Matthew T. O'Neill

Exported Proteins Required for Virulence and Rigidity of Plasmodium falciparum-Infected Human Erythrocytes

Summary A major part of virulence for Plasmodium falciparum malaria infection, the most lethal parasitic disease of humans, results from increased rigidity and adhesiveness of infected host red cells. These changes are caused by parasite proteins exported to the erythrocyte using novel trafficking machinery assembled in the host cell. To understand these unique modifications, we used a large-scale gene knockout strategy combined with functional screens to identify proteins exported into parasite-infected erythrocytes and involved in remodeling these cells. Eight genes were identified encoding proteins required for export of the parasite adhesin PfEMP1 and assembly of knobs that function as physical platforms to anchor the adhesin. Additionally, we show that multiple proteins play a role in generating increased rigidity of infected erythrocytes. Collectively these proteins function as a pathogen secretion system, similar to bacteria and may provide targets for antivirulence based therapies to a disease responsible for millions of deaths annually.

Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.

Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II‐deficient Plasmodium yoelii liver stages failed to form exo‐erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.

Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process.

A Set of Glycosylphosphatidyl Inositol-Anchored Membrane Proteins of Plasmodium falciparum Is Refractory to Genetic Deletion

ABSTRACT Targeted gene disruption has proved to be a powerful approach for studying the function of important ligands involved in erythrocyte invasion by the extracellular merozoite form of the human malaria parasite, Plasmodium falciparum. Merozoite invasion proceeds via a number of seemingly independent alternate pathways, such that entry can proceed with parasites lacking particular ligand-receptor interactions. To date, most focus in this regard has been on single-pass (type 1) membrane proteins that reside in the secretory organelles. Another class of merozoite proteins likely to include ligands for erythrocyte receptors are the glycosylphosphatidyl inositol (GPI)-anchored membrane proteins that coat the parasite surface and/or reside in the apical organelles. Several of these are prominent vaccine candidates, although their functions remain unknown. Here, we systematically attempted to disrupt the genes encoding seven of the known GPI-anchored merozoite proteins of P. falciparum by using a double-crossover gene-targeting approach. Surprisingly, and in apparent contrast to other merozoite antigen classes, most of the genes (six of seven) encoding GPI-anchored merozoite proteins are refractory to genetic deletion, with the exception being the gene encoding merozoite surface protein 5 (MSP-5). No distinguishable growth rate or invasion pathway phenotype was detected for the msp-5 knockout line, although its presence as a surface-localized protein was confirmed.

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites

A small molecule inhibitor of the malarial protease Plasmepsin V impairs protein export and cellular remodeling, reducing parasite survival in human erythrocytes.

Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites.

Malaria is caused by obligate intracellular parasites, of which Plasmodium falciparum is the most lethal species. In humans, P. falciparum merozoites (invasive forms of the parasite) employ a host of parasite proteins to rapidly invade erythrocytes. One of these is the P. falciparum apical membrane antigen 1 (PfAMA1) which forms a complex with rhoptry neck proteins at the tight junction. Here, we have placed the Pfama1 gene under conditional control using dimerizable Cre recombinase (DiCre) in P. falciparum. DiCre‐mediated excision of the loxP‐flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle. This reduces growth by 40%, due to decreased invasion efficiency characterized by a post‐invasion defect in sealing of the parasitophorous vacuole. These results show that PfAMA1 is an essential protein for merozoite invasion in P. falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event.

A Next-generation Genetically Attenuated Plasmodium falciparum Parasite Created by Triple Gene Deletion

Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

Export of virulence proteins by malaria-infected erythrocytes involves remodeling of host actin cytoskeleton

Following invasion of human red blood cells (RBCs) by the malaria parasite, Plasmodium falciparum, a remarkable process of remodeling occurs in the host cell mediated by trafficking of several hundred effector proteins to the RBC compartment. The exported virulence protein, P falciparum erythrocyte membrane protein 1 (PfEMP1), is responsible for cytoadherence of infected cells to host endothelial receptors. Maurer clefts are organelles essential for protein trafficking, sorting, and assembly of protein complexes. Here we demonstrate that disruption of PfEMP1 trafficking protein 1 (PfPTP1) function leads to severe alterations in the architecture of Maurers clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors. PfPTP1 functions in a large complex of proteins and is required for linking of Maurers clefts to the host actin cytoskeleton.

Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.

Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.