The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor.

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein ß-spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV-located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in ß-spectrin cleavage and RBC rupture. Drug-like inhibitors of SERA6 autoprocessing similarly prevent ß-spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum.

BACKGROUND: The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13. METHODS: The reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0-3 h ring survival assay (RSA0-3 h) was investigated. RESULTS AND CONCLUSION: Introducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA0-3 h. The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds.

The Plasmodium falciparum rhoptry bulb protein RAMA plays an essential role in rhoptry neck morphogenesis and host red blood cell invasion.

The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite.

Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites.

Malaria is caused by Plasmodium parasites, which invade and replicate in erythrocytes. For Plasmodium falciparum, the major cause of severe malaria in humans, a heterotrimeric complex comprised of the secreted parasite proteins, PfCyRPA, PfRIPR and PfRH5 is essential for erythrocyte invasion, mediated by the interaction between PfRH5 and erythrocyte receptor basigin (BSG). However, whilst CyRPA and RIPR are present in most Plasmodium species, RH5 is found only in the small Laverania subgenus. Existence of a complex analogous to PfRH5-PfCyRPA-PfRIPR targeting BSG, and involvement of CyRPA and RIPR in invasion, however, has not been addressed in non-Laverania parasites. Here, we establish that unlike P. falciparum, P. knowlesi and P. vivax do not universally require BSG as a host cell invasion receptor. Although we show that both PkCyRPA and PkRIPR are essential for successful invasion of erythrocytes by P. knowlesi parasites in vitro, neither protein forms a complex with each other or with an RH5-like molecule. Instead, PkRIPR is part of a different trimeric protein complex whereas PkCyRPA appears to function without other parasite binding partners. It therefore appears that in the absence of RH5, outside of the Laverania subgenus, RIPR and CyRPA have different, independent functions crucial for parasite survival.

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.

Regulation and Essentiality of the StAR-related Lipid Transfer (START) Domain-containing Phospholipid Transfer Protein PFA0210c in Malaria Parasites.

StAR-related lipid transfer (START) domains are phospholipid- or sterol-binding modules that are present in many proteins. START domain-containing proteins (START proteins) play important functions in eukaryotic cells, including the redistribution of phospholipids to subcellular compartments and delivering sterols to the mitochondrion for steroid synthesis. How the activity of the START domain is regulated remains unknown for most of these proteins. The Plasmodium falciparum START protein PFA0210c (PF3D7_0104200) is a broad-spectrum phospholipid transfer protein that is conserved in all sequenced Plasmodium species and is most closely related to the mammalian START proteins STARD2 and STARD7. PFA0210c is unusual in that it contains a signal sequence and a PEXEL export motif that together mediate transfer of the protein from the parasite to the host erythrocyte. The protein also contains a C-terminal extension, which is very uncommon among mammalian START proteins. Whereas the biochemical properties of PFA0210c have been characterized, the function of the protein remains unknown. Here, we provide evidence that the unusual C-terminal extension negatively regulates phospholipid transfer activity. Furthermore, we use the genetically tractable Plasmodium knowlesi model and recently developed genetic technology in P. falciparum to show that the protein is essential for growth of the parasite during the clinically relevant asexual blood stage life cycle. Finally, we show that the regulation of phospholipid transfer by PFA0210c is required in vivo, and we identify a potential second regulatory domain. These findings provide insight into a novel mechanism of regulation of phospholipid transfer in vivo and may have important implications for the interaction of the malaria parasite with its host cell.

Binding of Plasmodium falciparum Merozoite Surface Proteins DBLMSP and DBLMSP2 to Human Immunoglobulin M Is Conserved among Broadly Diverged Sequence Variants.

Diversity at pathogen genetic loci can be driven by host adaptive immune selection pressure and may reveal proteins important for parasite biology. Population-based genome sequencing of Plasmodium falciparum, the parasite responsible for the most severe form of malaria, has highlighted two related polymorphic genes called dblmsp and dblmsp2, which encode Duffy binding-like (DBL) domain-containing proteins located on the merozoite surface but whose function remains unknown. Using recombinant proteins and transgenic parasites, we show that DBLMSP and DBLMSP2 directly and avidly bind human IgM via their DBL domains. We used whole genome sequence data from over 400 African and Asian P. falciparum isolates to show that dblmsp and dblmsp2 exhibit extreme protein polymorphism in their DBL domain, with multiple variants of two major allelic classes present in every population tested. Despite this variability, the IgM binding function was retained across diverse sequence representatives. Although this interaction did not seem to have an effect on the ability of the parasite to invade red blood cells, binding of DBLMSP and DBLMSP2 to IgM inhibited the overall immunoreactivity of these proteins to IgG from patients who had been exposed to the parasite. This suggests that IgM binding might mask these proteins from the host humoral immune system.

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain.

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Plasmodium falciparum†SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle.

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.

Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5.

There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.

RON12, a novel Plasmodium-specific rhoptry neck protein important for parasite proliferation.

Apicomplexan parasites invade host cells by a conserved mechanism: parasite proteins are secreted from apical organelles, anchored in the host cell plasma membrane, and then interact with integral membrane proteins on the zoite surface to form the moving junction (MJ). The junction moves from the anterior to the posterior of the parasite resulting in parasite internalization into the host cell within a parasitophorous vacuole (PV). Conserved as well as coccidia-unique rhoptry neck proteins (RONs) have been described, some of which associate with the MJ. Here we report a novel RON, which we call RON12. RON12 is found only in Plasmodium and is highly conserved across the genus. RON12 lacks a membrane anchor and is a major soluble component of the nascent PV. The bulk of RON12 secretion happens late during invasion (after parasite internalization) allowing accumulation in the fully formed PV with a small proportion of RON12 also apparent occasionally in structures resembling the MJ. RON12, unlike most other RONs is not essential, but deletion of the gene does affect parasite proliferation. The data suggest that although the overall mechanism of invasion by Apicomplexan parasites is conserved, additional components depending on the parasite-host cell combination are required.

Population genomic scan for candidate signatures of balancing selection to guide antigen characterization in malaria parasites.

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study.

A multifunctional serine protease primes the malaria parasite for red blood cell invasion.

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in 'priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.

Targeted disruption of py235ebp-1: invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein.

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand.

Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6'-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognise endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

Sequential roles for red blood cell binding proteins enable phased commitment to invasion for malaria parasites.

Invasion of red blood cells (RBCs) by Plasmodium merozoites is critical to their continued survival within the host. Two major protein families, the Duffy binding-like proteins (DBPs/EBAs) and the reticulocyte binding like proteins (RBLs/RHs) have been studied extensively in P. falciparum and are hypothesized to have overlapping, but critical roles just prior to host cell entry. The zoonotic malaria parasite, P. knowlesi, has larger invasive merozoites and contains a smaller, less redundant, DBP and RBL repertoire than P. falciparum. One DBP (DBPα) and one RBL, normocyte binding protein Xa (NBPXa) are essential for invasion of human RBCs. Taking advantage of the unique biological features of P. knowlesi and iterative CRISPR-Cas9 genome editing, we determine the precise order of key invasion milestones and demonstrate distinct roles for each family. These distinct roles support a mechanism for phased commitment to invasion and can be targeted synergistically with invasion inhibitory antibodies.