Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis.

Babesia orientalis, a major infectious agent of water buffalo hemolytic babesiosis, is transmitted by Rhipicephalus haemaphysaloides. However, no effective vaccine is available. Essential antigens that are involved in parasite invasion of host red blood cells (RBCs) are potential vaccine candidates. Therefore, the identification and the conduction of functional studies of essential antigens are highly desirable. Here, we evaluated the function of B. orientalis merozoite surface antigen 2c1 (BoMSA-2c1), which belongs to the variable merozoite surface antigen (VMSA) family in B. orientalis. We developed a polyclonal antiserum against the purified recombinant (r)BoMSA-2c1 protein. Immunofluorescence staining results showed that BoMSA-2c1 was expressed only on extracellular merozoites, whereas the antigen was undetectable in intracellular parasites. RBC binding assays suggested that BoMSA-2c1 specifically bound to buffalo erythrocytes. Cytoadherence assays using a eukaryotic expression system in vitro further verified the binding and inhibitory ability of BoMSA-2c1. We found that BoMSA-2c1 with a GPI domain was expressed on the surface of HEK293T cells that bound to water buffalo RBCs, and that the anti-rBoMSA2c1 antibody inhibited this binding. These results indicated that BoMSA-2c1 was involved in mediating initial binding to host erythrocytes of B. orientalis. Identification of the occurrence of binding early in the invasion process may facilitate understanding of the growth characteristics, and may help in formulating strategies for the prevention and control of this parasite.

Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites.

Invasion of human erythrocytes by the malaria parasite Plasmodium falciparum is a multi-step process. Previously, a forward genetic screen for P. falciparum host factors identified erythrocyte CD55 as essential for invasion, but its specific role and how it interfaces with the other factors that mediate this complex process are unknown. Using CRISPR-Cas9 editing, antibody-based inhibition, and live cell imaging, here we show that CD55 is specifically required for parasite internalization. Pre-invasion kinetics, erythrocyte deformability, and echinocytosis were not influenced by CD55, but entry was inhibited when CD55 was blocked or absent. Visualization of parasites attached to CD55-null erythrocytes points to a role for CD55 in stability and/or progression of the moving junction. Our findings demonstrate that CD55 acts after discharge of the parasite's rhoptry organelles, and plays a unique role relative to all other invasion receptors. As the requirement for CD55 is strain-transcendent, these results suggest that CD55 or its interacting partners may hold potential as therapeutic targets for malaria.

Acquisition and decay of IgM and IgG responses to merozoite antigens after Plasmodium falciparum malaria in Ghanaian children.

Developing a vaccine against Plasmodium falciparum malaria has been challenging, primarily due to high levels of antigen polymorphism and a complex parasite lifecycle. Immunization with the P. falciparum merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 has been shown to give rise to growth inhibitory and synergistic antisera. Therefore, these five merozoite proteins are considered to be promising candidates for a second-generation multivalent malaria vaccine. Nevertheless, little is known about IgG and IgM responses to these antigens in populations that are naturally exposed to P. falciparum. In this study, serum samples from clinically immune adults and malaria exposed children from Ghana were studied to compare levels of IgG and IgM specific for PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5. All five antigens were found to be specifically recognized by both IgM and IgG in serum from clinically immune adults and from children with malaria. Longitudinal analysis of the latter group showed an early, transient IgM response that was followed by IgG, which peaked 14 days after the initial diagnosis. IgG levels and parasitemia did not correlate, whereas parasitemia was weakly positively correlated with IgM levels. These findings show that IgG and IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 are high in children during P. falciparum malaria, but that the IgM induction and decline occurs earlier in infection than that of IgG.

Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.

Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A.

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.

Calcium-dependent Protein Kinases in Malaria Parasite Development and Infection.

Apicomplexan parasites have challenged researchers for nearly a century. A major challenge to developing efficient treatments and vaccines is the parasite's ability to change its cellular and molecular makeup to develop intracellular and extracellular niches in its hosts. Ca2+ signaling is an important messenger for the egress of the malaria parasite from the infected erythrocyte, gametogenesis, ookinete motility in the mosquito, and sporozoite invasion of mammalian hepatocytes. Calcium-dependent protein kinases (CDPKs) have crucial functions in calcium signaling at various stages of the parasite's life cycle; this therefore makes them attractive drug targets against malaria. Here, we summarize the functions of the various CDPK isoforms in relation to the malaria life cycle by emphasizing the molecular mechanism of developmental progression within host tissues. We also discuss the current development of anti-malarial drugs, such as how specific bumped kinase inhibitors (BKIs) for parasite CDPKs have been shown to reduce infection in Toxoplasma gondii, Cryptosporidium parvum, and Plasmodium falciparum. Our suggested combinations of BKIs, artemisinin derivatives with peroxide bridge, and inhibitors on the Ca(2+)-ATPase PfATP6 as a potential target should be inspected further as a treatment against malaria.

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion.

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.

Proteomic Analysis of Plasmodium Merosomes: The Link between Liver and Blood Stages in Malaria.

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for antimalarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique data set will allow us to explore key questions about the biology of merosomes and hepatic merozoites.

SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion.

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich-domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

Cellular dissection of malaria parasite invasion of human erythrocytes using viable Plasmodium knowlesi merozoites.

Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P. knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P. knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan-Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.

Targeting a Reticulocyte Binding Protein and Duffy Binding Protein to Inhibit Reticulocyte Invasion by Plasmodium vivax.

Plasmodium vivax merozoite invasion is restricted to Duffy positive reticulocytes. Merozoite interaction with the Duffy antigen is mediated by the P. vivax Duffy binding protein (PvDBP). The receptor-binding domain of PvDBP maps to an N-terminal cysteine-rich region referred to as region II (PvDBPII). In addition, a family of P. vivax reticulocyte binding proteins (PvRBPs) mediates interactions with reticulocyte receptors. The receptor binding domain of P. vivax reticulocyte binding protein 1a (PvRBP1a) maps to a 30 kD region (PvRBP1a30). Antibodies raised against recombinant PvRBP1a30 and PvDBPII recognize the native P. vivax antigens and inhibit their binding to host receptors. Rabbit IgG purified from sera raised against PvRBP1a30 and PvDBPII were tested individually and in combination for inhibition of reticulocyte invasion by P. vivax field isolates. While anti-PvDBPII rabbit IgG inhibits invasion, anti-PvRBP1a30 rabbit IgG does not show significant invasion inhibitory activity. Combining antibodies against PvDBPII and PvRBP1a30 also does not increase invasion inhibitory activity. These studies suggest that although PvRBP1a mediates reticulocyte invasion by P. vivax merozoites, it may not be useful to include PvRBP1a30 in a blood stage vaccine for P. vivax malaria. In contrast, these studies validate PvDBPII as a promising blood stage vaccine candidate for P. vivax malaria.

Characterizing Blood-Stage Antimalarial Drug MIC Values In Vivo Using Reinfection Patterns.

The MIC is an essential quantitative measure of the asexual blood-stage effect of an antimalarial drug. In areas of high malaria transmission, and thus frequent individual infection, patients who are treated with slowly eliminated antimalarials become reinfected as drug concentrations decline. In the frequent relapse forms of Plasmodium vivax and in Plasmodium ovale malaria, recurrent infection occurs from relapses which begin to emerge from the liver approximately 2 weeks after the primary illness. An important determinant of the interval from starting treatment of a symptomatic infection to the patency of these recurrent infections is the in vivo concentration-response relationship and thus the in vivo MIC. Using mechanistic knowledge of parasite asexual replication and the pharmacokinetic and pharmacodynamic properties of the antimalarial drugs, a generative statistical model was derived which relates the concentration-response relationship to time of reinfection patency. This model was used to estimate the in vivo MIC of chloroquine in the treatment of Plasmodium vivax malaria.

Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5.

The malaria parasite Plasmodium falciparum proliferates in the blood stream where the host immune system is most active. To escape from host immunity, P. falciparum has developed a number of evasion mechanisms. Serine repeat antigen 5 (SERA5) is a blood stage antigen highly expressed at late trophozoite and schizont stages. The P47 N-terminal domain of SERA5, the basis of SE36 antigen of the blood stage vaccine candidate under clinical trials, covers the merozoite surface. Exploring the role of the P47 domain, screening of serum proteins showed that vitronectin (VTN) directly binds to 20 residues in the C-terminal region of SE36. VTN co-localized with P47 domain in the schizont and merozoite stages. Phagocytosis assay using THP-1 cells demonstrated that VTN bound to SE36 prevented engulfment of SE36-beads. In addition, several serum proteins localized on the merozoite surface, suggesting that host proteins camouflage merozoites against host immunity via binding to VTN.

Parasite Recognition and Signaling Mechanisms in Innate Immune Responses to Malaria.

Malaria caused by the Plasmodium family of parasites, especially P.falciparum and P. vivax, is a major health problem in many countries in the tropical and subtropical regions of the world. The disease presents a wide array of systemic clinical conditions and several life-threatening organ pathologies, including the dreaded cerebral malaria. Like many other infectious diseases, malaria is an inflammatory response-driven disease, and positive outcomes to infection depend on finely tuned regulation of immune responses that efficiently clear parasites and allow protective immunity to develop. Immune responses initiated by the innate immune system in response to parasites play key roles both in protective immunity development and pathogenesis. Initial pro-inflammatory responses are essential for clearing infection by promoting appropriate cell-mediated and humoral immunity. However, elevated and prolonged pro-inflammatory responses owing to inappropriate cellular programming contribute to disease conditions. A comprehensive knowledge of the molecular and cellular mechanisms that initiate immune responses and how these responses contribute to protective immunity development or pathogenesis is important for developing effective therapeutics and/or a vaccine. Historically, in efforts to develop a vaccine, immunity to malaria was extensively studied in the context of identifying protective humoral responses, targeting proteins involved in parasite invasion or clearance. The innate immune response was thought to be non-specific. However, during the past two decades, there has been a significant progress in understanding the molecular and cellular mechanisms of host-parasite interactions and the associated signaling in immune responses to malaria. Malaria infection occurs at two stages, initially in the liver through the bite of a mosquito, carrying sporozoites, and subsequently, in the blood through the invasion of red blood cells by merozoites released from the infected hepatocytes. Soon after infection, both the liver and blood stage parasites are sensed by various receptors of the host innate immune system resulting in the activation of signaling pathways and production of cytokines and chemokines. These immune responses play crucial roles in clearing parasites and regulating adaptive immunity. Here, we summarize the knowledge on molecular mechanisms that underlie the innate immune responses to malaria infection.

P. falciparum RH5-Basigin interaction induces changes in the cytoskeleton of the host RBC.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.