Characterization of sexual commitment and the early steps of sexual development in the human malaria parasite Plasmodium falciparum

Transmission of malaria requires that some parasites abandon asexual replication and develop into sexual stages termed gametocytes. The discovery of the transcription factor PfAP2-G as the master regulator of sexual conversion has boosted our understanding of sexual development. The pfap2-g locus is controlled by heterochromatin-based silencing, with only a few parasites activating the locus and committing to sexual development at each asexual cycle. How heterochromatin forms in pfap2-g is currently unknown. Moreover, the initial steps of sexual conversion after pfap2-g activation, together with the biology of sexually committed stages, remain poorly characterized. In this thesis, we identified a new route of sexual conversion involving direct conversion within the same cycle of initial PfAP2-G expression. We also developed a conditional activation system for PfAP2-G, achieving synchronous sexual conversion of the majority of parasites, which enables the characterization of sexually committed parasites and early sexual stages. Finally, in an attempt to elucidate the mechanisms behind heterochromatin formation in pfap2-g, we gained insight into the role of different elements in heterochromatin nucleation.Per a la transmissió de la malària, alguns paràsits han de deixar de replicar-se de forma asexual i desenvolupar-se com a estadis sexuals anomenats gametòcits. El descobriment del factor de transcripció PfAP2-G com a regulador clau de la conversió sexual ha permès entendre millor el procés. El locus pfap2-g està silenciat per la presència d’heterocromatina i només alguns paràsits activen el locus i inicien el desenvolupament sexual a cada cicle asexual. Actualment es desconeix com es forma heterocromatina a pfap2-g. A més a més, els primers passos de la conversió sexual després de l’activació de pfap2-g, juntament amb la biologia dels estadis que acabaran desenvolupant-se sexualment, estan molt poc caracteritzats. En aquesta tesis, hem identificat una nova ruta de conversió que consisteix en la conversió sexual directa, sense un nou cicle de replicació, just després de l’activació de PfAP2-G. També hem desenvolupat un sistema d’activació condicional de PfAP2-G que indueix la conversió sexual sincrònica de la majoria dels paràsits i que permet la caracterització dels estadis sexuals primerencs. Finalment, en un intent per entendre els mecanismes darrera la formació d’heterocromatina a pfap2-g, hem obtingut informació rellevant sobre el paper de diferents elements en la nucleació d’heterocromatina

Transcriptional variation in malaria parasites: why and how

Transcriptional differences enable the generation of alternative phenotypes from the same genome. In malaria parasites, transcriptional plasticity plays a major role in the process of adaptation to fluctuations in the environment. Multiple studies with culture-adapted parasites and field isolates are starting to unravel the different transcriptional alternatives available to Plasmodium falciparum and the underlying molecular mechanisms. Here we discuss how epigenetic variation, directed transcriptional responses and also genetic changes that affect transcript levels can all contribute to transcriptional variation and, ultimately, parasite survival. Some transcriptional changes are driven by stochastic events. These changes can occur spontaneously, resulting in heterogeneity within parasite populations that provides the grounds for adaptation by dynamic natural selection. However, transcriptional changes can also occur in response to external cues. A better understanding of the mechanisms that the parasite has evolved to alter its transcriptome may ultimately contribute to the design of strategies to combat malaria to which the parasite cannot adapt

Reporter lines based on the gexp02 promoter enable early quantification of sexual conversion rates in the malaria parasite Plasmodium falciparum

Transmission of malaria parasites from humans to mosquito vectors requires that some asexual parasites differentiate into sexual forms termed gametocytes. The balance between proliferation in the same host and conversion into transmission forms can be altered by the conditions of the environment. The ability to accurately measure the rate of sexual conversion under different conditions is essential for research addressing the mechanisms underlying sexual conversion, and to assess the impact of environmental factors. Here we describe new Plasmodium falciparum transgenic lines with genome-integrated constructs in which a fluorescent reporter is expressed under the control of the promoter of the gexp02 gene. Using these parasite lines, we developed a sexual conversion assay that shortens considerably the time needed for an accurate determination of sexual conversion rates, and dispenses the need to add chemicals to inhibit parasite replication. Furthermore, we demonstrate that gexp02 is expressed specifically in sexual parasites, with expression starting as early as the sexual ring stage, which makes it a candidate marker for circulating sexual rings in epidemiological studies

Conditional expression of PfAP2-G for controlled massive sexual conversion in Plasmodium falciparum.

Malaria transmission requires that some asexual parasites convert into sexual forms termed gametocytes. The initial stages of sexual development, including sexually committed schizonts and sexual rings, remain poorly characterized, mainly because they are morphologically identical to their asexual counterparts and only a small subset of parasites undergo sexual development. Here, we describe a system for controlled sexual conversion in the human malaria parasite Plasmodium falciparum, based on conditional expression of the PfAP2-G transcription factor. Using this system, ~90 percent of the parasites converted into sexual forms upon induction, enabling the characterization of committed and early sexual stages without further purification. We characterized sexually committed schizonts and sexual rings at the transcriptomic and phenotypic levels, which revealed down-regulation of genes involved in solute transport upon sexual commitment, among other findings. The new inducible lines will facilitate the study of early sexual stages at additional levels, including multiomic characterization and drug susceptibility assays

Conditional expression of PfAP2-G for controlled massive sexual conversion in Plasmodium falciparum

--- - i: - Plasmodium falciparum content: - "Malaria transmission requires that some asexual parasites convert into sexual forms termed gametocytes. The initial stages of sexual development, including sexually committed schizonts and sexual rings, remain poorly haracterized, mainly because they are morphologically identical to their sexual counterparts and only a small subset of parasites undergo sexual development. Here, we describe a system for controlled sexual conversion in the human malaria parasite " - ", based on conditional expression of the PfAP2-G transcription factor. Using this system, ~90 percent of the parasites converted into sexual forms upon induction, enabling the characterization of committed and early sexual stages without further purification. We characterized sexually committed schizonts and sexual rings at the transcriptomic and phenotypic levels, which revealed down-regulation of genes involved in solute transport upon sexual commitment, among other findings. The new inducible lines will facilitate the study of early sexual stages at additional levels, including multiomic characterization and drug susceptibility assays.

Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum

Human to vector transmission of malaria requires that some blood-stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here, we study this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually committed parasite stages that precede the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route in which PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon re-invasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Reanalysis of published single-cell RNA-sequencing (RNA-seq) data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in freshfrozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease

The private life of malaria parasites:Strategies for sexual reproduction

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector’s midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations (“strategies”) to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host

Characterization of sexual commitment and the early steps of sexual development in the human malaria parasite Plasmodium falciparum

Transmission of malaria requires that some parasites abandon asexual replication and develop into sexual stages termed gametocytes. The discovery of the transcription factor PfAP2-G as the master regulator of sexual conversion has boosted our understanding of sexual development. The pfap2-g locus is controlled by heterochromatin-based silencing, with only a few parasites activating the locus and committing to sexual development at each asexual cycle. How heterochromatin forms in pfap2-g is currently unknown. Moreover, the initial steps of sexual conversion after pfap2-g activation, together with the biology of sexually committed stages, remain poorly characterized. In this thesis, we identified a new route of sexual conversion involving direct conversion within the same cycle of initial PfAP2-G expression. We also developed a conditional activation system for PfAP2-G, achieving synchronous sexual conversion of the majority of parasites, which enables the characterization of sexually committed parasites and early sexual stages. Finally, in an attempt to elucidate the mechanisms behind heterochromatin formation in pfap2-g, we gained insight into the role of different elements in heterochromatin nucleation.Per a la transmissió de la malària, alguns paràsits han de deixar de replicar-se de forma asexual i desenvolupar-se com a estadis sexuals anomenats gametòcits. El descobriment del factor de transcripció PfAP2-G com a regulador clau de la conversió sexual ha permès entendre millor el procés. El locus pfap2-g està silenciat per la presència d’heterocromatina i només alguns paràsits activen el locus i inicien el desenvolupament sexual a cada cicle asexual. Actualment es desconeix com es forma heterocromatina a pfap2-g. A més a més, els primers passos de la conversió sexual després de l’activació de pfap2-g, juntament amb la biologia dels estadis que acabaran desenvolupant-se sexualment, estan molt poc caracteritzats. En aquesta tesis, hem identificat una nova ruta de conversió que consisteix en la conversió sexual directa, sense un nou cicle de replicació, just després de l’activació de PfAP2-G. També hem desenvolupat un sistema d’activació condicional de PfAP2-G que indueix la conversió sexual sincrònica de la majoria dels paràsits i que permet la caracterització dels estadis sexuals primerencs. Finalment, en un intent per entendre els mecanismes darrera la formació d’heterocromatina a pfap2-g, hem obtingut informació rellevant sobre el paper de diferents elements en la nucleació d’heterocromatina

Transcriptional variation in malaria parasites: why and how

Transcriptional differences enable the generation of alternative phenotypes from the same genome. In malaria parasites, transcriptional plasticity plays a major role in the process of adaptation to fluctuations in the environment. Multiple studies with culture-adapted parasites and field isolates are starting to unravel the different transcriptional alternatives available to Plasmodium falciparum and the underlying molecular mechanisms. Here we discuss how epigenetic variation, directed transcriptional responses and also genetic changes that affect transcript levels can all contribute to transcriptional variation and, ultimately, parasite survival. Some transcriptional changes are driven by stochastic events. These changes can occur spontaneously, resulting in heterogeneity within parasite populations that provides the grounds for adaptation by dynamic natural selection. However, transcriptional changes can also occur in response to external cues. A better understanding of the mechanisms that the parasite has evolved to alter its transcriptome may ultimately contribute to the design of strategies to combat malaria to which the parasite cannot adapt