Functional analysis of red blood cell determinants of Plasmodium vivax invasion

Background: Plasmodium vivax parasites have a unique tropism for infecting reticulocytes, the youngest red blood cells (RBCs). Reticulocyte maturation is accompanied by changes in abundance of surface membrane proteins such as the transferrin receptor (CD71). To date there is only one known P. vivax host receptor, the Duffy Antigen Receptor for Chemokines (DARC) which is found on reticulocytes and erythrocytes. In order to find essential P. vivax host receptors responsible for reticulocyte tropism, we used quantitative surface membrane proteomics to identify proteins unique to reticulocytes. We have developed an in vitro RBC culture system, using an erythroleukemia cell line, to enable functional analysis of reticulocyte specific gene knockouts. Materials and Methods: Reticulocytes and erythrocytes do not have nuclei precluding direct genetic analysis, therefore we exploited an immortalized cell line, JK-1, to produce nucleated RBCs (that we term jkRBCs) that enable production of in vitro genetic mutants. To overexpress DARC, we introduced a construct with DARC under the control of the β-globin promoter. We generated candidate gene knockouts via the CRISPR/Cas9 system, and knockouts from individual clones were validated by Sanger sequencing, flow cytometry and quantitative surface proteomics. P. vivax invasion assays were performed using isolates both from Brazil and India. To identify candidates that are lost during reticulocyte maturation, we sorted peripheral blood reticulocyte populations by co-staining with thiazole orange and anti-CD71 antibodies followed by quantitative surface membrane proteomics. Results and conclusions: We have established an erythroid based in vitro culture system which facilitates P. vivax invasion and enables the genetic manipulation of RBC determinants. JkRBCs expressed all known P. falciparum host receptors and we observed P. falciparum invasion at levels comparable to RBCs. P. vivax invasion into jkRBCs was increased >3-fold by overexpression of DARC. Using gene knockouts, we observed that the essential P. falciparum receptors (BSG, CD55) were not required for P. vivax invasion. By quantitative surface proteomics, we identified several high priority candidates that, in addition to CD71, are more abundant in reticulocytes than erythrocytes. Currently we are deleting these candidates in order to identify essential receptors for P. vivax invasion and tropism to inform vaccine development.