essential parasite target
molecules can be identified. A recent microarray analysis has identified a large number of transcripts
differentially expressed during the parasite’s developmental transitions. Genes differentially
regulated during the ookinete-to-oocyst transition may determine the development of the parasite
within the mosquito host, as well as, participating directly in parasite/mosquito interactions. Yet, the
function of the majority of such molecules is largely unknown.
This PhD thesis aims to identify and functionally characterise genes putatively involved in
ookinete development and/or the interactions between the parasite and the mosquito host in the model
system Plasmodium berghei. Thirty three proteins likely to be implicated in the parasite’s interaction
with the mosquito immune system and local epithelial response were identified based on their
expression pattern and predicted structural features. Generation of knock-out mutants through
targeted gene disruption by homologous recombination was the first step towards functional
characterization of these candidates.Successful mutants were assessed for their ability to complete
their sexual sporogonic development, as well as, their impact on mosquito immunity following
infection of Anopheline mosquitoes of various immune backgrounds. Interestingly, two of the
successful mutants were hampered in their ability to undergo normal differentiation during ookinete
development while the third one’s ability to invade the mosquito midgut epithelium was impaired.
The inability to invade implies a potential interaction of this gene product with mosquito midgut
ligands. Eventually malaria transmission through Anopheline mosquitoes was affected in all three
mutants. Moreover, challenging of a mosquito protein LRIM1, a major parasite antagonist, also
revealed potential involvement of the three mutants in mosquito/parasite immune response pathways.
Genetic crosses with parasite lines deficient in the production of either male or female fertile gametes
demonstrated in the case of two mutants that, this defect in ookinete development is sex dependent,
thus underlining the critical importance of maternal and/or paternal control during the first few hours
of parasite development in the mosquito
lincRNAs: Genomics, Evolution, and Mechanisms
Long intervening noncoding RNAs (lincRNAs) are transcribed from thousands of loci in mammalian genomes and might play widespread roles in gene regulation and other cellular processes. This Review outlines the emerging understanding of lincRNAs in vertebrate animals, with emphases on how they are being identified and current conclusions and questions regarding their genomics, evolution and mechanisms of action.National Institutes of Health (U.S.) (Grant GM067031
Deciphering causal genetic determinants of red blood cell traits
Les études d’association pan-génomiques ont révélé plusieurs variants génétiques associés à des traits complexes. Les mesures érythrocytaires ont souvent fait l’objet de ce genre d’études, étant mesurées de façon routinière et précise. Comprendre comment les variations génétiques influencent ces phénotypes est primordial étant donné leur importance comme marqueurs cliniques et leur influence sur la sévérité de plusieurs maladies. En particulier, des niveaux élevés d’hémoglobine fœtal chez les patients atteints d’anémie falciforme est associé à une réduction des complications et une augmentation de l’espérance de vie. Néanmoins, la majorité des variants génétiques identifiés par ces études tombent à l’intérieur de régions génétiques non-codantes, augmentant la difficulté d’identifier des gènes causaux.
L’objectif premier de ce projet est l’identification et la caractérisation de gènes influençant les traits complexes, et tout particulièrement les traits sanguins. Pour y arriver, j’ai tout d’abord développé une méthode permettant d’identifier et de tester l’effet de gènes knockouts sur les traits anthropométriques. Malgré un échantillon de grande taille, cette approche n’a révélé aucune association. Ensuite, j’ai caractérisé le méthylome et le transcriptome d’érythroblastes différentiés à partir de cellules souches hématopoïétiques et identifié plusieurs gènes potentiellement impliqués dans les programmes érythroïdes fœtaux et adultes. Par ailleurs, j’ai identifié plusieurs micro-ARNs montrant des motifs d’expression spécifiques entre les stages fœtaux et adultes et qui sont enrichis pour des cibles exprimées de façon opposée. Finalement, j’ai identifié plusieurs variants génétiques associés à l’expression de gènes dans les érythroblastes (eQTL). Cette étude a permis d’identifier des variants associés à l’expression du gène ATP2B4, qui encode le principal transporteur de calcium des érythrocytes. Ces variants, qui sont également associés à des traits sanguins et à la susceptibilité à la malaria, tombent dans un élément d’ADN spécifique aux cellules érythroïdes. La délétion de cet élément par le système CRISPR/Cas9 induit une forte diminution de l’expression du gène et une augmentation des niveaux de calcium intracellulaires.
En conclusion, des échantillons de génotypages exhaustifs seront nécessaires pour étudier l’effet de gènes knockouts sur les traits complexes. Les érythroblastes montrent de grandes différences au niveau de leur méthylome et transcriptome entre les différents stages développementaux. Ces différences influencent potentiellement la régulation de l’hémoglobine fœtale et impliquent de nombreux micro-ARNs et régions régulatrices non-codantes. Finalement, l’exemple d’ATP2B4 montre qu’intégrer des études épigénomiques, transcriptomiques et des expériences d’édition de génome est une approche puissante pour caractériser des variants génétiques non-codants. Par ailleurs, ces résultats impliquent ATP2B4 dans l’hydratation des érythroblastes, qui est associé à la susceptibilité à la malaria et la sévérité de l’anémie falciforme. Cibler ATP2B4 de façon thérapeutique pourrait avoir un impact majeur sur ces maladies qui affectent des millions d’individus à travers le monde.Genome-wide association studies (GWAS) have revealed several genetic variants associated with complex phenotypes. This is the case for red blood cell (RBC) traits, which are particularly amenable to GWAS as they are routinely and accurately measured. Understanding RBC trait variation is important given their significance as clinical markers and modifiers of disease severity. Notably, increased fetal hemoglobin (HbF) production in sickle cell disease (SCD) patients is associated with a higher life expectancy and decreased morbidity. Nonetheless, most variants identified through GWAS fall in non-coding regions of the human genome, increasing the difficulty of identifying causal links.
The main goal of this project was to identify and characterize genes influencing complex traits, and in particular RBC phenotypes. First, I developed an approach to identify and test potential gene knockouts affecting anthropometric traits in a large sample from the general population, which did not yield significant associations. Then, I characterized the DNA methylome and transcriptome of erythroblasts differentiated ex vivo from hematopoietic progenitor stem cells (HPSC), and identified several genes potentially implicated in fetal and adult-stage erythroid programs. I also identified microRNAs (miRNA) that show specific developmental expression patterns and that are enriched in inversely expressed targets. Finally, I mapped expression quantitative trait loci (eQTL) in erythroblasts, and identify erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of RBCs. These genetic variants are associated with RBC traits and malaria susceptibly, and overlap an erythroid-specific enhancer of ATP2B4. Deletion of this regulatory element using CRISPR/Cas9 experiments in human erythroid cells minimized ATP2B4 expression and increased intracellular calcium levels.
In conclusion, large and comprehensive genotyping datasets will be necessary to test the role of rare gene knockouts on complex phenotypes. The transcriptomes and DNA methylomes of erythroblasts show substantial differences correlating with their developmental stages and that may be implicated in HbF production. These results also suggest a strong implication of erythroid enhancers and miRNAs in developmental stage specificity. Finally, characterizing the erythroid-specific enhancer of ATP2B4 suggest that integrating epigenomic, transcriptomic and gene editing experiments can be a powerful approach to characterize non-coding genetic variants. These results implicate ATP2B4 in erythroid cell hydration, which is associated with malaria susceptibility and SCD severity, suggesting that therapies targeting this gene could impact diseases affecting millions of individuals worldwide
Optimization of the heterologous expression of folate metabolic enzymes of Plasmodium falciparum
Malaria is a fatal tropical disease affecting billions of people in impoverished countries world-wide. An alarming fact is that a child in Africa dies of malaria every 30 seconds that amounts to 2500 children per day (www.who.int/features/factfiles). Malaria is caused by the intraerythrocytic forms of Plasmodium species, notably P. falciparum, P. vivax, P. ovale and P. malariae (Hyde 2007). The spread of drug-resistant strains, failure of vector control programs, rapid growth rate of the parasite, and lack of a vaccine have further exacerbated the effects of malaria on economic development and human health. It is therefore imperative that novel drug targets are developed or current antimalarial drugs optimized (Foley and Tilley 1998). One such target is folate biosynthesis, given that folates and their derivatives are required for the survival of organisms (Muller et al. 2009). DHFR and DHPS are currently the only folate targets exploited however, their antifolate drugs are almost useless against parasite resistant strains. As such, guanosine-5’triphosphate cyclohydrolase I (GTPCHl) among other antifolate candidates are considered for intervention (Lee et al. 2001). Knock-out studies (of P. falciparum gtpchI) resulted in the suppression of DHPS activity (Nzila et al. 2005). Additionally, gtpchI amplified 11-fold in P. falciparum strains resistant to antifolates due to mutations in dhps and dhfr and this may be a mechanism for the compensation of reduced flux of folate intermediates (Kidgell et al. 2006; Nair et al. 2008). Over-expression of P. falciparum proteins in E. coli remains a challenge mainly due to the A+T rich Plasmodium genome resulting in a codon bias. This results in the expression of recombinant proteins as insoluble proteins sequestered in inclusion bodies (Carrio and Villaverde 2002; Mehlin et al. 2006; Birkholtz et al. 2008a). Comparative expression studies were conducted of native GTPCHI (nGTPCHI), codon optimized GTPCHI (oGTPCHI) and codon harmonized (hGTPCHI) in various E. coli cell lines, using alternative media compositions and co-expression with Pfhsp70. The nGTPCHI protein did not express because the gene consisted of codons rarely used by E. coli (codon bias). The expression levels of purified hGTPCHI were a greater in comparison to oGTPCHI using the different expression conditions. This is because codon-harmonization involves substituting codons to replicate the codon frequency preference of the target gene in P. falciparum, as such the translation machinery matches that of Plasmodium (Angov et al. 2008). Furthermore, greater expression levels of GTPCHI were achieved in the absence of Pfhsp70 due to expression of a possible Nterminal deletion product or E. coli protein. Purification conditions could be improved to obtain homogenous GTPCHI and further analysis (mass spectrometry and enzyme activity assays) would be required to determine the nature of soluble GTPCHI obtained. To improve the expression of soluble proteins the wheat germ expression system was used as an alternate host. However, GTPCHI expression was not effective, possibly due to degradation of mRNA template or the absence of translation enhancer elements.Dissertation (MSc)--University of Pretoria, 2010.Biochemistryunrestricte
Characterization of a new large family of extinct short retroposons in the protozoan parasite Leishmania and their role in post-transcriptional regulation of gene expression
Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010Les leishmanioses se caractérisent par divers symptômes allant de lésions cutanées guérissant spontanément, causées surtout par L. major, . à des infections viscérales potentiellement mortelles causées par le complexe L. donovani/L. infantum. Malgré des génomes presque identiques, des analyses comparatives sur biopuces ont révélé d'importantes différences d'expression génique entre l~s deux stades de vie, promastigotes et amastigotes chez ces deux espèces, ce qui pourrait contribuer aux différences dans les manifestations cliniques. Chez Leishmania, le contrôle de l'expression génique s' effectue au niveau posttranscriptionnel et implique surtout des séquences cis régulatrices situées dans les séquences 3' non-traduites (3 'UTRs) des transcrits. Jusqu'à maintenant, quelques séquences régulatrices seulement ont été caractérisées et les mécanismes contrôlant l'expression génique entre les différents stades et différentes espèces de Leishmania sont peu connus. Ce projet visait à mieux comprendre les voies exploitées par Leishmania pour pallier au manque .de contrôle transcriptionnel et contrôler globalement l'expression génique afin de s'adapter à ses différents environnements. Nous avons identifié deux nouvelles grandes familles de courts rétroposons éteints nommées SIDER1 (Short Interspersed DEgenerated Retroposons; 785 copies) et SIDER2 (1 073 copies), situés presqu'exclusivement dans les régions 3'UTRs. Nous avons caractérisé la famille SIDER2 et démontré qu'elle réprime l'expression génique en déstabilisant spécifiquement les transcrits portant ces éléments. La dégradation rapide des transcrits instables contenant un élément SIDER2 est amorcée par un clivage endonucléolytique séquence-spécifique sans raccourcissement préalable de la séquence poly(A). Le clivage se produit dans une région riche en C d'environ 20 nt) au début de la seconde séquence signature de 79 nt, qui est hautement conservée parmi les SIDER2 et essentielle pour l'instabilité de l'ARNm. Nous suggérons -l'hypothèse que Leishmania exploite les SIDER2 pour réprimer de manière coordonnée l'expression des transcrits devant être faiblement exprimés. L'effet répressif de SIDER2 est bloqué chez les amastigotes de L. infantum, possiblement pour produire une quantité suffisante de certains ARNm ou protéines. Cette inactivation sélective des SIDER2 chez L. infantum contribue aux différences d'expression génique chez les différentes espèces et différents stades de Leishmania. À notre connaissance, ceci est le premier exemple chez les eucaryotes d'une famille entière d' éléments mobiles domestiqués pour participer au contrôle posttranscriptionnel de l'expression génique
Investigation of conserved Flagellum proteins in Trypanosoma brucei
The single celled protozoan parasite Trypanosoma brucei is an excellent model organism to study eukaryotic cilia and flagella as it has a single flagellum that remains assembled throughout the cell cycle. The new flagellum assembles in a known position relative to the old flagellum, therefore creating a model system of identifiable organelle generations. In additional to a sequenced genome, there are many reverse genetics tools developed for T. brucei which makes the functional analysis of proteins possible. More than 300 proteins have been identified as components of the T. brucei flagellum but functional analysis of the majority of these proteins has not been carried out to date.
This project used a bioinformatics approach to identify potential flagellum proteins in T. brucei that were also conserved in Homo sapiens, thereby identifying potential ciliopathy candidates. Candidate proteins were confirmed as flagellum components through endogenous localisation techniques and co-localisation studies. Functional analysis was performed using inducible RNAi cell lines. Light and electron microscopy techniques were used for phenotypic analysis.
Through bioinformatics analysis a novel family of coiled-coil TPH domain-containing proteins were identified that are highly conserved in flagellated eukaryotes. There are three TPH domain-containing proteins conserved in T. brucei that all have a role in flagellum length control and cell morphogenesis. In all three cases protein ablation has a detrimental effect on cellular motility.
This work provides further understanding into the complexities of flagellum biogenesis in
T. brucei and the downstream effects on cell motility and morphogenesis
Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution
BACKGROUND: The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis. RESULTS: I elucidate nuclear and mitotic coevolution, explaining the origins of dicer and small centromeric RNAs for positionally controlling centromeric heterochromatin, and how 27 major features of the cell nucleus evolved in four logical stages, making both mechanisms and selective advantages explicit: two initial stages (origin of 30 nm chromatin fibres, enabling DNA compaction; and firmer attachment of endomembranes to heterochromatin) protected DNA and nascent RNA from shearing by novel molecular motors mediating vesicle transport, division, and cytoplasmic motility. Then octagonal nuclear pore complexes (NPCs) arguably evolved from COPII coated vesicle proteins trapped in clumps by Ran GTPase-mediated cisternal fusion that generated the fenestrated nuclear envelope, preventing lethal complete cisternal fusion, and allowing passive protein and RNA exchange. Finally, plugging NPC lumens by an FG-nucleoporin meshwork and adopting karyopherins for nucleocytoplasmic exchange conferred compartmentation advantages. These successive changes took place in naked growing cells, probably as indirect consequences of the origin of phagotrophy. The first eukaryote had 1-2 cilia and also walled resting cysts; I outline how encystation may have promoted the origin of meiotic sex. I also explain why many alternative ideas are inadequate. CONCLUSION: Nuclear pore complexes are evolutionary chimaeras of endomembrane- and mitosis-related chromatin-associated proteins. The keys to understanding eukaryogenesis are a proper phylogenetic context and understanding organelle coevolution: how innovations in one cell component caused repercussions on others. REVIEWERS: This article was reviewed by Anthony Poole, Gáspár Jékely and Eugene Koonin
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