Genetic manipulation of cell line\xa0derived reticulocytes enables dissection of host malaria invasion requirements

Jake Baum,Ashley M. Toye,Katherine E. Wright,Jan Frayne,Timothy J. Satchwell,Pedro L. Moura,Joseph Hawksworth,Katy L. Haydn-Smith,Fernando Sánchez-Román Terán

doi:10.1038/s41467-019-11790-w

Abstract

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.

Highlights

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell
The selectivity index, which measures the propensity of the cells to support multiple invasions, was approximately twofold higher in reticulocytes derived from either cell source compared with red blood cells (Fig. 1d and Supplementary Fig. 1)
To assess whether rings observed in the reticulocytes complete the intracellular replication cycle, cytospins of infected BEL-Aderived reticulocytes and red blood cells were taken at several timepoints post-invasion across the intra-erythrocytic cycle (Fig. 1a, b)

Summary

Introduction

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Through lentiviral transduction of immature nucleated erythroblast precursors prior to differentiation it is possible to generate enucleated reticulocytes with rare or novel phenotypes to study host cell protein requirements and involvement in invasion The power of this approach was demonstrated in 2015 in a forward genetic screen employing shRNA-mediated knockdown of blood group proteins in primary in vitro-derived reticulocytes. These cells display a nucleated polychromatic erythroblast-like morphology and despite supporting parasite invasion were not able to support further parasite development[15] These cells can provide insight into the requirement of receptors, such as basigin, for attachment, and entry[15], the significant membrane complex remodeling and reduction of membrane protein abundance (basigin and CD44 in particular) that occur prior to and during erythroblast enucleation[7,16,17] means that observations made using this model may not extrapolate well to anucleate red blood cells

Methods

Results

Conclusion