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The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum , adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite’s nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pf centrin-1 and −4, microtubule-associated protein, End-binding protein-1, kinetochore protein, Pf NDC80 and centromere-associated protein, Pf CENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes. The sexual blood stages of Plasmodium falciparum develop through five morphologically distinct stages culminating in mature crescent-shaped gametocytes that can be transmitted from the mammalian host to the mosquito vector. Here, Li et al. apply different microscopy and tomography approaches to characterize how the microtubule organizing center and cytoplasmic and nuclear microtubules are organized and oriented during these different stages in the absence of genome replication and mitosis.

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, Pf NDC80 and Pf Nuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites. Using Ultra-Expansion Microscopy of the malaria parasite, P. falciparum , Li et al. show that disruption of kinetochore components breaks a nexus between the mitotic spindle and the nascent apical organelles.

Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, Plasmodium falciparum, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC.

The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology. Rhoptries are essential organelles for invasion of erythrocytes by Plasmodium . Here, the authors characterize the rhoptry-associated protein CERLI1 using quantitative super-resolution microscopy, showing that it is important for parasite invasion and secretion of rhoptry proteins including vaccine antigens.

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins the parasite’s pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer’s cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer’s clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, identified only in the Laverania clade of Plasmodium , is critical for efficient virulence protein trafficking.

Plasmodium falciparum mediates adhesion of infected red blood cells (RBCs) to blood vessel walls by assembling a multi-protein complex at the RBC surface. This virulence-mediating structure, called the knob, acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). In this work we developed correlative STochastic Optical Reconstruction Microscopy-Scanning Electron Microscopy (STORM-SEM) to spatially and temporally map the delivery of the knob-associated histidine-rich protein (KAHRP) and PfEMP1 to the RBC membrane skeleton. We show that KAHRP is delivered as individual modules that assemble in situ, giving a ring-shaped fluorescence profile around a dimpled disk that can be visualized by SEM. Electron tomography of negatively-stained membranes reveals a previously observed spiral scaffold underpinning the assembled knobs. Truncation of the C-terminal region of KAHRP leads to loss of the ring structures, disruption of the raised disks and aberrant formation of the spiral scaffold, pointing to a critical role for KAHRP in assembling the physical knob structure. We show that host cell actin remodeling plays an important role in assembly of the virulence complex, with cytochalasin D blocking knob assembly. Additionally, PfEMP1 appears to be delivered to the RBC membrane, then inserted laterally into knob structures.

The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer’s clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

Pathogenesis of malaria infections is linked to remodeling of erythrocytes, a process dependent on the trafficking of hundreds of parasite-derived proteins into the host erythrocyte. Recent studies have demonstrated that the Plasmodium translocon of exported proteins (PTEX) serves as the central gateway for trafficking of these proteins, as inducible knockdown of the core PTEX constituents blocked the trafficking of all classes of cargo into the erythrocyte. However, the role of the auxiliary component PTEX88 in protein export remains less clear. Here we have used inducible knockdown technologies in P. falciparum and P. berghei to assess the role of PTEX88 in parasite development and protein export, which reveal that the in vivo growth of PTEX88-deficient parasites is hindered. Interestingly, we were unable to link this observation to a general defect in export of a variety of known parasite proteins, suggesting that PTEX88 functions in a different fashion to the core PTEX components. Strikingly, PTEX88-deficient P. berghei were incapable of causing cerebral malaria despite a robust pro-inflammatory response from the host. These parasites also exhibited a reduced ability to sequester in peripheral tissues and were removed more readily from the circulation by the spleen. In keeping with these findings, PTEX88-deficient P. falciparum-infected erythrocytes displayed reduced binding to the endothelial cell receptor, CD36. This suggests that PTEX88 likely plays a specific direct or indirect role in mediating parasite sequestration rather than making a universal contribution to the trafficking of all exported proteins.

Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC.