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Even with the best available treatment, the mortality from severe Plasmodium falciparum malaria remains high. Typical features at death are high parasite loads and obstructed micro- vasculature. Infected erythrocytes (IE) containing mature parasites bind to the host receptor heparan sulfate, which is also an important receptor for merozoite invasion. To block merozoite invasion has not previously been proposed as an adjunctive therapeutic approach but it may preclude the early expansion of an infection that else leads to exacerbated sequestration and death. The drug sevuparin was developed from heparin because heparan sulfate and heparin are nearly identical, so the rationale was that sevuparin would act as a decoy receptor during malaria infection. A phase I study was performed in healthy male volunteers and sevuparin was found safe and well tolerated. A phase I/II clinical study was performed in which sevuparin was administered via short intravenous infusions to malaria patients with uncomplicated malaria who were also receiving atovaquone/proguanil treatment. This was a Phase I/II, randomized, open label, active control, parallel assignment study. Sevuparin was safe and well tolerated in the malaria patients. The mean relative numbers of ring-stage IEs decreased after a single sevuparin infusion and mature parasite IEs appeared transiently in the circulation. The effects observed on numbers of merozoites and throphozoites in the circulation, were detected already one hour after the first sevuparin injection. Here we report the development of a candidate drug named sevuparin that both blocks merozoite invasion and transiently de-sequesters IE in humans with P. falciparum malaria. ClinicalTrials.gov NCT01442168.

Egress of the malaria parasite Plasmodium falciparum from its host red blood cell is a rapid, highly regulated event that is essential for maintenance and completion of the parasite life cycle. Egress is protease-dependent and is temporally associated with extensive proteolytic modification of parasite proteins, including a family of papain-like proteins called SERA that are expressed in the parasite parasitophorous vacuole. Previous work has shown that the most abundant SERA, SERA5, plays an important but non-enzymatic role in asexual blood stages. SERA5 is extensively proteolytically processed by a parasite serine protease called SUB1 as well as an unidentified cysteine protease just prior to egress. However, neither the function of SERA5 nor the role of its processing is known. Here we show that conditional disruption of the SERA5 gene, or of both the SERA5 and related SERA4 genes simultaneously, results in a dramatic egress and replication defect characterised by premature host cell rupture and the failure of daughter merozoites to efficiently disseminate, instead being transiently retained within residual bounding membranes. SERA5 is not required for poration (permeabilization) or vesiculation of the host cell membrane at egress, but the premature rupture phenotype requires the activity of a parasite or host cell cysteine protease. Complementation of SERA5 null parasites by ectopic expression of wild-type SERA5 reversed the egress defect, whereas expression of a SERA5 mutant refractory to processing failed to rescue the phenotype. Our findings implicate SERA5 as an important regulator of the kinetics and efficiency of egress and suggest that proteolytic modification is required for SERA5 function. In addition, our study reveals that efficient egress requires tight control of the timing of membrane rupture.

The potential threat of parasite resistance to current antimalarials begs further research into antimalarial drug discovery to control disease progression. In addition, even when effective drugs are used, severe malaria symptoms still pose an important risk for death and cerebral residual disease in children. Further understanding of the pathophysiology of malaria and the biology of the parasite will open doors to new antimalarial treatments. Plasmodium falciparum malaria, an infectious disease caused by a parasitic protozoan, claims the lives of nearly a million children each year in Africa alone and is a top public health concern. Evidence is accumulating that resistance to artemisinin derivatives, the frontline therapy for the asexual blood stage of the infection, is developing in southeast Asia. Renewed initiatives to eliminate malaria will benefit from an expanded repertoire of antimalarials, including new drugs that kill circulating P. falciparum gametocytes, thereby preventing transmission. Our current understanding of the biology of asexual blood-stage parasites and gametocytes and the ability to culture them in vitro lends optimism that high-throughput screenings of large chemical libraries will produce a new generation of antimalarial drugs. There is also a need for new therapies to reduce the high mortality of severe malaria. An understanding of the pathophysiology of severe disease may identify rational targets for drugs that improve survival.

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways.

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.

Malaria pathogenesis and parasite multiplication depend on the ability of Plasmodium merozoites to invade human erythrocytes. Invasion is a complex multi-step process involving multiple parasite proteins which can differ between species and has been most extensively studied in P . falciparum . However, dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed P . falciparum merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified strains including some generated specifically for this work, we quantitated the contribution of individual P . falciparum proteins to these merozoite-erythrocyte attachment interactions. Conditional deletion of the major P . falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. By comparing over 4000 individual merozoite-erythrocyte interactions in a range of conditions and strains, we establish that the PfEBA/PfRH families play a central role in P . falciparum merozoite attachment, not the major merozoite surface protein PfMSP1.

During blood-stage infection by Plasmodium falciparum, merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts. We show that the half-life of merozoite invasive capacity after rupture is 5 min at 37 °C, and 15 min at room temperature. Studying the kinetics of invasion revealed that 80% of invasion events occur within 10 min of mixing merozoites and RBCs. Invasion efficiency was maximum at low merozoite-to-RBC ratios and occurred efficiently in the absence of serum and with high concentrations of dialyzed nonimmune serum. We developed and optimized an invasion assay by using purified merozoites that enabled invasion-inhibitory activity of antibodies and compounds to be measured separately from other mechanisms of growth inhibition; the assay was more sensitive for detecting inhibitory activity than established growth-inhibition assays. Furthermore, with the use of purified merozoites it was possible to capture and fix merozoites at different stages of invasion for visualization by immunofluorescence microscopy and EM. We thereby demonstrate that processing of the major merozoite antigen merozoite surface protein-1 occurs at the time of RBC invasion. These findings have important implications for defining invasion events and molecular interactions, understanding immune interactions, and identifying and evaluating inhibitors to advance vaccine and drug development.

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Malaria, a life-threatening disease caused by Plasmodium parasites, relies on a specialized stage, the merozoite to invade red blood cells and multiply in the bloodstream. While much research has focused on the molecular interactions between merozoite and host cell surface, invasion is a complex process that also requires the parasite to move actively, sense its environment, and respond to signals such as changes in ions or contact with the red blood cell. These cues trigger precise biological responses, including the secretion of specialized organelles and activation of gliding motility, enabling the parasite to successfully enter the host cell. This review provides an overview of current understanding of merozoite physiology, highlighting how motility, signal sensing, and secretory organelle function are coordinated to drive invasion. By exploring these processes, we gain insights into potential strategies for malaria control and intervention.