Genetic manipulation of stem cell derived red blood cells: dissecting the role of host receptor context and cytoskeletal disruption in malaria parasite invasion

The Plasmodium falciparum parasites that cause malaria survive by attaching to and invading circulating RBCs in which they subsequently multiply. Understanding the dynamic remodelling of the (RBC) membrane that occurs during malaria merozoite invasion and dissecting the involvement of host cell proteins specifically is a challenging proposition complicated by the genetic intractability of the anucleate RBC.

My work exploits an in vitro culture system that enables the expansion and terminal erythroid differentiation of haematopoietic stem cells and immortalised sources of erythroid precursors to generate reticulocytes (young RBCs) that support invasion by and intracellular development of P. falciparum.

By using CRISPR mediated gene editing or lentiviral transduction of erythroblasts prior to differentiation we are able to generate reticulocytes with unique phenotypes and characteristics that enable exploration of a variety of aspects of RBC biology including membrane biogenesis and protein interdependency during erythropoiesis, the identification of new blood group systems and the role of host proteins in malaria invasion.

Having established the invasive susceptibility of in vitro derived reticulocytes I am currently exploiting this system through generation of receptor knockouts alongside reticulocytes expressing novel domain swap hybrid surface receptor proteins, generating novel cellular models to specifically explore the role of membrane context of host receptor proteins including basigin and CD55 in invasion and will present ongoing work to this end.

Since extracellular attachment of the merozoite to the RBC potentiates transient cytoskeletal disruption events required for invasion, in addition to the role of surface receptors we are further investigating the basis of this host remodelling event, investigating parallels with capillary deformation and the role of phosphorylation of cytoskeletal adaptor proteins.