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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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PI3P-binding module disturbs apicoplast biogenesis.(A) The expression of ddFYVE in intracellular FNR-RFP/ddFYVE expressing parasites was induced by the addition of 1 µM Shield-1 and the localization of both markers was analyzed at the indicated time of incubation. Arrowheads indicate the formation of a PI3P-containing compartment next to the apicoplast and at the base of V-shaped dividing apicoplasts. DNA-, ddFYVE- and FNR labels in residual bodies (arrows) and tachyzoites that have lost their apicoplast, but retain the PI3P-containing compartment at the sub-apical pole (open triangles) are indicated. The lowest panel shows the cytosolic staining of ddFYVEm and the persistence of the apicoplast 18 h after Shield-1 induction. (B) Time-lapse microscopy of FNR-RFP/ddFYVE parasites. Elapsed time after addition of 1 µM Shield-1 is indicated. Early during apicoplast replication, ddFYVE remained concentrated near the apicoplast (190 min). When apicoplasts started to separate (240 min), a portion of FRN label remained associated with the ddFYVE compartment (open triangle). After completion of daughter cell formation (300 min), all ddFYVE label and part of FNR label accumulated in the residual body (arrow). The arrowhead points to the parasite that has lost the FNR label. (C) Two hours after Shield-1 induction, the accumulation of ddFYVE is observed at the base of the V-shaped elongated apicoplasts.
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ppat-1001286-g002: PI3P-binding module disturbs apicoplast biogenesis.(A) The expression of ddFYVE in intracellular FNR-RFP/ddFYVE expressing parasites was induced by the addition of 1 µM Shield-1 and the localization of both markers was analyzed at the indicated time of incubation. Arrowheads indicate the formation of a PI3P-containing compartment next to the apicoplast and at the base of V-shaped dividing apicoplasts. DNA-, ddFYVE- and FNR labels in residual bodies (arrows) and tachyzoites that have lost their apicoplast, but retain the PI3P-containing compartment at the sub-apical pole (open triangles) are indicated. The lowest panel shows the cytosolic staining of ddFYVEm and the persistence of the apicoplast 18 h after Shield-1 induction. (B) Time-lapse microscopy of FNR-RFP/ddFYVE parasites. Elapsed time after addition of 1 µM Shield-1 is indicated. Early during apicoplast replication, ddFYVE remained concentrated near the apicoplast (190 min). When apicoplasts started to separate (240 min), a portion of FRN label remained associated with the ddFYVE compartment (open triangle). After completion of daughter cell formation (300 min), all ddFYVE label and part of FNR label accumulated in the residual body (arrow). The arrowhead points to the parasite that has lost the FNR label. (C) Two hours after Shield-1 induction, the accumulation of ddFYVE is observed at the base of the V-shaped elongated apicoplasts.

Mentions: Despite the presence of a destabilization domain, a faint ddFYVE signal could already be observed by western blot analysis in the absence of Shield-1 treatment of the parasites (Figure 1D). The signal increased upon addition of the Shield-1 ligand, reaching a maximum after about 1 hour, and remaining stable thereafter (Figure 1D). The level of ddFYVE expression was considerably lower than that of the mutated version ddFYVEm, suggesting that it would be less tolerated by the parasite and might be degraded. IFA analysis detected ddFYVE in the absence of Shield-1 as a dot in the sub-apical part of the parasite (Figure 1E), similar to the one described above with transient transfection (Figure 1C). In other organisms PI3P is concentrated on early endosomes and co-localizes with Rab5 [17]. In T. gondii, however, we found that ddFYVE did not co-localize with the early endosome marker TgRab5.1 [12] (Figure 1E), but most unexpectedly co-localized instead with the apicoplast, whose DNA can be stained and appears as a small dot next to the nucleus (Figure 1E). This was subsequently confirmed by co-localization of ddFYVE with the FRN-RFP apicoplast marker [25] transfected in the ddFYVE strain (Figure 1F). Shield-1 treatment induced a progressive increase in fluorescence and some ddFYVE staining also appeared to surround the apicoplast (Figure 2A, see below). In striking contrast, the mutated form ddFYVEm was found throughout the cytoplasm (Figure 1G), confirming that the specific localization of the native construct was due to functional PI3P-binding domains. To confirm PI3P localization in T. gondii, we probed infected cells with anti-PI3P antibody and observed in each parasite a bright dot of fluorescence that partially co-localized with apicoplast (Figure S2). We therefore concluded that PI3P-enriched compartments are localized at the apicoplast and in its vicinity.


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)

PI3P-binding module disturbs apicoplast biogenesis.(A) The expression of ddFYVE in intracellular FNR-RFP/ddFYVE expressing parasites was induced by the addition of 1 µM Shield-1 and the localization of both markers was analyzed at the indicated time of incubation. Arrowheads indicate the formation of a PI3P-containing compartment next to the apicoplast and at the base of V-shaped dividing apicoplasts. DNA-, ddFYVE- and FNR labels in residual bodies (arrows) and tachyzoites that have lost their apicoplast, but retain the PI3P-containing compartment at the sub-apical pole (open triangles) are indicated. The lowest panel shows the cytosolic staining of ddFYVEm and the persistence of the apicoplast 18 h after Shield-1 induction. (B) Time-lapse microscopy of FNR-RFP/ddFYVE parasites. Elapsed time after addition of 1 µM Shield-1 is indicated. Early during apicoplast replication, ddFYVE remained concentrated near the apicoplast (190 min). When apicoplasts started to separate (240 min), a portion of FRN label remained associated with the ddFYVE compartment (open triangle). After completion of daughter cell formation (300 min), all ddFYVE label and part of FNR label accumulated in the residual body (arrow). The arrowhead points to the parasite that has lost the FNR label. (C) Two hours after Shield-1 induction, the accumulation of ddFYVE is observed at the base of the V-shaped elongated apicoplasts.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040667&req=5

ppat-1001286-g002: PI3P-binding module disturbs apicoplast biogenesis.(A) The expression of ddFYVE in intracellular FNR-RFP/ddFYVE expressing parasites was induced by the addition of 1 µM Shield-1 and the localization of both markers was analyzed at the indicated time of incubation. Arrowheads indicate the formation of a PI3P-containing compartment next to the apicoplast and at the base of V-shaped dividing apicoplasts. DNA-, ddFYVE- and FNR labels in residual bodies (arrows) and tachyzoites that have lost their apicoplast, but retain the PI3P-containing compartment at the sub-apical pole (open triangles) are indicated. The lowest panel shows the cytosolic staining of ddFYVEm and the persistence of the apicoplast 18 h after Shield-1 induction. (B) Time-lapse microscopy of FNR-RFP/ddFYVE parasites. Elapsed time after addition of 1 µM Shield-1 is indicated. Early during apicoplast replication, ddFYVE remained concentrated near the apicoplast (190 min). When apicoplasts started to separate (240 min), a portion of FRN label remained associated with the ddFYVE compartment (open triangle). After completion of daughter cell formation (300 min), all ddFYVE label and part of FNR label accumulated in the residual body (arrow). The arrowhead points to the parasite that has lost the FNR label. (C) Two hours after Shield-1 induction, the accumulation of ddFYVE is observed at the base of the V-shaped elongated apicoplasts.
Mentions: Despite the presence of a destabilization domain, a faint ddFYVE signal could already be observed by western blot analysis in the absence of Shield-1 treatment of the parasites (Figure 1D). The signal increased upon addition of the Shield-1 ligand, reaching a maximum after about 1 hour, and remaining stable thereafter (Figure 1D). The level of ddFYVE expression was considerably lower than that of the mutated version ddFYVEm, suggesting that it would be less tolerated by the parasite and might be degraded. IFA analysis detected ddFYVE in the absence of Shield-1 as a dot in the sub-apical part of the parasite (Figure 1E), similar to the one described above with transient transfection (Figure 1C). In other organisms PI3P is concentrated on early endosomes and co-localizes with Rab5 [17]. In T. gondii, however, we found that ddFYVE did not co-localize with the early endosome marker TgRab5.1 [12] (Figure 1E), but most unexpectedly co-localized instead with the apicoplast, whose DNA can be stained and appears as a small dot next to the nucleus (Figure 1E). This was subsequently confirmed by co-localization of ddFYVE with the FRN-RFP apicoplast marker [25] transfected in the ddFYVE strain (Figure 1F). Shield-1 treatment induced a progressive increase in fluorescence and some ddFYVE staining also appeared to surround the apicoplast (Figure 2A, see below). In striking contrast, the mutated form ddFYVEm was found throughout the cytoplasm (Figure 1G), confirming that the specific localization of the native construct was due to functional PI3P-binding domains. To confirm PI3P localization in T. gondii, we probed infected cells with anti-PI3P antibody and observed in each parasite a bright dot of fluorescence that partially co-localized with apicoplast (Figure S2). We therefore concluded that PI3P-enriched compartments are localized at the apicoplast and in its vicinity.

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