Abstract
Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.
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Acknowledgements
We would like to thank Dr. Toyoshi Fujimoto (Juntendo University) for kindly gifting the wild-type yeast (SEY6210), and Editage (www.editage.com) for English language editing. This study was supported by JSPS KAKENHI [Grant No. JP22K19252, JP20H03154, JP17H03935, and JP16K15056], research grants from the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, Takeda Science Foundation, the Naito Foundation, ONO Medical Research Foundation, The NOVARTIS Foundation (Japan) for the Promotion of Science, the Uehara Memorial Foundation (to A.F.), and the Cooperation Research Grant of the National Research Center for Protozoan Diseases at Obihiro University of Agriculture and Veterinary Medicine (to A.F.). The funding sources had no involvement in the study design; the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the article for publication.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by RK, KF, SK, TM, XX, and AF. The first draft of the manuscript was written by AF, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Supplementary file1. Fig. S1 Lack of GST-MFGE8-C2 labeling in cho1∆ yeast. Absence of GST-MFGE8-C2 labeling in the plasma membrane (A, Pp), endoplasmic reticulum (ER) (yellow, Eo and pink, Pi in B), nuclear (C), and vacuolar (D) membranes of cho1∆ yeast lacking phosphatidylserine (PtdSer) synthesis. Eo, E-face of the outer ER membrane; Pp, P-face of the plasma membrane; Pi, P-face of the inner ER membrane. Scale bar, 200 nm (EPS 6770 KB)
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Supplementary file2. Fig. S2 Gold labeling densities of GST-MFGE8-C2 increased concomitantly with the concentration of phosphatidylserine (PtdSer) in liposomes. Labeling of GST-MFG8-C2 in the unilamellar liposomes containing 0.1–5 mol% PtdSer (PS) by the quick-freeze and freeze-fracture labeling method. The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than 80 randomly selected liposomes. The values for all concentrations were statistically different between the various conditions (e.g., 1 mol vs. 2 mol%; mean ± SEM; Student’s t test; P < 0.05). Scale bar, 100 nm (EPS 6921 KB)
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Supplementary file3. Fig. S3 Distributions of phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) in the plasma membrane of HFF-1 and the plasma and ER membranes of yeast cells. Replicas of HFF-1 and budding yeast cells were labeled with GST-MFGE8-C2 (Aa, Ca, Da) and biotin-duramycin (Ab, Cb, Db). Data are presented as means ± SEM of three independent experiments (n > 30). B and E Gold labeling densities of PtdSer (a) and PtdEtn (b). In the plasma membrane, both PtdSer and PtdEtn were predominantly located in the cytoplasmic leaflets (PF) of the HFF-1 as well as the yeast cells. The gold labeling densities of both PtdSer and PtdEtn on the EF (yellow) were similar to the PF (pink) of the ER membrane, and the expression levels of PtdSer and PtdEtn in the plasma membrane PF were higher and similar, respectively, when compared to those on the EF in the WT yeast (D). Scale bar, 200 nm (A) and 500 nm (C, D) (EPS 21482 KB)
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Supplementary file4. Fig. S4 Distribution of phosphatidylserine (PtdSer) in the cortical endoplasmic reticulum membrane of wild-type (WT) yeast cells. PtdSer labeling was observed in the cytoplasmic leaflets of both the outer (Po, blue in B) and inner (Pi, pink in A) membranes in the cortical endoplasmic reticulum of WT yeast. Eo, E-face of the outer ER membrane; Ep, E-face of the plasma membrane; Po, P-face of the outer ER membrane; Pp, P-face of the plasma membrane; Pi, P-face of the inner ER membrane. Scale bar, 200 nm (EPS 9222 KB)
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Konishi, R., Fukuda, K., Kuriyama, S. et al. Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis. Histochem Cell Biol 160, 279–291 (2023). https://doi.org/10.1007/s00418-023-02218-0
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DOI: https://doi.org/10.1007/s00418-023-02218-0