Red blood cell invasion by Plasmodium vivax: structural basis for DBP engagement of DARC.

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.

Malaria adhesins: structure and function.

The malaria parasite Plasmodium utilizes specialized proteins for adherence to cellular receptors in its mosquito vector and human host. Adherence is critical for parasite development, host cell traversal and invasion, and protection from vector and host immune mechanisms. These vital roles have identified several adhesins as vaccine candidates. A deficiency in current adhesin-based vaccines is induction of antibodies targeting non-conserved, non-functional and decoy epitopes due to the use of full length proteins or binding domains. To alleviate the elicitation of non-inhibitory antibodies, conserved functional regions of proteins must be identified and exploited. Structural biology provides the tools necessary to achieve this goal, and has succeeded in defining biologically functional receptor binding and oligomerization interfaces for a number of promising malaria vaccine candidates. We describe here the current knowledge of Plasmodium adhesin structure and function, and how it has illuminated elements of parasite biology and defined interactions at the host/vector and parasite interface.

Molecular basis for sialic acid-dependent receptor recognition by the Plasmodium falciparum invasion protein erythrocyte-binding antigen-140/BAEBL.

Plasmodium falciparum erythrocyte invasion is dependent on high affinity recognition of sialic acid on cell surface receptors. The erythrocyte binding-like (EBL) family of invasion ligands mediates recognition of sialic acid on erythrocyte glycoproteins. Erythrocyte-binding antigen-140 (PfEBA-140/BAEBL) is a critical EBL ligand that binds sialic acid on its receptor glycophorin C. We present here the crystal structure of the two-domain receptor-binding region of PfEBA-140 in complex with a glycan containing sialic acid. The structure identifies two glycan-binding pockets unique to PfEBA-140 and not shared by other EBL ligands. Specific molecular interactions that enable receptor engagement are identified and reveal that the glycan binding mode is distinct from that of apicomplexan and viral cell surface recognition ligands as well as host immune factors that bind sialic acid. Erythrocyte binding experiments elucidated essential glycan contact residues and identified divergent functional roles for each receptor-binding site. One of four polymorphisms proposed to affect receptor binding was localized to a glycan-binding site, providing a structural basis for altered erythrocyte engagement. The studies described here provide the first full description of sialic acid-dependent molecular interactions at the P. falciparum erythrocyte invasion interface and define a framework for development of PfEBA-140-based therapeutics, vaccines, and diagnostics assessing vaccine efficacy and natural immunity to infection.

Crystal and solution structures of Plasmodium falciparum erythrocyte-binding antigen 140 reveal determinants of receptor specificity during erythrocyte invasion.

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.

Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion.

The most lethal form of malaria in humans is caused by Plasmodium falciparum. These parasites invade erythrocytes, a complex process involving multiple ligand-receptor interactions. The parasite makes initial contact with the erythrocyte followed by dramatic deformations linked to the function of the Erythrocyte binding antigen family and P. falciparum reticulocyte binding-like families. We show EBA-175 mediates substantial changes in the deformability of erythrocytes by binding to glycophorin A and activating a phosphorylation cascade that includes erythrocyte cytoskeletal proteins resulting in changes in the viscoelastic properties of the host cell. TRPM7 kinase inhibitors FTY720 and waixenicin A block the changes in the deformability of erythrocytes and inhibit merozoite invasion by directly inhibiting the phosphorylation cascade. Therefore, binding of P. falciparum parasites to the erythrocyte directly activate a signaling pathway through a phosphorylation cascade and this alters the viscoelastic properties of the host membrane conditioning it for successful invasion.

Reading, Writing, and Presenting Original Scientific Research: A Nine-Week Course in Scientific Communication for High School Students.

High school students are not often given opportunities to communicate scientific findings to their peers, the general public, and/or people in the scientific community, and therefore they do not develop scientific communication skills. We present a nine-week course that can be used to teach high school students, who may have no previous experience, how to read and write primary scientific articles and how to discuss scientific findings with a broad audience. Various forms of this course have been taught for the past 10 years as part of an intensive summer research program for rising high school seniors that is coordinated by the Young Scientist Program at Washington University in St. Louis. The format presented here includes assessments for efficacy through both rubric-based methods and student self-assessment surveys.