Calcium-Dependent Protein Kinase 5 Is Required for Release of Egress-Specific Organelles in Plasmodium falciparum

ABSTRACT The human malaria parasite Plasmodium falciparum requires efficient egress out of an infected red blood cell for pathogenesis. This egress event is highly coordinated and is mediated by several signaling proteins, including the plant-like P. falciparum calcium-dependent protein kinase 5 (PfCDPK5). Knockdown of PfCDPK5 results in an egress block where parasites are trapped inside their host cells. The mechanism of this PfCDPK5-dependent block, however, remains unknown. Here, we show that PfCDPK5 colocalizes with a specialized set of parasite organelles known as micronemes and is required for their discharge, implicating failure of this step as the cause of the egress defect in PfCDPK5-deficient parasites. Furthermore, we show that PfCDPK5 cooperates with the P. falciparum cGMP-dependent kinase (PfPKG) to fully activate the protease cascade critical for parasite egress. The PfCDPK5-dependent arrest can be overcome by hyperactivation of PfPKG or by physical disruption of the arrested parasite, and we show that both treatments facilitate the release of the micronemes required for egress. Our results define the molecular mechanism of PfCDPK5 function and elucidate the complex signaling pathway of parasite egress

Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis.

New techniques for obtaining electron microscopy data through the cell volume are being increasingly utilized to answer cell biologic questions. Here, we present a three-dimensional atlas of Plasmodium falciparum ultrastructure throughout parasite cell division. Multiple wild type schizonts at different stages of segmentation, or budding, were imaged and rendered, and the 3D structure of their organelles and daughter cells are shown. Our high-resolution volume electron microscopy both confirms previously described features in 3D and adds new layers to our understanding of Plasmodium nuclear division. Interestingly, we demonstrate asynchrony of the final nuclear division, a process that had previously been reported as synchronous. Use of volume electron microscopy techniques for biological imaging is gaining prominence, and there is much we can learn from applying them to answer questions about Plasmodium cell biology. We provide this resource to encourage readers to consider adding these techniques to their cell biology toolbox

Essential function of alveolin PfIMC1g in the Plasmodium falciparum asexual blood stage

ABSTRACT The cytoskeleton of Plasmodium parasites is essential for replication, motility, and infectivity. Plasmodium falciparum leverages a family of cytoskeletal proteins known as alveolins to meet these diverse needs. The functional role of individual alveolins in Plasmodium blood stages, however, remains unexplored. Here, we demonstrate that the alveolin PfIMC1g (PF3D7_0525800) is essential for P. falciparum asexual replication. Unlike alveolins studied in mosquito stages, PfIMC1g does not play an important role in determining cell shape. PfIMC1g-deficient parasites exhibit only minor defects during segmentation, with most merozoites being indistinguishable from wild type by super-resolution fluorescence microscopy, ultrastructure expansion microscopy, and electron microscopy. In the current study, we demonstrate that the absence of PfIMC1g leads to parasite death shortly after merozoite internalization into red blood cells (RBCs). PfIMC1g-deficient parasites egress and enter new RBCs but fail to develop into rings and die. We hypothesize that the primary role of PfIMC1g is to maintain structural integrity, protecting parasites from incurring damage during the process of internalization. Along with the dispensability of PfIMC1g for merozoite cell shape, we report new findings about the architecture of Plasmodium alveolins including the localization of PfIMC1e and 1f to the basal complex. Importance Infection by the Plasmodium falciparum parasite is responsible for the most severe form of human malaria. The asexual blood stage of the parasite, which occurs inside human red blood cells, is responsible for the symptoms of malaria and is the target of most antimalarial drugs. Plasmodium spp. rely on their highly divergent cytoskeletal structures to scaffold their cell division, sustain the mechanical stress of invasion, and survive in both the human bloodstream and the mosquito. We investigate the function of a class of divergent intermediate filament-like proteins called alveolins in the clinically important blood stage. The functional role of individual alveolins in Plasmodium remains poorly understood due to pleiotropic effects of gene knockouts and redundancy among alveolins. We evaluate the localization and essentiality of the four asexual-stage alveolins and find that PfIMC1g and PfIMC1c are essential. Furthermore, we demonstrate that PfIMC1g is critical for survival of the parasite post-invasion