Sickle-trait hemoglobin reduces adhesion to both CD36 and EPCR by Plasmodium falciparum-infected erythrocytes

Abstract

Sickle-trait hemoglobin protects against severe Plasmodium falciparum malaria. Severe malaria is governed in part by the expression of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that are encoded by var genes, specifically those variants that bind Endothelial Protein C Receptor (EPCR). In this study, we investigate the effect of sickle-trait on parasite var gene expression and function in vitro and in field-collected parasites. We mapped var gene reads generated from RNA sequencing in parasite cultures in normal and sickle-cell trait blood throughout the asexual lifecycle. We investigated sickle-trait effect on PfEMP1 interactions with host receptors CD36 and EPCR using static adhesion assays and flow cytometry. Var expression in vivo was compared by assembling var domains sequenced from total RNA in parasites infecting Malian children with HbAA and HbAS. Sickle-trait did not alter the abundance or type of var gene transcripts in vitro, nor the abundance of overall transcripts or of var functional domains in vivo. In adhesion assays using recombinant host receptors, sickle-trait reduced adhesion by 73–86% to CD36 and 83% to EPCR. Similarly, sickle-trait reduced the surface expression of EPCR-binding PfEMP1. In conclusion, Sickle-cell trait does not directly affect var gene transcription but does reduce the surface expression and function of PfEMP1. This provides a direct mechanism for protection against severe malaria conferred by sickle-trait hemoglobin.

Trial Registration: ClinicalTrials.gov Identifier: NCT02645604.

Author summary

Mutations that confer protection against severe malaria have evolved in humans, while malaria parasites have evolved extremely diverse var genes that encode proteins that enable the adhesion of infected red blood cells to host receptors to ensure parasite survival. In this study, we investigated the interplay between these two sides by examining the effect of the malaria protective sickle-cell trait hemoglobin mutations (HbAS) on the expression of parasite var genes, including those that cause severe malaria by mediating infected erythrocyte adhesion to host EPCR. We found that although var gene transcription was unaffected in vitro, HbAS reduced parasites ability to adhere to both CD36 and EPCR in vitro, by attenuating the expression of parasite proteins on the erythrocyte surface. Through RNA-sequencing we show that HbAS does not appear to cause any changes in overall var gene transcription, nor in the types of var genes expressed, and that children with HbAS mainly expressed var genes not associated with severe disease. Our results support a model in which HbAS confers protection from severe malaria by attenuating adhesion to host receptors.