Receptor-based identification of an inhibitory peptide against blood stage malaria.

Plasmodium falciparum uses multiple host receptors to attach and invade human erythrocytes. Glycophorins have been implicated as receptors for parasite invasion in human erythrocytes. Here, we screened a phage display cDNA library of P. falciparum (FCR3, a sialic acid-dependent strain) using purified glycophorins and erythrocytes as bait. Several phage clones were identified that bound to immobilized glycophorins and contained the same 74 bp insert encoding the 7-amino acids sequence ETTLKSF. A similar screen using intact human erythrocytes in solution identified additional phage clones containing the same 7-amino acids sequence. Using ELISA and immunofluorescence, direct binding of ETTLKSF peptide to glycophorins and erythrocytes was confirmed. Pull-down and protease treatment assays suggest that ETTLKSF peptide specifically interacts with glycophorin C. The synthetic ETTLKSF peptide partially blocks merozoite invasion in human erythrocytes. Further characterization of ETTLKSF peptide could lead to the development of a novel class of inhibitors against the blood stage malaria.

Phage display: a useful tool for malaria research?

Defining the molecular intricacies of malaria pathogenesis is a vital area of medical and scientific research. Sophisticated methods have been developed to identify and characterise host-parasite interactions that are important in infection. Phage display involves the combinatorial display of proteins or peptides on the surface of bacteriophage. The technology provides an invaluable tool for screening diverse libraries for polypeptides that have a high affinity for a given target. Phage display in malaria research has proven successful, not only in mapping the protein-protein interactions that are important in Plasmodium biology, but also in the identification of molecules that might be exploited in the design of therapeutic agents or vaccines.

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein.

BACKGROUND: Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins. METHODS: 4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R. RESULTS: Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R. CONCLUSION: The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.

Myosin-like sequences in the malaria parasite Plasmodium falciparum bind human erythrocyte membrane protein 4.1.

BACKGROUND AND OBJECTIVES: Plasmodium falciparum malaria is one of the most lethal infectious diseases afflicting humanity. During development within the erythrocyte, P. falciparum induces significant modifications to the structure and function of the human erythrocyte membrane. This study focused on the identification of new protein-protein interactions between host and parasite. DESIGN AND METHODS: A novel application of in vitro display technology was used: P. falciparum phage display expression libraries were screened against purified human erythrocyte protein 4.1. DNA sequencing and bioinformatic analyses were used to identify parasite proteins that bind protein 4.1. RESULTS: P. falciparum proteins displaying strong binding specificity toward protein 4.1 included five hypothetical proteins, erythrocyte binding antigen-175, erythrocyte binding ligand-1 like protein and a putative serine/threonine kinase. A common binding motif displaying homology to muscle myosin and neurofilament sequences was also identified in four of the eight proteins. INTERPRETATION AND CONCLUSIONS: These proteins are potentially involved in the invasion and/or release, as well as the growth and survival of malaria parasites during development with the red blood cell. The characterization of novel protein interactions between P. falciparum and erythrocyte membrane protein 4.1 will lead to a better understanding of malaria pathogenesis and parasite biology.

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions.

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.