Phage Display cDNA Screens Reveal a Specific Region of Plasmodium falciparum Glutamic Acid‐rich Protein that Binds to Human Red Blood Cells. A Functional Role in Malaria Pathogenesis

Abstract

Severe malaria caused by Plasmodium falciparum is a serious disease that affects a large number of people regardless of the mode of treatment with currently existing antimalarial drugs. In 2015, the Centers for Disease Control and Prevention (CDC) estimated nearly 214 million cases of malaria infection worldwide resulting in ~400,000 deaths mainly in sub‐Saharan Africa. Infection of red blood cells (RBCs) by P. falciparum, the most lethal species among the human malaria parasites, leads to several complications such as cytoadherence, which involves binding of infected red blood cells (iRBCs) to endothelial receptors, and rosetting, which is the binding of iRBCs to uninfected RBCs. These features contribute to specific organ damage and exacerbate disease pathogenesis. To elucidate the biological mechanisms of P. falciparum infection in human RBCs, we are currently investigating the virulence phenomena including parasite invasion and rosetting. Using phage display technology, we previously identified several novel ligand‐receptor interactions at the blood stage of malaria infection. Here, we used two independent phage display cDNA library screens and identified P. falciparum glutamic acid‐rich protein (Pf‐GARP) as a novel parasite ligand that binds to the surface of human RBCs. Specifically, we screened phage display cDNA libraries constructed from P. falciparum FCR‐3 and 3D7 strains and identified several overlapping Pf‐GARP clones expressing peptides that bind to intact RBCs. Two phage clones encoding Pf‐GARP392–437 and Pf‐GARP356–552 revealed a specific binding region located nearly in the middle of full length Pf‐GARP1–673. Using bioinformatics tools, we chemically synthesized a codon‐optimized segment, Pf‐GARP370–444, located within the overlapping phage clones. The phage cDNA inserts were cloned into pET‐32b vector, and recombinant peptides were expressed in E. coli. Recombinant fusion proteins encoded by all three Pf‐GARP cDNA were confirmed to bind human RBCs in vitro. The codon‐optimized Pf‐GARP370–444 was cloned into the pRE4 vector, and expressed on the surface of Chinese hamster ovary (CHO) cells. The transfected CHO cells formed rosette‐like clusters in the presence of uninfected human RBCs in vitro. Importantly, direct biochemical interaction between purified Pf‐GARP370–444 protein and human RBCs was abolished upon treatment of RBCs with chymotrypsin but not with trypsin and neuraminidase. These results indicate that a centrally‐located segment of Pf‐GARP recognizes a specific chymotrypsin‐sensitive receptor(s) on the surface of host RBCs. Our findings suggest a functional role of Pf‐GARP in the rosetting and invasion phenomena with implications in inter‐membrane transport and vaccine development against malaria.Support or Funding InformationFunding source: NIH‐HL060961