SummaryThe malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny merozoites that are released from infected cells via a poorly understood process called egress. The most abundant merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress.
In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress. malaria | egress | electron tomography | soft X-ray microscopy | electron energy loss spectroscopy T he major cause of severe human malaria is Plasmodium falciparum, and its asexual blood cycle is the source of all clinical disease (1). Egress is an important step in the blood life cycle, as it allows daughter merozoites produced by intracellular parasite replication to escape and invade new erythrocytes, thereby continuing and amplifying the infection. Merozoites develop within a parasitophorous vacuole (PV), a membrane-bound compartment that forms during invasion (2-4), so the daughter parasites have two compartments to escape (5, 6).Blood-stage malaria parasites replicate by schizogony, in which several rounds of nuclear division form a multinucleated syncytium called a schizont. Individual merozoites are then produced by an unusual form of cytokinesis called budding or segmentation, which involves invagination of the single plasma membrane of the schizont. Minutes before egress, the segmented schizont suddenly transforms from an irregular to a relatively symmetrical structure with the merozoites arranged around the central digestive vacuole (5). This process, referred to as "flower formation" or rounding up, is usually accompanied by noticeable swelling of the PV and apparent shrinkage of the host cell (4, 5, 7-9). The first membrane to rupture at egress is the parasitophorous vacuole membrane (PVM) (5,6,8). When the PV does not occupy the entire infected cell, the individual merozoites can be seen to be expelled into the blood cell cytosol seconds before they escape fr...
Key Points• Plasmodium falciparumgenerated cytoadherent knobs on infected erythrocytes contain a spiral framework linked to the red cell cytoskeleton.• The findings suggest a structural basis for transmission of shear forces in adhesion of infected cells.Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells. (Blood. 2016;127(3):343-351) IntroductionPlasmodium falciparum malaria remains one of the leading causes of child deaths globally, with the majority of cases occurring in subSaharan Africa and Southeast Asia. Although chemopreventive and vector control initiatives led to an estimated 42% reduction in mortality rates between 2000 and 2012, the emergence of artemisinin resistance highlights the importance of continued efforts to understand and interfere with the biology of the parasite. 1Of the 5 Plasmodium species capable of infecting humans, P falciparum and P vivax are the most prevalent, with P falciparum causing 90% of malaria-related deaths. P falciparum-infected erythrocytes become cytoadherent, causing erythrocyte sequestration in the microvasculature and avoiding clearance of infected cells by the spleen.2 Much of the virulence of P falciparum malaria has been attributed to this cytoadherence, which impedes blood circulation and results in severe syndromes such as cerebral or placental malaria. 2-4The dominant ligand mediating cytoadherence is PfEMP1, a major variable erythrocyte...
Summary Many large biological macromolecules have inherent structural symmetry, being composed of a few distinct subunits, repeated in a symmetric array. These complexes are often not amenable to traditional high-resolution structural determination methods, but can be imaged in functionally relevant states using cryo-electron microscopy (cryo-EM). A number of methods for fitting atomic-scale structures into cryo-EM maps have been developed, including the molecular dynamics flexible fitting (MDFF) method. However, quality and resolution of the cryo-EM map are the major determinants of a method’s success. In order to incorporate knowledge of structural symmetry into the fitting procedure, we developed the symmetry-restrained MDFF method. The new method adds to the cryo-EM map-derived potential further restraints on the allowed conformations of a complex during fitting, thereby improving the quality of the resultant structure. The benefit of using symmetry-based restraints during fitting, particularly for medium to low-resolution data, is demonstrated for three different systems.
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