An unusual trafficking domain in MSRP6 defines a complex needed for Maurer’s clefts anchoring and maintenance in P. falciparum infected red blood cells

Intracellular malaria blood stage parasites remodel their host cell, a process essential for parasite survival and a cause of pathology in malaria infections. Host cell remodeling depends on the export of different classes of exported parasite proteins into the infected red blood cell (RBC). Here we show that members of a recently discovered group of difficult to predict exported proteins harbor an N-terminal export domain, similar to other classes of exported proteins, indicating that this is a common theme among all classes of exported proteins. For one such protein, MSRP6 (MSP-7 related protein 6), we identified a second, untypical export-mediating domain that corresponded to its MSP7-like region. In addition to its function in export, this domain also mediated attachment to the Maurer’s clefts, prominent parasite-induced structures in the host cell where MSRP6 is located. Using BioID with the Maurer’s clefts attachment domain of MSRP6 to identify interactors and compartment neighbors in live parasites we discovered a novel complex of proteins at the Maurer’s clefts. We show that this complex is necessary for the anchoring and maintaining the structural integrity of the Maurer’s clefts. The Maurer’s clefts are believed to be involved in the transport of the major virulence factor PfEMP1 to the host cell surface where it mediates cytoadherence of infected RBCs to endothelial cells, a main reason for the importance of host cell modifications for parasite virulence in the human host. Taking advantage of MSRP6 complex mutants and IT4 parasites that we modified to express only one specific PfEMP1 we find that abolishing Maurer’s clefts anchoring was neither needed for PfEMP1 transport to the host cell surface nor for cytoadherence. Altogether, this work reveals parasite proteins involved in Maurer’s clefts anchoring and maintenance and unexpectedly finds that these functions are dispensable for virulence factor transport and surface display.

Virulence determinant, PTP7, controls vesicle budding from the Maurer’s clefts, adhesin protein trafficking and host cell remodeling in Plasmodium falciparum

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins P. falciparum malaria pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer’s cleft, and the red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer’s clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, found only in the Laverania clade of Plasmodium , is critical for efficient virulence protein trafficking.

Trafficking and Association of Plasmodium falciparum MC-2TM with the Maurer’s Clefts

In this study, we investigated stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in P. falciparum (3D7) infected erythrocytes. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence with antibodies reactive with PfMC-2TM. PfMC-2TM is sensitive to BFA treatment and permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associated with the MC and infected erythrocyte membrane and was resistant to extraction with alkaline sodium carbonate, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM is an additional marker of the nascent MCs.

Role of Plasmodium falciparum Protein GEXP07 in Maurer’s Cleft Morphology, Knob Architecture, and P. falciparum EMP1 Trafficking

ABSTRACT The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer’s clefts. We developed a method for efficient enrichment of Maurer’s clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer’s cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer’s cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer’s clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer’s clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer’s cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile. IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

Host Cytoskeleton Remodeling throughout the Blood Stages ofPlasmodium falciparum

SUMMARYThe asexual intraerythrocytic development ofPlasmodium falciparum, causing the most severe form of human malaria, is marked by extensive host cell remodeling. Throughout the processes of invasion, intracellular development, and egress, the erythrocyte membrane skeleton is remodeled by the parasite as required for each specific developmental stage. The remodeling is facilitated by a plethora of exported parasite proteins, and the erythrocyte membrane skeleton is the interface of most of the observed interactions between the parasite and host cell proteins. Host cell remodeling has been extensively described and there is a vast body of information on protein export or the description of parasite-induced structures such as Maurer’s clefts or knobs on the host cell surface. Here we specifically review the molecular level of each host cell-remodeling step at each stage of the intraerythrocytic development ofP. falciparum. We describe key events, such as invasion, knob formation, and egress, and identify the interactions between exported parasite proteins and the host cell cytoskeleton. We discuss each remodeling step with respect to time and specific requirement of the developing parasite to explain host cell remodeling in a stage-specific manner. Thus, we highlight the interaction with the host membrane skeleton as a key event in parasite survival.

The Plasmodium falciparum protein VCAP1 controls Maurer’s cleft morphology, knob architecture and PfEMP1 trafficking

The malaria parasite, Plasmodium falciparum, traffics the virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer’s clefts. We developed a method for efficient enrichment of Maurer’s clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterised or poorly characterised Maurer’s cleft proteins. We generated transfectants expressing GFP-fusions of 7 proteins and confirmed their Maurer’s cleft location. Using co-immunoprecipitation and mass spectrometry we have generated a protein interaction map of proteins at the Maurer’s clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer’s clefts. We focus on a putative PfEMP1 loading complex that includes the newly characterised virulence complex assembly protein 1 (VCAP1). Disruption of VCAP1 causes Maurer’s cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression and loss of the PfEMP1 directed adhesion. ΔVCAP1 parasite lines have a growth advantage compared to wildtype parasites; and the infected RBCs are more deformable and more osmotically fragile.ImportanceThe trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite infected RBCs is central to severe adhesion related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. This work adds significantly to our understanding of virulence protein trafficking and will provide crucial knowledge that will be required to determine the mechanisms underpinning parasite driven host cell remodelling, parasite survival within the host and virulence mechanisms.

A network-based approach reveals novel invasion and Maurer's clefts-related proteins in Plasmodium falciparum

To understand disease pathogenesis, all the disease-related proteins must be identified. In this work, known proteins were used to identify related novel proteins using RWR method on a dynamic P. falciparum protein–protein interaction network.

Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate that had been adapted to in vitro culture for a relatively shorter period, and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). Results: We show that P. falciparum extracellular vesicles (PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer’s clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that PfEVs may play a role in parasite-host interactions. Comparison of this dataset with previously published datasets helps to define a core secretome present in PfEVs. Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify new targets for interventions or diagnostics.