Spatial and temporal mapping of the PfEMP1 export pathway inPlasmodium falciparum

Abstract

Summary The human malaria parasite, Plasmodium falciparum, modifies the red blood cells (RBCs) that it infects by exporting proteins to the host cell. One key virulence protein, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), is trafficked to the surface of the infected RBC, where it mediates adhesion to the vascular endothelium. We have investigated the organization and development of the exomembrane system that is used for PfEMP1 trafficking. Maurer’s cleft cisternae are formed early after invasion and proteins are delivered to these (initially mobile) structures in a temporally staggered and spatially segregated manner. Membrane-Associated Histidine-Rich Protein-2 (MAHRP2)-containing tether-like structures are generated as early as 4 h post-invasion and become attached to Maurer’s clefts. The tether/ Maurer’s cleft complex docks onto the RBC membrane at ~20 h post-invasion via a process that is not affected by cytochalasin D treatment. We have examined the trafficking of a GFP chimera of PfEMP1 expressed in transfected parasites. PfEMP1B-GFP accumulates near the parasite surface, within membranous structures exhibiting a defined ultrastructure, before being transferred to pre-formed mobile Maurer’s clefts. Endogenous PfEMP1 and PfEMP1B-GFP are associated with Electron-Dense Vesicles that may be responsible for trafficking PfEMP1 from the Maurer’s clefts to the RBC membrane.

Maurer's clefts, the enigma of Plasmodium falciparum

“…In contrast, GFP-tagged Maurer's clefts resident proteins were found only in a punctuate manner at Maurer's clefts, indicating distinct, independent structures. In the case of a continuous network, fluorescent signal would have been observed throughout the network (38)(39)(40)(41)(42)(43)(44)(45), but in photo-bleaching experiments no lateral diffusion of Maurer's clefts resident proteins or lipids (41,44,75,76) was observed. In contrast, the erythrocyte membrane showed a fast recovery after photo-bleaching when BODIPYceramide was used (44).…”

“…Maurer's clefts look highly convoluted and interconnected in whole-cell imaging (21) (21). Fluorescence microscopy of erythrocytes infected with transfectant parasites expressing GFPchimera of resident proteins shows discrete puncta (38)(39)(40)(41)(42)(43)(44)(45), which cannot be replenished by lateral diffusion (41).…”

“…It is also unclear if Maurer's clefts resident proteins are loaded into the clefts because they are formed at the PVM, or if there is continuous transport of cargo to already existing Maurer's clefts. Recent live-imaging studies demonstrated that some proteins arrived at Maurer's clefts after these had been formed (45,81). …”

“…Other Maurer's clefts resident proteins, skeleton binding protein 1 (SBP1) and MAHRP1, appear somewhat later at 4-6 hpi (45,81), and at 6 hpi all three proteins are detected in most of the Maurer's clefts (45). All clefts seem to be formed at the early ring stage because no new clefts were observed after invasion (81) and the number of clefts remained constant as early as 8 hpi (45) 45). Taken together, these data indicate a constant flow of cargo from the parasite to the Maurer's clefts, the means by which, however, remains elusive.…”

“…Taken together, these data indicate a constant flow of cargo from the parasite to the Maurer's clefts, the means by which, however, remains elusive. Although Maurer's clefts are formed very early after invasion, they occur as highly mobile structures (41,45,81). Live imaging over the 48-h life cycle using REX2-GFPexpressing parasites revealed a movement of Maurer's clefts over three phases.…”