Integrative transcriptome and phenome analysis reveals unique regulatory cascades controlling the intraerythrocytic asexual and sexual development of human malaria parasites

Abstract

The Plasmodium falciparum parasite, the major causative agent of malaria on the African continent, has evolved numerous cellular adaptations to effectively propagate its species. The parasite can proliferate asexually, producing mass amounts of progeny to subsist in the human host or differentiate into sexual forms (gametocytes) that, once mature, can transmit to a feeding Anopheles mosquito. Key to our ability to effectively develop chemical candidates that interfere with either of these processes is the identification and understanding of critical factors that regulate parasite development. This is particularly true for the development of antimalarials that can be used in malaria elimination strategies by targeting both parasite proliferation and transmission. We therefore hypothesized that parasite proliferation and differentiation use divergent mechanisms for gene expression that could be observed through a thorough investigation of the functional genome of these different parasite forms. This doctoral study therefore set out to increase our knowledge base on three crucial aspects of parasite development: 1) the atypical cell cycle that allows the rapid proliferation of asexual parasites; 2) the full molecular profile of gametocytogenesis enabling the cellular differentiation that allows the parasite to transmit; and 3) the metabolic differences between these proliferating and differentiating parasites that results from their strategy-specific mechanisms of developmental control. The atypical cell cycle of the parasite, associated with the massive cell number expansion in asexual development, is notoriously difficult to study. Here, we contributed a novel system by developing a cell cycle synchronization tool that reversibly blocks the development of asexual parasites at the G1/S transition. This results in an inescapable arrest of the cell cycle that is completely and functionally reversible; parasites re-initiate cell cycle progression and continue to S phase within 6 h. This system provided the opportunity to characterize cell cycle phases in the parasite and additionally evaluate molecular mechanisms associated with cell cycle arrest or re-initiation. During cell cycle arrest, the parasite enters a quiescent state reminiscent of a mitogen-activated restriction point. This arrest is unique and solely attributed to the removal of the specific mitogens within this system, polyamines. These analyses indicate the close interaction between transcriptional regulation and signal transduction cascades in the progression through the parasite’s cell cycle and for the first time highlight aspects of controlled cell cycle regulation in Plasmodium. In contrast to proliferation, the process of sexual differentiation only started receiving attention in the past few years. As such, we lack fundamental understanding of the mechanisms driving the unique gametocyte differentiation of P. falciparum parasites. This study contributes a detailed analysis of gametocyte differentiation that revealed distinct developmental transitions demarcating the start of gametocytogenesis, intermediate gametocyte development and finally maturation to produce the transmissible mature gametocytes. The study provides evidence for coordinated regulation of gene expression on a transcriptional level. We propose a model for regulation of gametocytogenesis in malaria parasites that involves active repression of gene sets mediated through epigenetics and RNA destabilization as well as active transcription of gene sets through successive ApiAP2 transcription factor activity. This data provides the most detailed framework of coordinated gene regulation events underlying development of P. falciparum gametocytes to date, a unique resource for the malaria community. The comprehensive and complex transcriptional regulation described for the proliferation and differentiation of the parasite led us to evaluate the functional consequence thereof. A whole cell phenotype microarray system was evaluated for its ability to measure the metabolic processes that define asexual and sexual stage metabolism as a functional consequence of changed gene expression profiles during proliferation and differentiation. The study provided metabolic profiles detailing carbon and nitrogen metabolism in asexual parasites, mature and immature gametocyte stages. The data highlighted dipeptide metabolism as a distinguishing feature in mature gametocytes and showed the presence of a low, delayed metabolic state concurrent with reduced transcriptional activity observed in this stage. These results show that gene expression changes associated with differentiation compared to proliferation translate to an observable metabolic phenotype and that transcriptional regulation shapes the molecular landscape underlying crucial events that enable the parasite’s intraerythrocytic asexual and sexual development.