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Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton.

ABSTRACT: Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein-protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites.

SUBMITTER: Shakya B

PROVIDER: S-EPMC9320996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. The malaria parasite exports hundreds of proteins into the erythrocyte compartment. However, for most of these proteins, their physiological function is unknown. In this study, we investigate two "hypothetical" proteins of the α/β-hydrolase fold family that share sequence similarity with epoxide hydrolases (EHs)-enzymes that destroy bioactive epoxides. Altering EH expression in parasite-infected erythrocytes resulted in a significant change in the epoxide fatty acids stored in the host cell. We propose that these EH enzymes may help the parasite to manipulate host blood vessel opening and inflame the vessel walls as they pass through the circulation system. Understanding how the malaria parasite interacts with its host RBCs will aid in our ability to combat this deadly disease.

Project description:The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane.IMPORTANCE The malarial parasite within the erythrocyte is surrounded by two membranes. Plasmodium translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We now describe a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We find that RON3 is required for the protein transport function of the PTEX and for glucose transport from the RBC cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2.

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Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton.

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