Mutant/mutation
To confirm functionally that the C741Y substitution plays a key role in control of parasitemia and parasite virulence, we constructed two linear DNA fragments to replace C741 with Y741 in PyEBL of N67C (N67C_C-Y) parasites and Y741 with C741 in N67 (N67_Y-C) parasites (see RMgm-4705). We also constructed synonymous controls, replacing C741 with C741 in N67C (N67C_C-C) parasites and Y741 with Y741 in N67 (N67_Y-Y) parasites.

Protein (function)
Erythrocyte-binding-like (EBL) proteins are a family of type I transmembrane proteins that localize to micronemes of a malaria merozoite and, following microneme discharge, play a crucial role during the invasion of red blood cells (RBCs). An EBL protein generally has an N-terminal cysteine (Cys)-rich binding domain (region 2) and a C-terminal Cys-rich trafficking domain (C-Cys domain or region 6), along with a signal sequence and a transmembrane domain. The N-terminal Cys-rich region consists of one or two Duffy-binding-like (DBL) domains that bind host proteins like the Duffy antigen receptor for chemokines (DARC) or glycophorin A on the erythrocyte surface during invasion of RBCs. The C-terminal C-Cys domain directs the protein for microneme trafficking. Plasmodium yoelii has one EBL gene (Pyebl), encoding a protein (PyEBL) with a single DBL domain. A cysteine-to-arginine substitution at residue 713 (C713R) in the PyEBL C-Cys domain has been linked to parasite growth and virulence in vivo in the 17X lineage. The parasite line 17XL (or YM) has the R713 variant C-Cys domain, grows fast, and kills its host by day 7, whereas the 17X (17XNL) line has the C713 variant, grows slowly, and is cured by the host. The 17X, 17XL, 17XNL, and YM lines are isogenic parasites derived from a parasite isolated from a thicket rat. In addition, EBL protein with C713 is localized in merozoite micronemes, whereas the R713 protein is mostly mistargeted to dense granules (DGs). Although these results suggest that PyEBL has a direct role in parasite invasion and virulence, it is difficult to explain the observation that abnormal localization of the variant PyEBL can greatly improve the parasite growth rate. Recently, PyEBL was found to be translocated to the merozoite surface as a confined patch in both the 17XL and 17XNL lines of P. yoelii, which may help maintain normal invasion of RBCs but cannot explain the enhanced growth rate of 17XL.

We previously performed genetic crosses between Plasmodium yoelii nigeriensis strain N67 and 17XNL parasites and found a significant linkage of a growth-related virulence phenotype to genetic loci on chromosomes 7, 10, and 13. The chromosome 13 locus contains the gene encoding PyEBL, in which a Cys-to-Tyr substitution at residue 741 (C741Y) in the C-Cys domain was implicated in the differences of virulence between N67 and P. yoellii nigeriensis strain N67C parasite lines.

In C57BL/6 mice, infection with N67C resulted in 40 to 50% parasitemia and host death on day 7 postinfection (p.i.), whereas mice infected with N67 suppressed the parasitemia to under 5% on day 7 p.i. The suppression of N67 parasitemia was associated with N67 parasite stimulation of a strong early type I interferon (IFN-I) response.

Phenotype
In this study, we exchanged the C741 and Y741 alleles of PyEBL between N67 and N67C parasites and showed that allelic replacements altered parasite growth and virulence and host immune responses but not invasion of RBCs. 

 

- The C741Y substitution does not affect parasite invasion of RBCs in vitro.
- C741Y substitution alters PyEBL protein trafficking in merozoites and iRBCs
- The C741Y substitution affects PyEBL processing and host band 3 distribution
- The C741Y substitution increases iRBC osmotic fragility
- The C741Y substitution changes host spleen pathology and production of cytokines/chemokines
- Y741 linked to elevated IFN-I response, isotype switching, and T-helper cell differentiation
- Y741 iRBCs trigger strong PS-CD36-mediated phagocytosis

Additional information
Our study also reveals a previously unknown mechanism of host-parasite interaction mediated by PyEBL, showing increased phosphatidylserine (PS) exposure on infected RBCs (iRBCs), altered osmotic fragility, enhanced phagocytosis of iRBCs, an elevated IFN-I response, and improved parasitemia control after the C741Y substitution. This study demonstrates that a single amino acid substitution in a pathogen genome can result in major alterations in the host-parasite interface, with significant consequences for host viability, and provides a paradigm shift in our understanding of the roles of PyEBL in host-pathogen interaction during malarial-parasite infection.

N67C was originally named 33X(Pr3) and was deposited in MR4 under that name (MRA-754) by David Walliker, University of Edinburgh. Genotyping of 33X(Pr3), 33X, YM, 17XNL, and N67 parasite lines with microsatellites suggested that the 33X(Pr3) and N67 strains were closely related; therefore, 33X(Pr3) was renamed N67C

Other mutants