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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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Related in: MedlinePlus

Delayed death phenotype of parasites expressing ddFYVE.(A) Expression of ddFYVE does not reduce invasion of HFF cells. Confluent monolayers of HFF cells were incubated with tachyzoites previously incubated with or without Shield-1 for 48 h and invasion was assessed as described in materials and methods. Results are presented as invaded tachyzoite numbers per field. Values are the mean ± SEM (10 fields per assay, n = 4). (B) Expression of ddFYVE reduced intracellular growth after reinvasion. The effect of expression of ddFYVE on intracellular growth was measured (A) after 24 h of growth in presence or not of Shield-1, (B) on parasites pre-incubated intracellularly for 2 days in the presence or absence of Shield-1, then purified and allowed to reinvade HFF for 24 h. The number of vacuoles containing only one or two parasites was statistically higher for parasites having expressed ddFYVE during the previous cycle (P<0.01 for one parasite per vacuole and P<0.05 for two parasites per vacuole). Conversely, the number of vacuoles containing eight parasites was statistically lower as compared to wild type or to untreated ddFYVE transfectants (P<0.001). No effect was observed with untransfected (RH) parasites. Values are the mean ± SEM (100 vacuoles were counted per assay, n = 4) of one representative experiment. Graphs and statistical analysis were done with GraphPad Prism. Two-tailed P values were determined by unpaired t-test.
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ppat-1001286-g004: Delayed death phenotype of parasites expressing ddFYVE.(A) Expression of ddFYVE does not reduce invasion of HFF cells. Confluent monolayers of HFF cells were incubated with tachyzoites previously incubated with or without Shield-1 for 48 h and invasion was assessed as described in materials and methods. Results are presented as invaded tachyzoite numbers per field. Values are the mean ± SEM (10 fields per assay, n = 4). (B) Expression of ddFYVE reduced intracellular growth after reinvasion. The effect of expression of ddFYVE on intracellular growth was measured (A) after 24 h of growth in presence or not of Shield-1, (B) on parasites pre-incubated intracellularly for 2 days in the presence or absence of Shield-1, then purified and allowed to reinvade HFF for 24 h. The number of vacuoles containing only one or two parasites was statistically higher for parasites having expressed ddFYVE during the previous cycle (P<0.01 for one parasite per vacuole and P<0.05 for two parasites per vacuole). Conversely, the number of vacuoles containing eight parasites was statistically lower as compared to wild type or to untreated ddFYVE transfectants (P<0.001). No effect was observed with untransfected (RH) parasites. Values are the mean ± SEM (100 vacuoles were counted per assay, n = 4) of one representative experiment. Graphs and statistical analysis were done with GraphPad Prism. Two-tailed P values were determined by unpaired t-test.

Mentions: We thus evaluated whether ddFYVE expression would induce a delayed death phenotype. We first compared the number of parasites per vacuole 18 h after infection between ddFYVE expressing parasites grown in the presence of Shield-1 and parasites grown without drug. No significant reduction in parasite proliferation was found (Figure 4B). Then, intracellular ddFYVE parasites were treated or not with Shield-1 for 48 h and mechanically released from host cells before adding them to new host cells. We found no significant difference in the ability of untreated tachyzoites versus ddFYVE-expressing parasites to invade host cells (Figure 4A), showing that the invasion process was not affected. In contrast, pre-incubation of intracellular parasites with Shield-1 for 48 h before mechanical release and reinvasion of fresh cells followed by counting the number of parasites 24 h after invasion demonstrated a reduction in parasite growth of tachyzoites that had been grown in the presence of Shield-1 during the previous intracellular cycle (Figure 4B). The number of vacuoles containing one or two parasites was significantly higher for ddFYVE-expressing parasites grown in presence of Shield-1 while, conversely, the number of vacuoles containing eight parasites was significantly lower. Thus, the expression of a PI3P-binding module induced a delayed intracellular growth defect without affecting invasion as expected for apicoplast deprived parasites. This result also offered an explanation for our failure to generate and maintain stable parasite lines constitutively expressing high levels of GFP-2xFYVE.


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)

Delayed death phenotype of parasites expressing ddFYVE.(A) Expression of ddFYVE does not reduce invasion of HFF cells. Confluent monolayers of HFF cells were incubated with tachyzoites previously incubated with or without Shield-1 for 48 h and invasion was assessed as described in materials and methods. Results are presented as invaded tachyzoite numbers per field. Values are the mean ± SEM (10 fields per assay, n = 4). (B) Expression of ddFYVE reduced intracellular growth after reinvasion. The effect of expression of ddFYVE on intracellular growth was measured (A) after 24 h of growth in presence or not of Shield-1, (B) on parasites pre-incubated intracellularly for 2 days in the presence or absence of Shield-1, then purified and allowed to reinvade HFF for 24 h. The number of vacuoles containing only one or two parasites was statistically higher for parasites having expressed ddFYVE during the previous cycle (P<0.01 for one parasite per vacuole and P<0.05 for two parasites per vacuole). Conversely, the number of vacuoles containing eight parasites was statistically lower as compared to wild type or to untreated ddFYVE transfectants (P<0.001). No effect was observed with untransfected (RH) parasites. Values are the mean ± SEM (100 vacuoles were counted per assay, n = 4) of one representative experiment. Graphs and statistical analysis were done with GraphPad Prism. Two-tailed P values were determined by unpaired t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040667&req=5

ppat-1001286-g004: Delayed death phenotype of parasites expressing ddFYVE.(A) Expression of ddFYVE does not reduce invasion of HFF cells. Confluent monolayers of HFF cells were incubated with tachyzoites previously incubated with or without Shield-1 for 48 h and invasion was assessed as described in materials and methods. Results are presented as invaded tachyzoite numbers per field. Values are the mean ± SEM (10 fields per assay, n = 4). (B) Expression of ddFYVE reduced intracellular growth after reinvasion. The effect of expression of ddFYVE on intracellular growth was measured (A) after 24 h of growth in presence or not of Shield-1, (B) on parasites pre-incubated intracellularly for 2 days in the presence or absence of Shield-1, then purified and allowed to reinvade HFF for 24 h. The number of vacuoles containing only one or two parasites was statistically higher for parasites having expressed ddFYVE during the previous cycle (P<0.01 for one parasite per vacuole and P<0.05 for two parasites per vacuole). Conversely, the number of vacuoles containing eight parasites was statistically lower as compared to wild type or to untreated ddFYVE transfectants (P<0.001). No effect was observed with untransfected (RH) parasites. Values are the mean ± SEM (100 vacuoles were counted per assay, n = 4) of one representative experiment. Graphs and statistical analysis were done with GraphPad Prism. Two-tailed P values were determined by unpaired t-test.
Mentions: We thus evaluated whether ddFYVE expression would induce a delayed death phenotype. We first compared the number of parasites per vacuole 18 h after infection between ddFYVE expressing parasites grown in the presence of Shield-1 and parasites grown without drug. No significant reduction in parasite proliferation was found (Figure 4B). Then, intracellular ddFYVE parasites were treated or not with Shield-1 for 48 h and mechanically released from host cells before adding them to new host cells. We found no significant difference in the ability of untreated tachyzoites versus ddFYVE-expressing parasites to invade host cells (Figure 4A), showing that the invasion process was not affected. In contrast, pre-incubation of intracellular parasites with Shield-1 for 48 h before mechanical release and reinvasion of fresh cells followed by counting the number of parasites 24 h after invasion demonstrated a reduction in parasite growth of tachyzoites that had been grown in the presence of Shield-1 during the previous intracellular cycle (Figure 4B). The number of vacuoles containing one or two parasites was significantly higher for ddFYVE-expressing parasites grown in presence of Shield-1 while, conversely, the number of vacuoles containing eight parasites was significantly lower. Thus, the expression of a PI3P-binding module induced a delayed intracellular growth defect without affecting invasion as expected for apicoplast deprived parasites. This result also offered an explanation for our failure to generate and maintain stable parasite lines constitutively expressing high levels of GFP-2xFYVE.

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