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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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Traffic of apicoplast proteins in the presence of ddFYVE.(A) The traffic of outermost apicoplast proteins is disturbed in the presence of ddFYVE. Immunofluorescence analysis of ddFYVE transfected parasites treated for 24 h with Shield-1 revealed a more diffuse labelling of ATrx1 (blue) around the apicoplast FNR-RFP dot (red) after Shield-1 treatment compared to untreated parasites (enlarged in the white framed box of the ATrx1 and FNR-RFP merged image). Single plane apotome sections are shown. (B) PI3P-containing vesicles and outermost apicoplast membrane proteins are still detected in the absence of the apicoplast. Open triangles indicate a diffuse staining of membranous ATrx1 (magenta) or FtsH1 (magenta) in FNR-RFP negative parasites. (C) Both ddFYVE (green) and ATrx1 (blue) accumulate and co-localize at the base of V-shaped dividing apicoplasts (FNR-RFP, red) in parasites treated for 6 h with Shield-1.
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ppat-1001286-g006: Traffic of apicoplast proteins in the presence of ddFYVE.(A) The traffic of outermost apicoplast proteins is disturbed in the presence of ddFYVE. Immunofluorescence analysis of ddFYVE transfected parasites treated for 24 h with Shield-1 revealed a more diffuse labelling of ATrx1 (blue) around the apicoplast FNR-RFP dot (red) after Shield-1 treatment compared to untreated parasites (enlarged in the white framed box of the ATrx1 and FNR-RFP merged image). Single plane apotome sections are shown. (B) PI3P-containing vesicles and outermost apicoplast membrane proteins are still detected in the absence of the apicoplast. Open triangles indicate a diffuse staining of membranous ATrx1 (magenta) or FtsH1 (magenta) in FNR-RFP negative parasites. (C) Both ddFYVE (green) and ATrx1 (blue) accumulate and co-localize at the base of V-shaped dividing apicoplasts (FNR-RFP, red) in parasites treated for 6 h with Shield-1.

Mentions: We thus analysed the distribution of the three apicoplast outermost membrane proteins FtsH1 [33], APT1 [34] and the thioredoxin ATrx1 [32] following disturbance of PI3P function. For this purpose, ddFYVE expressing parasites were stably transfected with plasmids coding for V5-FtsH1 or APT1-HA, and subsequently transiently transfected with FNR-RFP and the cells were then treated or not with Shield-1. IFA analysis of FtsH1, APT1 and ATrx1 was performed using anti-V5, anti-HA and anti-ATrx1 (Mab 11G8) antibodies respectively. As described previously, the localization of outermost apicoplast proteins varies during the cell cycle ([35], data not shown). During plastid elongation, these apicoplast proteins localized around the length of the elongated apicoplast as well as on vesicles or tubules nearby, while during the interphase, the label was restricted to the plastid, co-localizing with the FNR-RFP marker. After one day of Shield-1 disturbance of PI3P function, we observed a diffuse staining of FtsH1, APT1 and ATrx1 around the organelle still labelled by FNR-RFP (Figure 6A and Figure S8), suggesting that the fusion of the vesicles containing membranous proteins with the apicoplast was inhibited. Moreover, when the apicoplast had disappeared from some parasites as a consequence of Shield-1 treatment (as detected by the loss of the FNR-RFP marker), the vesicular staining of outermost apicoplast proteins was still detected in all apicoplast-deprived parasites (Figure 6B and Figure S8).


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)

Traffic of apicoplast proteins in the presence of ddFYVE.(A) The traffic of outermost apicoplast proteins is disturbed in the presence of ddFYVE. Immunofluorescence analysis of ddFYVE transfected parasites treated for 24 h with Shield-1 revealed a more diffuse labelling of ATrx1 (blue) around the apicoplast FNR-RFP dot (red) after Shield-1 treatment compared to untreated parasites (enlarged in the white framed box of the ATrx1 and FNR-RFP merged image). Single plane apotome sections are shown. (B) PI3P-containing vesicles and outermost apicoplast membrane proteins are still detected in the absence of the apicoplast. Open triangles indicate a diffuse staining of membranous ATrx1 (magenta) or FtsH1 (magenta) in FNR-RFP negative parasites. (C) Both ddFYVE (green) and ATrx1 (blue) accumulate and co-localize at the base of V-shaped dividing apicoplasts (FNR-RFP, red) in parasites treated for 6 h with Shield-1.
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Related In: Results  -  Collection

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ppat-1001286-g006: Traffic of apicoplast proteins in the presence of ddFYVE.(A) The traffic of outermost apicoplast proteins is disturbed in the presence of ddFYVE. Immunofluorescence analysis of ddFYVE transfected parasites treated for 24 h with Shield-1 revealed a more diffuse labelling of ATrx1 (blue) around the apicoplast FNR-RFP dot (red) after Shield-1 treatment compared to untreated parasites (enlarged in the white framed box of the ATrx1 and FNR-RFP merged image). Single plane apotome sections are shown. (B) PI3P-containing vesicles and outermost apicoplast membrane proteins are still detected in the absence of the apicoplast. Open triangles indicate a diffuse staining of membranous ATrx1 (magenta) or FtsH1 (magenta) in FNR-RFP negative parasites. (C) Both ddFYVE (green) and ATrx1 (blue) accumulate and co-localize at the base of V-shaped dividing apicoplasts (FNR-RFP, red) in parasites treated for 6 h with Shield-1.
Mentions: We thus analysed the distribution of the three apicoplast outermost membrane proteins FtsH1 [33], APT1 [34] and the thioredoxin ATrx1 [32] following disturbance of PI3P function. For this purpose, ddFYVE expressing parasites were stably transfected with plasmids coding for V5-FtsH1 or APT1-HA, and subsequently transiently transfected with FNR-RFP and the cells were then treated or not with Shield-1. IFA analysis of FtsH1, APT1 and ATrx1 was performed using anti-V5, anti-HA and anti-ATrx1 (Mab 11G8) antibodies respectively. As described previously, the localization of outermost apicoplast proteins varies during the cell cycle ([35], data not shown). During plastid elongation, these apicoplast proteins localized around the length of the elongated apicoplast as well as on vesicles or tubules nearby, while during the interphase, the label was restricted to the plastid, co-localizing with the FNR-RFP marker. After one day of Shield-1 disturbance of PI3P function, we observed a diffuse staining of FtsH1, APT1 and ATrx1 around the organelle still labelled by FNR-RFP (Figure 6A and Figure S8), suggesting that the fusion of the vesicles containing membranous proteins with the apicoplast was inhibited. Moreover, when the apicoplast had disappeared from some parasites as a consequence of Shield-1 treatment (as detected by the loss of the FNR-RFP marker), the vesicular staining of outermost apicoplast proteins was still detected in all apicoplast-deprived parasites (Figure 6B and Figure S8).

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