Empowering the Experimental Biology of Plasmodium Vivax Through Elucidating Requirements for Ex Vivo Culture
Rangel, Gabriel W.
MetadataShow full item record
CitationRangel, Gabriel W. 2019. Empowering the Experimental Biology of Plasmodium Vivax Through Elucidating Requirements for Ex Vivo Culture. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractFor over 100 years, various research groups have been attempting to establish a continuous and robust in vitro culture system for the blood stages of various Plasmodium species, apicomplexan parasites that cause malaria, which is a testament to the potential importance of in vitro culture. Indeed, for P. falciparum, in vitro culture of blood stages has enabled countless experimental capabilities including large-scale antimalarial screens, vaccine development and production, and malaria transmission studies. However, there still exists no comparable in vitro culture system for P. vivax, greatly setting back the fight against this malaria parasite that puts one third of the global human population at risk for infection. Thus, this dissertation aimed to better understand and overcome various aspects of P. vivax intraerythrocytic biology that act as hurdles on the path toward culture.
In Chapter One, we review the current state of knowledge surrounding P. vivax, its impact on global health and malaria eradication efforts, the path toward in vitro culture of Plasmodium parasites and why this is a particularly difficult but necessary goal for P. vivax. In Chapter Two, attempting to maximize the experimental utility of cryopreserved P. vivax isolates, we initially focused on increasing the quantifiability of our assays by enriching parasites and increasing their survival through one round of maturation. We successfully applied reticulocyte enrichment techniques that minimize cellular dehydration to robustly enrich P. vivax-infected cells. We also identified Iscove’s Modified Dulbecco’s Medium from a culture media screen as a formulation that doubled P. vivax survival to mature gametocytes and schizonts. These advancements enabled the adaptation of an isotopic metabolic labelling assay for rapid and sensitive antimalarial assays. In Chapter Three, the enhanced enrichment and survival of P. vivax also enabled the application of low-input RNAseq technologies to generate robust transcriptome data from minimal parasite material. From this we establish the first transcriptome from purified P. vivax gametocytes, identifying novel markers and potential vaccine candidates. We also conclude that culture media has a relatively small effect on parasite transcriptome signature, suggesting the key effect of the culture media may be on host cells rather than the parasite itself. In Chapter Four, we focus on the host cells and utilize a surface proteomics approach to identify novel markers of very young members of the heterogeneous circulating reticulocyte population. P. vivax parasites within host cells positive for one of these markers, SLC12A6, are more likely to survive intraerythrocytic maturation, and this appears to be dependent on the potassium chloride cotransporter function of this protein. Finally, Chapter Five highlights the implications of the findings of this dissertation and suggests future directions the field of P. vivax molecular biology should take. Overall, this work has enhanced the capacity for molecular experimentation on P. vivax, revealed some important interactions between parasite and host-cell biology, and moved the field closer to a continuous and robust in vitro culture system.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:42029545
- FAS Theses and Dissertations