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Identification of a Plasmodium falciparum phospholipid transfer protein.

van Ooij C, Withers-Martinez C, Ringel A, Cockcroft S, Haldar K, Blackman MJ - J. Biol. Chem. (2013)

Bottom Line: Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes.In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles.Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

ABSTRACT
Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.

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PFA0210c is an exported P. falciparum protein.A, the PFA0210c-His6-HA3-GFP fusion can be exported into the erythrocyte cytosol. In P. falciparum strain PfBLD390, which produces the same GFP fusion as PfBLD397, but where expression is controlled by the stronger calmodulin promoter, the GFP fusion is detected in the erythrocyte cytosol, indicating export beyond the PVM boundary. B and C, PfBLD397, expressing a fusion of PFA0210c to GFP controlled by its native promoter (see supplemental Fig. S2 for description of the fusion), was imaged at the early trophozoite stage (28 h after invasion; panels in B) and the schizont stage (46 h after invasion; panels in C). In the trophozoite stage, the protein is present in the characteristic circular pattern of the PV. In the schizont stage, the protein is recruited to organelles within the parasite.
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Figure 6: PFA0210c is an exported P. falciparum protein.A, the PFA0210c-His6-HA3-GFP fusion can be exported into the erythrocyte cytosol. In P. falciparum strain PfBLD390, which produces the same GFP fusion as PfBLD397, but where expression is controlled by the stronger calmodulin promoter, the GFP fusion is detected in the erythrocyte cytosol, indicating export beyond the PVM boundary. B and C, PfBLD397, expressing a fusion of PFA0210c to GFP controlled by its native promoter (see supplemental Fig. S2 for description of the fusion), was imaged at the early trophozoite stage (28 h after invasion; panels in B) and the schizont stage (46 h after invasion; panels in C). In the trophozoite stage, the protein is present in the characteristic circular pattern of the PV. In the schizont stage, the protein is recruited to organelles within the parasite.

Mentions: To obtain insight into the possible role of PFA0210c in the parasite, we sought to determine its subcellular localization. Our previous study that reported export of PFA0210c used a C-terminal fusion to GFP expressed under control of the strong P. falciparum calmodulin promoter (11). To confirm that the PFA0210c-His6-HA3-GFP fusion can also be exported, we produced a new construct designed to express PFA0210c-His6-HA3-GFP under control of the calmodulin promoter and introduced this into P. falciparum. GFP fluorescence was readily detected in the cytosol of the resulting parasite line (Fig. 6A), confirming that PFA0210c is an exported parasite protein. Examination of PFA0210c-His6-HA3-GFP expressed in synchronized PfBLD397 parasites at ∼28 h post-invasion (early trophozoite stage) revealed a much lower level of fluorescence (likely due to its expression from the native PFA0210c promoter in these parasites) and the erythrocyte cytosol signal was not obvious, likely due to fluorescence quenching by host cell hemoglobin. However, the visible GFP signal was still concentrated in a circular pattern indicative of localization in the PV (Fig. 6B), again confirming export beyond the parasite plasma membrane. Interestingly, examination of mature PfBLD397 schizonts revealed the fusion protein to be localized primarily in small compartments surrounding the edge of the parasite, suggesting that it could be transferred to an apical organelle in mature developmental stages (Fig. 6C). This is consistent with the finding that PFA0210c can be detected in merozoites (39).


Identification of a Plasmodium falciparum phospholipid transfer protein.

van Ooij C, Withers-Martinez C, Ringel A, Cockcroft S, Haldar K, Blackman MJ - J. Biol. Chem. (2013)

PFA0210c is an exported P. falciparum protein.A, the PFA0210c-His6-HA3-GFP fusion can be exported into the erythrocyte cytosol. In P. falciparum strain PfBLD390, which produces the same GFP fusion as PfBLD397, but where expression is controlled by the stronger calmodulin promoter, the GFP fusion is detected in the erythrocyte cytosol, indicating export beyond the PVM boundary. B and C, PfBLD397, expressing a fusion of PFA0210c to GFP controlled by its native promoter (see supplemental Fig. S2 for description of the fusion), was imaged at the early trophozoite stage (28 h after invasion; panels in B) and the schizont stage (46 h after invasion; panels in C). In the trophozoite stage, the protein is present in the characteristic circular pattern of the PV. In the schizont stage, the protein is recruited to organelles within the parasite.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814793&req=5

Figure 6: PFA0210c is an exported P. falciparum protein.A, the PFA0210c-His6-HA3-GFP fusion can be exported into the erythrocyte cytosol. In P. falciparum strain PfBLD390, which produces the same GFP fusion as PfBLD397, but where expression is controlled by the stronger calmodulin promoter, the GFP fusion is detected in the erythrocyte cytosol, indicating export beyond the PVM boundary. B and C, PfBLD397, expressing a fusion of PFA0210c to GFP controlled by its native promoter (see supplemental Fig. S2 for description of the fusion), was imaged at the early trophozoite stage (28 h after invasion; panels in B) and the schizont stage (46 h after invasion; panels in C). In the trophozoite stage, the protein is present in the characteristic circular pattern of the PV. In the schizont stage, the protein is recruited to organelles within the parasite.
Mentions: To obtain insight into the possible role of PFA0210c in the parasite, we sought to determine its subcellular localization. Our previous study that reported export of PFA0210c used a C-terminal fusion to GFP expressed under control of the strong P. falciparum calmodulin promoter (11). To confirm that the PFA0210c-His6-HA3-GFP fusion can also be exported, we produced a new construct designed to express PFA0210c-His6-HA3-GFP under control of the calmodulin promoter and introduced this into P. falciparum. GFP fluorescence was readily detected in the cytosol of the resulting parasite line (Fig. 6A), confirming that PFA0210c is an exported parasite protein. Examination of PFA0210c-His6-HA3-GFP expressed in synchronized PfBLD397 parasites at ∼28 h post-invasion (early trophozoite stage) revealed a much lower level of fluorescence (likely due to its expression from the native PFA0210c promoter in these parasites) and the erythrocyte cytosol signal was not obvious, likely due to fluorescence quenching by host cell hemoglobin. However, the visible GFP signal was still concentrated in a circular pattern indicative of localization in the PV (Fig. 6B), again confirming export beyond the parasite plasma membrane. Interestingly, examination of mature PfBLD397 schizonts revealed the fusion protein to be localized primarily in small compartments surrounding the edge of the parasite, suggesting that it could be transferred to an apical organelle in mature developmental stages (Fig. 6C). This is consistent with the finding that PFA0210c can be detected in merozoites (39).

Bottom Line: Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes.In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles.Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

ABSTRACT
Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.

Show MeSH
Related in: MedlinePlus