Limits...
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.

Show MeSH

Related in: MedlinePlus

The p120 form of PfA-M1 is present in the PV space. Identical amounts of synchronized trophozoite stages were treated with streptolysin O (SLO) (lanes 1, 2, 3) or 0.1% saponin (SAPO) (lanes 4, 5, 3) as described in the Methods section. Lane 1 corresponds to SLO supernatant (SNT) i.e. the cytoplasm of iRBC while lane 4 corresponds to SAPO supernatant, i.e. the cytoplasm of iRBC plus PV content. Remaining pellets containing intact parasites devoid of host cytosol (SLO treatment) or devoid of host cytosol plus PV content (SAPO treatment) were washed twice in PBS, further lysed in ice-cold water in presence of protease inhibitors and separated by centrifugation into soluble fractions (SF, lanes 2 and 5, for the SLO and SAPO treatments, respectively) and membrane fractions (MF, lanes 3 and 6, for the SLO and SAPO treatments, respectively). Processed samples were separated by SDS-PAGE and blotted with antibodies corresponding to PfA-M1 (A. anti-MAP1 antibodies [6]) and several control markers: anti-PfPV1 antibodies (B), used as PV markers [22], anti-PfBip (C) [21] and anti-PfAldolase (D) antibodies [15] used as parasite reticulum endoplasmic and cytoplasm markers, respectively. The molecular masses kDa are shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2914058&req=5

Figure 2: The p120 form of PfA-M1 is present in the PV space. Identical amounts of synchronized trophozoite stages were treated with streptolysin O (SLO) (lanes 1, 2, 3) or 0.1% saponin (SAPO) (lanes 4, 5, 3) as described in the Methods section. Lane 1 corresponds to SLO supernatant (SNT) i.e. the cytoplasm of iRBC while lane 4 corresponds to SAPO supernatant, i.e. the cytoplasm of iRBC plus PV content. Remaining pellets containing intact parasites devoid of host cytosol (SLO treatment) or devoid of host cytosol plus PV content (SAPO treatment) were washed twice in PBS, further lysed in ice-cold water in presence of protease inhibitors and separated by centrifugation into soluble fractions (SF, lanes 2 and 5, for the SLO and SAPO treatments, respectively) and membrane fractions (MF, lanes 3 and 6, for the SLO and SAPO treatments, respectively). Processed samples were separated by SDS-PAGE and blotted with antibodies corresponding to PfA-M1 (A. anti-MAP1 antibodies [6]) and several control markers: anti-PfPV1 antibodies (B), used as PV markers [22], anti-PfBip (C) [21] and anti-PfAldolase (D) antibodies [15] used as parasite reticulum endoplasmic and cytoplasm markers, respectively. The molecular masses kDa are shown on the left.

Mentions: To validate these observations and to determine which forms of PfA-M1 were present in the PV lumen, the pore forming toxin streptolysin O (SLO) and the detergent saponin (SAPO) were used on identical samples of synchronized trophozoite stage P. falciparum infected red blood cells (iRBC). SLO releases soluble erythrocyte components but leaves the parasitophorous vacuole membrane (PVM) intact while saponin disrupts the erythrocyte membrane and the PVM thus releasing the contents of the parasitophorous vacuole [15]. Using antibodies directed against marker proteins for defined compartments within the infected erythrocytes (PfPV1, a marker of parasitophorous vacuole [22]; PfBip, a marker of the parasite endoplasmic reticulum (ER) [21] and PfAldolase, a marker of the parasite cytosol; [15]) it was confirmed that SLO permeabilization did not affect the integrity of the PVM (the soluble proteins of the parasitophorous vacuole, PfPV1 is absent from lane 1, Figure 2B). In this assay, the control parasite PfBip and PfAldolase as well as PfA-M1 were not released into the supernatant fraction (they are absent from lane 1, Figure 2A, C, D). Conversely, the 0.1% saponin treatment released soluble proteins of the parasitophorous vacuole (such as PfPV1, lane 4, Figure 2B) without affecting internal parasite proteins (PfBip and PfAldolase, lane 4, Figure 2C, D). Interestingly, this 0.1% saponin treatment also released a proportion of p120 form of PfA-M1 (lane 4, Figure 2A) confirming its presence in the PV. The p68 form of PfA-M1 remained exclusively within the parasite, together with an internal population of the p120 (lane 5, Figure 2A). Note also that only p120 is, in part, associated with membrane fractions (Figure 2A, lanes 3 and 6). These results show that PfA-M1 is not translocated beyond the parasitophorous vacuole membrane to the cytoplasm of the infected red blood cell. In addition, PfA-M1 does not contain PEXEL, VTS, or the HTS sequences or motifs that have been proposed to be responsible for the targeting of parasite proteins across the PVM into the erythrocyte cytosol [31,32]. However, PfA-M1 appears to have the capacity to be exported or secreted into the parasitophorous vacuole, most likely by using its N-terminal hydrophobic domain which has been shown to act as a recessed signal sequence in some other P. falciparum proteins [33].


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)

The p120 form of PfA-M1 is present in the PV space. Identical amounts of synchronized trophozoite stages were treated with streptolysin O (SLO) (lanes 1, 2, 3) or 0.1% saponin (SAPO) (lanes 4, 5, 3) as described in the Methods section. Lane 1 corresponds to SLO supernatant (SNT) i.e. the cytoplasm of iRBC while lane 4 corresponds to SAPO supernatant, i.e. the cytoplasm of iRBC plus PV content. Remaining pellets containing intact parasites devoid of host cytosol (SLO treatment) or devoid of host cytosol plus PV content (SAPO treatment) were washed twice in PBS, further lysed in ice-cold water in presence of protease inhibitors and separated by centrifugation into soluble fractions (SF, lanes 2 and 5, for the SLO and SAPO treatments, respectively) and membrane fractions (MF, lanes 3 and 6, for the SLO and SAPO treatments, respectively). Processed samples were separated by SDS-PAGE and blotted with antibodies corresponding to PfA-M1 (A. anti-MAP1 antibodies [6]) and several control markers: anti-PfPV1 antibodies (B), used as PV markers [22], anti-PfBip (C) [21] and anti-PfAldolase (D) antibodies [15] used as parasite reticulum endoplasmic and cytoplasm markers, respectively. The molecular masses kDa are shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914058&req=5

Figure 2: The p120 form of PfA-M1 is present in the PV space. Identical amounts of synchronized trophozoite stages were treated with streptolysin O (SLO) (lanes 1, 2, 3) or 0.1% saponin (SAPO) (lanes 4, 5, 3) as described in the Methods section. Lane 1 corresponds to SLO supernatant (SNT) i.e. the cytoplasm of iRBC while lane 4 corresponds to SAPO supernatant, i.e. the cytoplasm of iRBC plus PV content. Remaining pellets containing intact parasites devoid of host cytosol (SLO treatment) or devoid of host cytosol plus PV content (SAPO treatment) were washed twice in PBS, further lysed in ice-cold water in presence of protease inhibitors and separated by centrifugation into soluble fractions (SF, lanes 2 and 5, for the SLO and SAPO treatments, respectively) and membrane fractions (MF, lanes 3 and 6, for the SLO and SAPO treatments, respectively). Processed samples were separated by SDS-PAGE and blotted with antibodies corresponding to PfA-M1 (A. anti-MAP1 antibodies [6]) and several control markers: anti-PfPV1 antibodies (B), used as PV markers [22], anti-PfBip (C) [21] and anti-PfAldolase (D) antibodies [15] used as parasite reticulum endoplasmic and cytoplasm markers, respectively. The molecular masses kDa are shown on the left.
Mentions: To validate these observations and to determine which forms of PfA-M1 were present in the PV lumen, the pore forming toxin streptolysin O (SLO) and the detergent saponin (SAPO) were used on identical samples of synchronized trophozoite stage P. falciparum infected red blood cells (iRBC). SLO releases soluble erythrocyte components but leaves the parasitophorous vacuole membrane (PVM) intact while saponin disrupts the erythrocyte membrane and the PVM thus releasing the contents of the parasitophorous vacuole [15]. Using antibodies directed against marker proteins for defined compartments within the infected erythrocytes (PfPV1, a marker of parasitophorous vacuole [22]; PfBip, a marker of the parasite endoplasmic reticulum (ER) [21] and PfAldolase, a marker of the parasite cytosol; [15]) it was confirmed that SLO permeabilization did not affect the integrity of the PVM (the soluble proteins of the parasitophorous vacuole, PfPV1 is absent from lane 1, Figure 2B). In this assay, the control parasite PfBip and PfAldolase as well as PfA-M1 were not released into the supernatant fraction (they are absent from lane 1, Figure 2A, C, D). Conversely, the 0.1% saponin treatment released soluble proteins of the parasitophorous vacuole (such as PfPV1, lane 4, Figure 2B) without affecting internal parasite proteins (PfBip and PfAldolase, lane 4, Figure 2C, D). Interestingly, this 0.1% saponin treatment also released a proportion of p120 form of PfA-M1 (lane 4, Figure 2A) confirming its presence in the PV. The p68 form of PfA-M1 remained exclusively within the parasite, together with an internal population of the p120 (lane 5, Figure 2A). Note also that only p120 is, in part, associated with membrane fractions (Figure 2A, lanes 3 and 6). These results show that PfA-M1 is not translocated beyond the parasitophorous vacuole membrane to the cytoplasm of the infected red blood cell. In addition, PfA-M1 does not contain PEXEL, VTS, or the HTS sequences or motifs that have been proposed to be responsible for the targeting of parasite proteins across the PVM into the erythrocyte cytosol [31,32]. However, PfA-M1 appears to have the capacity to be exported or secreted into the parasitophorous vacuole, most likely by using its N-terminal hydrophobic domain which has been shown to act as a recessed signal sequence in some other P. falciparum proteins [33].

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