Building and maintaining the ultrastructure of Plasmodium falciparum parasites during the asexual blood stage.

Abstract

Apicomplexan parasites exhibit great diversity in cellular and subcellular morphology that is key to their ability to infect diverse host species and target cells. The study of this distinctive cell biology is often hindered by the small size and complex lifecycles of these organisms. This is especially true in the study of Plasmodium parasites, the causative agents of malaria. This thesis surveys the changing ultrastructure of Plasmodium falciparum during its asexual blood stages using cutting-edge microscopy techniques, and uses molecular methods to characterize cytoskeletal structures and protein-protein interactions responsible for building and maintaining this ultrastructure. In Chapter 2, we apply ultrastructural expansion microscopy (U-ExM) to describe the three-dimensional organization of P. falciparum parasites in the asexual blood stages. We catalogue 13 different P. falciparum structures and organelles across the intraerythrocytic development of this parasite with a focus on schizogony, the simultaneous division of a parasite into dozens of daughter cells. We use this dataset to trace the biogenesis and organization of each of these 13 structures, shedding light on multiple poorly understood but fundamental aspects of P. falciparum cell biology. The cytoskeleton of Plasmodium parasites is essential for cell structure, replication, motility, and infectivity. P. falciparum leverages a divergent family of cytoskeletal proteins known as alveolins to meet some of these diverse needs. In Chapter 3, we demonstrate that the alveolin PfIMC1g (PF3D7_0525800) is essential for P. falciparum asexual replication. We characterize nonviable PfIMC1g-deficient parasites and hypothesize that the primary role of PfIMC1g is to maintain structural integrity, protecting parasites from incurring damage during the process of invasion. We also report new findings on the architecture of Plasmodium alveolins, including an interaction between PfIMC1g and 1c and the localization of PfIMC1e and 1f to the basal complex. In Chapter 4, we extend our characterization of alveolin architecture and essentiality to map how these proteins are recruited to the parasite cytoskeleton. Specifically, we characterize alveolin-alveolin interactions mediated by the alveolin domain conserved within this family. We find that blood-stage alveolins have variable dependence on their alveolin domains and alveolin binding partners for function and localization. Overall, this work provides a more detailed picture of how P. falciparum parasites organize their cytoplasmic contents during cell division, along with an in-depth analysis of cytoskeletal structures assembled during this process to protect parasite integrity. Our study of alveolin function and dissection of relevant domains is the first of its kind in Plasmodium and will increase our understanding of these unique filaments, whose physiological importance and divergence from human proteins make them candidates for future drug development.