An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes

Steven Batinovic,Kathryn Matthews,Scott A Chisholm,Emma Mchugh,Paul R Gilson,Tania F De Koning-Ward,Molly Parkyn Schneider,Matthew W A Dixon,Leann Tilley,Laure Dumont,Sarah C Charnaud,Boiyin Liu

doi:10.1038/ncomms16044

Abstract

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer’s clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo.

Highlights

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs)
The green fluorescent protein (GFP) and V5 epitope tags on PfEMP1B are recognized in transgenic parasites expressing the chimera in a compartment with a ‘necklace of beads’ profile at the parasite periphery (Fig. 1b; white arrows), as well as in punctate structures in the RBC cytoplasm (Fig. 1b; blue arrows), in agreement with a previous report[13]
Dual immunofluorescence labelling with heat shock protein-101 (HSP101), ring-exported protein-1 (REX1) and membraneassociated histidine-rich protein-2 (MAHRP2) showed patterns consistent with those observed for endogenous PfEMP1 (Supplementary Fig. 1a)[14,17,18,19]

Summary

Introduction

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). Conditional knockdown of the PTEX components heat shock protein-101 (HSP101) or PTEX-150 (PTEX150) prevents PfEMP1 export beyond the PVM10,11. It remains to be determined whether PfEMP1 is a direct substrate for PTEX or is dependent on other proteins that are exported via PTEX12. In transfectants expressing PfEMP1B, a proportion of the chimeric protein is delivered to the RBC surface[13], while some remains associated with intermediate trafficking compartments, including the PV and the Maurer’s clefts—a pattern reminiscent of the distribution of endogenous PfEMP1 (refs 14–17)

Methods

Results

Conclusion