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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., Author summary The human malaria parasite Plasmodium falciparum causes severe disease, which is initiated by the adhesion of parasite-infected RBCs to receptors on the walls of the host’s capillaries. Adhesion is mediated by a structure called the knob, which acts as a scaffold for the presentation of the virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). In this work we investigate the assembly of this complex at different stages of parasite development using a multimodal imaging approach that combines dSTORM localization microscopy and scanning electron microscopy (STORM-SEM). We show that the knob-associated histidine-rich protein (KAHRP) is delivered to the RBC membrane skeleton as individual protein modules that assemble into a ring-shaped complex. We provide evidence that host cell remodeling, driven by association of KAHRP with spectrin and the reorganization of actin, is required for assembly of the ring complex, which in turn supports a spiral scaffold that is required for correct knob morphology. Finally, we provide evidence that PfEMP1 is delivered to the RBC membrane before associating with knob complexes.

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., Author summary Malaria occurs when Plasmodium parasites replicate inside red blood cells, with the number of parasitised cells (pRBC) correlating with disease severity. Antibodies are highly effective at controlling pRBC numbers in the bloodstream, and yet we know very little about how they function in vivo. Human in vitro studies predict that antibodies may function in a number of ways, including via phagocytes or different complement mechanisms. However, to date it has been challenging to explore how antibodies might control parasite numbers in vivo. Here, we have used a unique method in mice, where clearance and replication of a single cohort of pRBC was closely tracked in the presence of protective antibodies. Surprisingly, antibodies played no role whatsoever in accelerating the removal of pRBC. Instead, antibodies were highly effective at preventing parasites from progressing from one generation of pRBC to the next. This process partly depended on host phagocytes. However, we found no role for complement-mediated direct killing. Together, our in vivo data suggest in mouse models that naturally-acquired antibodies do not clear pRBC, and instead prevent transition from one red blood cell to the next.

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., Author summary Transmission of the malaria parasite from humans to mosquitoes relies on the formation of the specialised blood stage gametocyte. Plasmodium falciparum gametocytes mature over about 10 days, during which time they undergo a remarkable morphological transformation, eventually adopting a characteristic crescent shape. The shape changes are thought to facilitate the mechanical sequestration of maturing gametocytes within the bone marrow and spleen, as well as the eventual release into the circulation. Failure to mature correctly leads to a failure to transmit. Despite the importance of this process, little is known about the molecular basis of elongation. In this work, we introduce 3D Electron Microscopy of P. falciparum gametocytes and use it, in a combination with super-resolution optical microscopy, to elucidate the genesis and expansion of the molecular structures that drive gametocyte elongation. We use protein interaction profiling to identify some of the proteins that help drive the shape change and employ inducible gene knockdown strategies to show that these proteins play a role in remodeling membranes, and are needed for gametocyte elongation. This work points to potential targets for the development of transmission-blocking therapies.