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Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

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BFA blocks the p120 form of PfA-M1 in a compartment close to the nucleus. Synchronized parasites were treated for 19 hours in presence of 5 μM BFA (B) or in presence (E) or absence (O) of ethanol, in control cultures. A. Parasites were harvested, isolated by using 0.1% saponin and soluble fractions were blotted with anti-MAP1 antibodies to detect PfA-M1. The p120 form of PfA-M1 is present in BFA-treated cultures while p120, p96 and p68 forms are present in both control cultures, E and O. B. BFA-treated (a) and control parasites in presence of ethanol (c) were analysed by immunofluorescence as previously described [4,6] by using anti-MAP1 antibodies [6] to detect PfA-M1 (red) and a mouse anti-exp2 [19] serum as positive control (green). Nuclei, stained by using Hoechst 33342 appear in blue. Corresponding phase contrasts are in (b, d). Scale bar, 5 μm.
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Figure 3: BFA blocks the p120 form of PfA-M1 in a compartment close to the nucleus. Synchronized parasites were treated for 19 hours in presence of 5 μM BFA (B) or in presence (E) or absence (O) of ethanol, in control cultures. A. Parasites were harvested, isolated by using 0.1% saponin and soluble fractions were blotted with anti-MAP1 antibodies to detect PfA-M1. The p120 form of PfA-M1 is present in BFA-treated cultures while p120, p96 and p68 forms are present in both control cultures, E and O. B. BFA-treated (a) and control parasites in presence of ethanol (c) were analysed by immunofluorescence as previously described [4,6] by using anti-MAP1 antibodies [6] to detect PfA-M1 (red) and a mouse anti-exp2 [19] serum as positive control (green). Nuclei, stained by using Hoechst 33342 appear in blue. Corresponding phase contrasts are in (b, d). Scale bar, 5 μm.

Mentions: Brefeldin A (BFA) is a fungal metabolite that specifically inhibits anterograde transport from the ER to Golgi in many eukaryotic cells [34,35] and has been used as a tool to investigate protein trafficking in P. falciparum [19,33]. Here, BFA treatments were used to investigate PfA-M1 maturation and trafficking. Synchronized early ring stages were treated with 5 μM BFA for 19 hours before parasite harvest and analysis. The immunoblot analysis (Figure 3A) shows that in BFA-treated parasites only the p120 form is present in contrast to the control cultures grown in the presence (E) or absence (O) of ethanol, the solvent used to solubilize the BFA. In these latter cultures, as expected, p120, p96 and p68 forms were present, as previously observed [4]. The immuno-fluorescence analysis revealed that, in BFA treated parasites, PfA-M1 was retained in a compartment close to the nucleus that appears to be distinct from the compartment where exp-2 is retained upon BFA treatment (Figure 3B). Exp-2 is a protein exported to the PVM [19,36]. In the control culture, exp-2 was targeted as expected to the PVM and PfA-M1 was diffuse in the parasite (but outside the FV) as previously shown [4]. Therefore, it appears that BFA blocks the p120 form PfA-M1 in a compartment close to the nucleus that could correspond to the ER, and this blockage prevents its maturation to the p96 and p68 forms.


Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

BFA blocks the p120 form of PfA-M1 in a compartment close to the nucleus. Synchronized parasites were treated for 19 hours in presence of 5 μM BFA (B) or in presence (E) or absence (O) of ethanol, in control cultures. A. Parasites were harvested, isolated by using 0.1% saponin and soluble fractions were blotted with anti-MAP1 antibodies to detect PfA-M1. The p120 form of PfA-M1 is present in BFA-treated cultures while p120, p96 and p68 forms are present in both control cultures, E and O. B. BFA-treated (a) and control parasites in presence of ethanol (c) were analysed by immunofluorescence as previously described [4,6] by using anti-MAP1 antibodies [6] to detect PfA-M1 (red) and a mouse anti-exp2 [19] serum as positive control (green). Nuclei, stained by using Hoechst 33342 appear in blue. Corresponding phase contrasts are in (b, d). Scale bar, 5 μm.
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Related In: Results  -  Collection

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Figure 3: BFA blocks the p120 form of PfA-M1 in a compartment close to the nucleus. Synchronized parasites were treated for 19 hours in presence of 5 μM BFA (B) or in presence (E) or absence (O) of ethanol, in control cultures. A. Parasites were harvested, isolated by using 0.1% saponin and soluble fractions were blotted with anti-MAP1 antibodies to detect PfA-M1. The p120 form of PfA-M1 is present in BFA-treated cultures while p120, p96 and p68 forms are present in both control cultures, E and O. B. BFA-treated (a) and control parasites in presence of ethanol (c) were analysed by immunofluorescence as previously described [4,6] by using anti-MAP1 antibodies [6] to detect PfA-M1 (red) and a mouse anti-exp2 [19] serum as positive control (green). Nuclei, stained by using Hoechst 33342 appear in blue. Corresponding phase contrasts are in (b, d). Scale bar, 5 μm.
Mentions: Brefeldin A (BFA) is a fungal metabolite that specifically inhibits anterograde transport from the ER to Golgi in many eukaryotic cells [34,35] and has been used as a tool to investigate protein trafficking in P. falciparum [19,33]. Here, BFA treatments were used to investigate PfA-M1 maturation and trafficking. Synchronized early ring stages were treated with 5 μM BFA for 19 hours before parasite harvest and analysis. The immunoblot analysis (Figure 3A) shows that in BFA-treated parasites only the p120 form is present in contrast to the control cultures grown in the presence (E) or absence (O) of ethanol, the solvent used to solubilize the BFA. In these latter cultures, as expected, p120, p96 and p68 forms were present, as previously observed [4]. The immuno-fluorescence analysis revealed that, in BFA treated parasites, PfA-M1 was retained in a compartment close to the nucleus that appears to be distinct from the compartment where exp-2 is retained upon BFA treatment (Figure 3B). Exp-2 is a protein exported to the PVM [19,36]. In the control culture, exp-2 was targeted as expected to the PVM and PfA-M1 was diffuse in the parasite (but outside the FV) as previously shown [4]. Therefore, it appears that BFA blocks the p120 form PfA-M1 in a compartment close to the nucleus that could correspond to the ER, and this blockage prevents its maturation to the p96 and p68 forms.

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Show MeSH
Related in: MedlinePlus