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Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

Tawk L, Dubremetz JF, Montcourrier P, Chicanne G, Merezegue F, Richard V, Payrastre B, Meissner M, Vial HJ, Roy C, Wengelnik K, Lebrun M - PLoS Pathog. (2011)

Bottom Line: Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles.These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast.This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

View Article: PubMed Central - PubMed

Affiliation: UMR 5235 CNRS, Université Montpellier 1 & 2, Montpellier, France.

ABSTRACT
Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

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The PI3-kinase inhibitor LY294002 interferes with apicoplast biogenesis.(A) ddFYVE/FRN-RFP expressing parasites were treated with 100 µM LY294002 for 4 h or mock treated before detection of PI3P by ddFYVE stabilization for 20 min with 1 µM Shield-1. Note the efficient depletion of PI3P by the inhibitor, revealed by the cytosolic localization of ddFYVE. Seven of the eight parasites have lost their apicoplast while FNR- and DNA label accumulate in the residual body. Scale bar  = 2 µm. (B) Immunofluorescence analysis of a parasite treated with 50 µM LY294002 for 4 h using antibodies against the stromal protein Hsp60 shows swollen apicoplasts. (C, D) EM analysis of wild-type parasites fixed 4 hours after addition of 100 µM LY294002. (C). The parasite ultrastructure is not altered, except at the level of the apicoplast, which displays abnormal membrane whorls and myelinic profiles accumulating under the outer membrane of the organelle. a, apicoplast; IMC, inner membrane complex; m, mitochondrion; n, nucleus; PM, parasite plasma membrane; mic, micronemes; rh, rhoptrie; g, Golgi. (D) Accumulation of membranous whorls in the outer membranes of the apicoplast. Bar  = 0.5 µm.
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ppat-1001286-g003: The PI3-kinase inhibitor LY294002 interferes with apicoplast biogenesis.(A) ddFYVE/FRN-RFP expressing parasites were treated with 100 µM LY294002 for 4 h or mock treated before detection of PI3P by ddFYVE stabilization for 20 min with 1 µM Shield-1. Note the efficient depletion of PI3P by the inhibitor, revealed by the cytosolic localization of ddFYVE. Seven of the eight parasites have lost their apicoplast while FNR- and DNA label accumulate in the residual body. Scale bar  = 2 µm. (B) Immunofluorescence analysis of a parasite treated with 50 µM LY294002 for 4 h using antibodies against the stromal protein Hsp60 shows swollen apicoplasts. (C, D) EM analysis of wild-type parasites fixed 4 hours after addition of 100 µM LY294002. (C). The parasite ultrastructure is not altered, except at the level of the apicoplast, which displays abnormal membrane whorls and myelinic profiles accumulating under the outer membrane of the organelle. a, apicoplast; IMC, inner membrane complex; m, mitochondrion; n, nucleus; PM, parasite plasma membrane; mic, micronemes; rh, rhoptrie; g, Golgi. (D) Accumulation of membranous whorls in the outer membranes of the apicoplast. Bar  = 0.5 µm.

Mentions: To confirm the implication of PI3P in apicoplast biogenesis, we used an alternative strategy by inhibiting the PI3P-producing enzyme PI3-kinase. LY294002 and wortmannin are well established inhibitors of the PI3-kinase [28]. LY294002 showing the advantage of substantially higher stability in complex culture media, was used throughout the study. Intracellular ddFYVE- and FNR-RFP co-expressing parasites were treated with 100 µM LY294002 for four hours and depletion of PI3P as a consequence of PI3-kinase inhibition was evaluated by adding Shield-1 for 20 min at the end of the drug treatment. In comparison to mock-treated controls, LY294002 reduced the prominent subapical ddFYVE dot in T. gondii tachyzoites and led to diffuse labelling throughout the cytoplasm, reflecting efficient depletion of PI3P (Figure 3A). Depletion was also observed to a lesser extent at 50 and 25 µM (data not shown). Concomitantly, the localization of the FNR-RFP marker was disturbed and in many vacuoles most of it was now found in the residual body (Fig. 3A) while other cellular markers appeared not affected (Figure S5). Apicoplast loss was not significantly observed at dilutions below 100 µM LY294002 (Figure S6) but the apicoplast appeared rounder (Figure 3B) and the number of parasites showing a typical V-shaped elongated apicoplast was reduced (Figure S6), a morphological change that was not significantly observed in ddFYVE expressing parasites. Because 100 µM LY294002 is a high concentration compared to what is normally used on mammalian cells, we evaluated the structural integrity of LY294002 treated parasites by electron microscopy. As shown in Figure 3C, after 4 hours of 100 µM LY294002, the parasites displayed a normal organization, except that the apicoplast appeared misshaped with prominent internal myelinic profiles, reflecting membranous disorder (Figure 3C, D). In addition, and confirming the FNR-RFP observations, discarding of apicoplasts in the residual body was a common feature in LY294002 treated parasites (Figure S7). Taken together, these results strongly indicate that in T. gondii, PI3P is involved in cellular functions necessary for apicoplast biogenesis and inheritance in daughter cells.


Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

Tawk L, Dubremetz JF, Montcourrier P, Chicanne G, Merezegue F, Richard V, Payrastre B, Meissner M, Vial HJ, Roy C, Wengelnik K, Lebrun M - PLoS Pathog. (2011)

The PI3-kinase inhibitor LY294002 interferes with apicoplast biogenesis.(A) ddFYVE/FRN-RFP expressing parasites were treated with 100 µM LY294002 for 4 h or mock treated before detection of PI3P by ddFYVE stabilization for 20 min with 1 µM Shield-1. Note the efficient depletion of PI3P by the inhibitor, revealed by the cytosolic localization of ddFYVE. Seven of the eight parasites have lost their apicoplast while FNR- and DNA label accumulate in the residual body. Scale bar  = 2 µm. (B) Immunofluorescence analysis of a parasite treated with 50 µM LY294002 for 4 h using antibodies against the stromal protein Hsp60 shows swollen apicoplasts. (C, D) EM analysis of wild-type parasites fixed 4 hours after addition of 100 µM LY294002. (C). The parasite ultrastructure is not altered, except at the level of the apicoplast, which displays abnormal membrane whorls and myelinic profiles accumulating under the outer membrane of the organelle. a, apicoplast; IMC, inner membrane complex; m, mitochondrion; n, nucleus; PM, parasite plasma membrane; mic, micronemes; rh, rhoptrie; g, Golgi. (D) Accumulation of membranous whorls in the outer membranes of the apicoplast. Bar  = 0.5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040667&req=5

ppat-1001286-g003: The PI3-kinase inhibitor LY294002 interferes with apicoplast biogenesis.(A) ddFYVE/FRN-RFP expressing parasites were treated with 100 µM LY294002 for 4 h or mock treated before detection of PI3P by ddFYVE stabilization for 20 min with 1 µM Shield-1. Note the efficient depletion of PI3P by the inhibitor, revealed by the cytosolic localization of ddFYVE. Seven of the eight parasites have lost their apicoplast while FNR- and DNA label accumulate in the residual body. Scale bar  = 2 µm. (B) Immunofluorescence analysis of a parasite treated with 50 µM LY294002 for 4 h using antibodies against the stromal protein Hsp60 shows swollen apicoplasts. (C, D) EM analysis of wild-type parasites fixed 4 hours after addition of 100 µM LY294002. (C). The parasite ultrastructure is not altered, except at the level of the apicoplast, which displays abnormal membrane whorls and myelinic profiles accumulating under the outer membrane of the organelle. a, apicoplast; IMC, inner membrane complex; m, mitochondrion; n, nucleus; PM, parasite plasma membrane; mic, micronemes; rh, rhoptrie; g, Golgi. (D) Accumulation of membranous whorls in the outer membranes of the apicoplast. Bar  = 0.5 µm.
Mentions: To confirm the implication of PI3P in apicoplast biogenesis, we used an alternative strategy by inhibiting the PI3P-producing enzyme PI3-kinase. LY294002 and wortmannin are well established inhibitors of the PI3-kinase [28]. LY294002 showing the advantage of substantially higher stability in complex culture media, was used throughout the study. Intracellular ddFYVE- and FNR-RFP co-expressing parasites were treated with 100 µM LY294002 for four hours and depletion of PI3P as a consequence of PI3-kinase inhibition was evaluated by adding Shield-1 for 20 min at the end of the drug treatment. In comparison to mock-treated controls, LY294002 reduced the prominent subapical ddFYVE dot in T. gondii tachyzoites and led to diffuse labelling throughout the cytoplasm, reflecting efficient depletion of PI3P (Figure 3A). Depletion was also observed to a lesser extent at 50 and 25 µM (data not shown). Concomitantly, the localization of the FNR-RFP marker was disturbed and in many vacuoles most of it was now found in the residual body (Fig. 3A) while other cellular markers appeared not affected (Figure S5). Apicoplast loss was not significantly observed at dilutions below 100 µM LY294002 (Figure S6) but the apicoplast appeared rounder (Figure 3B) and the number of parasites showing a typical V-shaped elongated apicoplast was reduced (Figure S6), a morphological change that was not significantly observed in ddFYVE expressing parasites. Because 100 µM LY294002 is a high concentration compared to what is normally used on mammalian cells, we evaluated the structural integrity of LY294002 treated parasites by electron microscopy. As shown in Figure 3C, after 4 hours of 100 µM LY294002, the parasites displayed a normal organization, except that the apicoplast appeared misshaped with prominent internal myelinic profiles, reflecting membranous disorder (Figure 3C, D). In addition, and confirming the FNR-RFP observations, discarding of apicoplasts in the residual body was a common feature in LY294002 treated parasites (Figure S7). Taken together, these results strongly indicate that in T. gondii, PI3P is involved in cellular functions necessary for apicoplast biogenesis and inheritance in daughter cells.

Bottom Line: Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles.These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast.This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

View Article: PubMed Central - PubMed

Affiliation: UMR 5235 CNRS, Université Montpellier 1 & 2, Montpellier, France.

ABSTRACT
Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

Show MeSH
Related in: MedlinePlus