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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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Expression and localization of PI3P in T. gondii.(A) Extracellular tachyzoites were labelled with [32P]orthophosphate, and phosphatidylinositol-monophosphates were analyzed by a combination of thin layer chromatography and HPLC. Retention times of 36 and 45.5 min identified the peaks as PI3P and PI4P, respectively. (B) Schematic representation of constructs expressed in T. gondii. PI3P-binding capacity of the constructs, the ability to generate stable parasite clones, and the observed localizations are indicated. (C) Fluorescence microscopy analysis of parasites transiently transfected with GFP-2xFYVE. Arrowheads indicate the PI3P-enriched compartment at the sub-apical pole of the parasite and arrows point to the accumulation of the FYVE marker in the residual body. Scale bar  = 2 µm. (D) Western blot analysis of ddFYVE and ddFYVEm proteins. Intracellular parasites grown with or without Shield-1 (1 µM) were lysed from host cells by passage through a 26-gauge needle and boiled in reducing sample buffer and then separated by SDS PAGE, before probing with anti-GFP. Equal numbers of parasites were loaded in each lane. Time of Shield-1 incubation is indicated. The difference of mobility between ddFYVE and ddFYVEm has also been observed for GST-FYVE and GST-FYVEm recombinant proteins produced in E. coli (our unpublished data), and thus appears to be a consequence of these mutations. Anti-SAG1 antibody was used as a loading control. (E) ddFYVE expressing parasites were transiently transfected with Rab51-HA and processed for IFA in absence of Shield-1 using the anti-HA antibodies. ddFYVE is enriched in a compartment that is distinct from the one labelled with the endosomal marker Rab51-HA. (F) Fluorescence analysis of a parasite clone expressing both ddFYVE and FNR-RFP shows co-localization of both markers in the absence of Shield-1. (G) Stable transfected parasites with the mutated construct ddFYVEm show diffuse cytosolic staining after 20 min of Shield-1 induction (fluorescence of ddFYVEm was not observed in the absence of Shield-1). Scale bar  = 2 µm.
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ppat-1001286-g001: Expression and localization of PI3P in T. gondii.(A) Extracellular tachyzoites were labelled with [32P]orthophosphate, and phosphatidylinositol-monophosphates were analyzed by a combination of thin layer chromatography and HPLC. Retention times of 36 and 45.5 min identified the peaks as PI3P and PI4P, respectively. (B) Schematic representation of constructs expressed in T. gondii. PI3P-binding capacity of the constructs, the ability to generate stable parasite clones, and the observed localizations are indicated. (C) Fluorescence microscopy analysis of parasites transiently transfected with GFP-2xFYVE. Arrowheads indicate the PI3P-enriched compartment at the sub-apical pole of the parasite and arrows point to the accumulation of the FYVE marker in the residual body. Scale bar  = 2 µm. (D) Western blot analysis of ddFYVE and ddFYVEm proteins. Intracellular parasites grown with or without Shield-1 (1 µM) were lysed from host cells by passage through a 26-gauge needle and boiled in reducing sample buffer and then separated by SDS PAGE, before probing with anti-GFP. Equal numbers of parasites were loaded in each lane. Time of Shield-1 incubation is indicated. The difference of mobility between ddFYVE and ddFYVEm has also been observed for GST-FYVE and GST-FYVEm recombinant proteins produced in E. coli (our unpublished data), and thus appears to be a consequence of these mutations. Anti-SAG1 antibody was used as a loading control. (E) ddFYVE expressing parasites were transiently transfected with Rab51-HA and processed for IFA in absence of Shield-1 using the anti-HA antibodies. ddFYVE is enriched in a compartment that is distinct from the one labelled with the endosomal marker Rab51-HA. (F) Fluorescence analysis of a parasite clone expressing both ddFYVE and FNR-RFP shows co-localization of both markers in the absence of Shield-1. (G) Stable transfected parasites with the mutated construct ddFYVEm show diffuse cytosolic staining after 20 min of Shield-1 induction (fluorescence of ddFYVEm was not observed in the absence of Shield-1). Scale bar  = 2 µm.

Mentions: The T. gondii genome database, ToxoDB [15], features a single putative PI3-kinase (TGME49_015700) belonging to the class III, Vps34–type enzymes [16] as expected in a unicellular eukaryotic organism. To confirm the presence of PI3-kinase activity in T. gondii, purified extracellular tachyzoites were metabolically labelled with [32P]-orthophosphate and phosphoinositides were extracted and analysed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The phosphoinositide profile revealed the presence of two phosphatidylinositol monophosphates representing PI3P and PI4P (Figure 1A). The amount of PI3P was important representing about a quarter of total phosphatidylinositol monophosphates (23.4% +/−8.6% (SD), n = 3).


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)

Expression and localization of PI3P in T. gondii.(A) Extracellular tachyzoites were labelled with [32P]orthophosphate, and phosphatidylinositol-monophosphates were analyzed by a combination of thin layer chromatography and HPLC. Retention times of 36 and 45.5 min identified the peaks as PI3P and PI4P, respectively. (B) Schematic representation of constructs expressed in T. gondii. PI3P-binding capacity of the constructs, the ability to generate stable parasite clones, and the observed localizations are indicated. (C) Fluorescence microscopy analysis of parasites transiently transfected with GFP-2xFYVE. Arrowheads indicate the PI3P-enriched compartment at the sub-apical pole of the parasite and arrows point to the accumulation of the FYVE marker in the residual body. Scale bar  = 2 µm. (D) Western blot analysis of ddFYVE and ddFYVEm proteins. Intracellular parasites grown with or without Shield-1 (1 µM) were lysed from host cells by passage through a 26-gauge needle and boiled in reducing sample buffer and then separated by SDS PAGE, before probing with anti-GFP. Equal numbers of parasites were loaded in each lane. Time of Shield-1 incubation is indicated. The difference of mobility between ddFYVE and ddFYVEm has also been observed for GST-FYVE and GST-FYVEm recombinant proteins produced in E. coli (our unpublished data), and thus appears to be a consequence of these mutations. Anti-SAG1 antibody was used as a loading control. (E) ddFYVE expressing parasites were transiently transfected with Rab51-HA and processed for IFA in absence of Shield-1 using the anti-HA antibodies. ddFYVE is enriched in a compartment that is distinct from the one labelled with the endosomal marker Rab51-HA. (F) Fluorescence analysis of a parasite clone expressing both ddFYVE and FNR-RFP shows co-localization of both markers in the absence of Shield-1. (G) Stable transfected parasites with the mutated construct ddFYVEm show diffuse cytosolic staining after 20 min of Shield-1 induction (fluorescence of ddFYVEm was not observed in the absence of Shield-1). Scale bar  = 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001286-g001: Expression and localization of PI3P in T. gondii.(A) Extracellular tachyzoites were labelled with [32P]orthophosphate, and phosphatidylinositol-monophosphates were analyzed by a combination of thin layer chromatography and HPLC. Retention times of 36 and 45.5 min identified the peaks as PI3P and PI4P, respectively. (B) Schematic representation of constructs expressed in T. gondii. PI3P-binding capacity of the constructs, the ability to generate stable parasite clones, and the observed localizations are indicated. (C) Fluorescence microscopy analysis of parasites transiently transfected with GFP-2xFYVE. Arrowheads indicate the PI3P-enriched compartment at the sub-apical pole of the parasite and arrows point to the accumulation of the FYVE marker in the residual body. Scale bar  = 2 µm. (D) Western blot analysis of ddFYVE and ddFYVEm proteins. Intracellular parasites grown with or without Shield-1 (1 µM) were lysed from host cells by passage through a 26-gauge needle and boiled in reducing sample buffer and then separated by SDS PAGE, before probing with anti-GFP. Equal numbers of parasites were loaded in each lane. Time of Shield-1 incubation is indicated. The difference of mobility between ddFYVE and ddFYVEm has also been observed for GST-FYVE and GST-FYVEm recombinant proteins produced in E. coli (our unpublished data), and thus appears to be a consequence of these mutations. Anti-SAG1 antibody was used as a loading control. (E) ddFYVE expressing parasites were transiently transfected with Rab51-HA and processed for IFA in absence of Shield-1 using the anti-HA antibodies. ddFYVE is enriched in a compartment that is distinct from the one labelled with the endosomal marker Rab51-HA. (F) Fluorescence analysis of a parasite clone expressing both ddFYVE and FNR-RFP shows co-localization of both markers in the absence of Shield-1. (G) Stable transfected parasites with the mutated construct ddFYVEm show diffuse cytosolic staining after 20 min of Shield-1 induction (fluorescence of ddFYVEm was not observed in the absence of Shield-1). Scale bar  = 2 µm.
Mentions: The T. gondii genome database, ToxoDB [15], features a single putative PI3-kinase (TGME49_015700) belonging to the class III, Vps34–type enzymes [16] as expected in a unicellular eukaryotic organism. To confirm the presence of PI3-kinase activity in T. gondii, purified extracellular tachyzoites were metabolically labelled with [32P]-orthophosphate and phosphoinositides were extracted and analysed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The phosphoinositide profile revealed the presence of two phosphatidylinositol monophosphates representing PI3P and PI4P (Figure 1A). The amount of PI3P was important representing about a quarter of total phosphatidylinositol monophosphates (23.4% +/−8.6% (SD), n = 3).

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