Trafficking and function of Maurer's clefts proteins and Maurer's clefts biogenesis
Date
2020
Authors
Zhang, Meng
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Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature human red blood cell (RBC) upon invasion for its own survival. The severe pathology of P. falciparum infection is associated with the display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected RBC and the subsequent cytoadhesion of infected cells in the microvasculature of host organs. This trafficking process is dependent on parasite-derived membranous cellular structures, Maurer's clefts (MCs). Currently, the underlying mechanism of how MCs aid trafficking and establishing the virulence complex is not well understood. The genesis and proper function of these compartments relies on a range of proteins and potentially lipids residing in MCs, but the underlying mechanisms remain elusive.
Here we characterised PFE60, a MCs resident protein that interacts with PfEMP1 trafficking protein 1 (PTP1) and is part of the virulence complex. We determined its role in the biogenesis of MCs and its involvement in trafficking of other parasite exported proteins. Based on these phenotypic analyses we propose a model on the function of this protein and how it interacts with other proteins in MCs. We demonstrated that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected RBCs. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore - unlike all other described resident MC membrane proteins - PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a RBC surface antigen.
Furthermore, we investigated the presence of different lipids in infected RBC, with particular focus on MCs. Membrane lipids are crucial for survival and stability of the iRBC and MCs are the most prominent membranous compartments that P. falciparum establishes in its host cell. By conducting a series of microscopic analyses on lipid distribution using lipid dyes, we detected polar lipids (e.g. phospholipids) and neutral lipids in the PVM, MCs and the iRBC membrane. We also developed APEX2-NT-Lys and APEX2-theta D4 molecular probes which can bind specifically to sphingomyelin and cholesterol for direct visualisation of distribution using electron microscopy. Using these probes we detected the presence of sphingomyelin and cholesterol in MCs and the iRBC membrane using transmission electron microscopy. In addition, we investigated the fate of MCs after intraerythrocytic development and their potential roles in the sexual development and transmission of the parasite. To this end, we characterised a protein (PFA0670c) which is expressed during early gametocyte stages and localised in MCs-like punctate structures.
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