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Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process.

Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, Struck NS, Engelberg K, Haase S, Frischknecht F, Miura K, Spielmann T, Gilberger TW - PLoS Pathog. (2009)

Bottom Line: We identify several residues in the cytoplasmic tail that are essential for AMA-1 function.We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes.We show that the cytoplasmic domain of AMA-1 is phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Parasitology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.

ABSTRACT
A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

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Phosphorylation of the cytoplasmic domain.(A) After invasion, secondary processing of AMA-1-GFP N- and C-terminal of domain III results in two additional AMA-1 GFP fragments of 44 and 49 kDa. Both fragments are indicated by coloured asterisks (49 kDa blue; 44 kDa red). (B) Live images showing AMA-1-GFP distribution (green) in two young ring stage or reinvaded merozoites. Blue: DAPI-stained nucleus. (C) Western blot analysis of AMA-1-GFP wild type and AMA-1-PM. Mutation of all putative phosphorylation sites to alanine within the cytoplasmic domain (PM, reducing the calculated MW by 0.6 kDa) leads to a reduced molecular weight of approximately 3 kDa of both fragments. (D) Western blot analysis of phosphatase-treated AMA-1-GFP. AMA-1-GFP was extracted and either treated with (+) or without (−) lambda-phosphatase followed by precipitation and then subjected to Western analysis. The pellet fraction (P) and the precipitated lysate supernatant (SN) without any treatment was loaded as a control. Treatment with lambda-phosphatase leads to a reduced molecular weight of approximately 3 kDa of both fragments, which is not seen in the control (−). (E) The treatment of the phosphorylation mutant (PM) with phosphatase does not affect the mobility of AMA-1-PM (with (+) and without (−) phosphatase treatment). Supernatant and pellet fraction served as controls. The size of all fragments is identical to that of the phosphatase-treated wild type AMA-1-GFP.
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ppat-1000322-g006: Phosphorylation of the cytoplasmic domain.(A) After invasion, secondary processing of AMA-1-GFP N- and C-terminal of domain III results in two additional AMA-1 GFP fragments of 44 and 49 kDa. Both fragments are indicated by coloured asterisks (49 kDa blue; 44 kDa red). (B) Live images showing AMA-1-GFP distribution (green) in two young ring stage or reinvaded merozoites. Blue: DAPI-stained nucleus. (C) Western blot analysis of AMA-1-GFP wild type and AMA-1-PM. Mutation of all putative phosphorylation sites to alanine within the cytoplasmic domain (PM, reducing the calculated MW by 0.6 kDa) leads to a reduced molecular weight of approximately 3 kDa of both fragments. (D) Western blot analysis of phosphatase-treated AMA-1-GFP. AMA-1-GFP was extracted and either treated with (+) or without (−) lambda-phosphatase followed by precipitation and then subjected to Western analysis. The pellet fraction (P) and the precipitated lysate supernatant (SN) without any treatment was loaded as a control. Treatment with lambda-phosphatase leads to a reduced molecular weight of approximately 3 kDa of both fragments, which is not seen in the control (−). (E) The treatment of the phosphorylation mutant (PM) with phosphatase does not affect the mobility of AMA-1-PM (with (+) and without (−) phosphatase treatment). Supernatant and pellet fraction served as controls. The size of all fragments is identical to that of the phosphatase-treated wild type AMA-1-GFP.

Mentions: During the invasion process AMA-1 is shed by a protease from the merozoite surface releasing the extracellular domain into the supernatant (Figure 6A). The remaining small C-terminus of AMA-1 is carried into the host cell [24] (Figure 6B). A bioinformatics screen predicted six amino acids within the cytoplasmic domain to be phosphorylated (www.cbs.dtu.dk/services/NetPhos) (Figure 4B). To validate phosphorylation, all conserved putative phosphorylation sites (and those that might be phosphorylated due to the substitutions) were mutated to generate the parasite line AMA-1PM-GFP (Figure 4A). Taking advantage of the increased length of the processed C-terminal fragment due to the GFP tag, the released C-terminal fragments were easily detectable as 45 and 49 kDa fragments (Figure 6A and 6C). The introduced mutations (calculated to alter the MW by 0.6 kDa) led to a clearly reduced molecular weight (significantly more than 0.6 kDa) of both the 45 and the 49 kDa fragments (Figure 6C). In order to test if the observed shift in mobility was due to phosphorylation, AMA-1-GFP was treated with lambda-phosphatase (Figure 6D). This reduced the apparent molecular weight of wild type AMA-1-GFP, resulting in a size identical to that of the phosphorylation mutant AMA-1PM-GFP, whereas AMA-1PM-GFP itself showed no shift after the phosphatase treatment (Figure 6E). Thus the observed size difference between the mutated and wild type cytoplasmic domains of AMA1 is due to phosphorylation. Interestingly, this phosphorylation mutant (AMA-1PM) led to a functional inactivation of AMA-1 as shown in the complementation assays (Figures 4E and 5C).


Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process.

Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, Struck NS, Engelberg K, Haase S, Frischknecht F, Miura K, Spielmann T, Gilberger TW - PLoS Pathog. (2009)

Phosphorylation of the cytoplasmic domain.(A) After invasion, secondary processing of AMA-1-GFP N- and C-terminal of domain III results in two additional AMA-1 GFP fragments of 44 and 49 kDa. Both fragments are indicated by coloured asterisks (49 kDa blue; 44 kDa red). (B) Live images showing AMA-1-GFP distribution (green) in two young ring stage or reinvaded merozoites. Blue: DAPI-stained nucleus. (C) Western blot analysis of AMA-1-GFP wild type and AMA-1-PM. Mutation of all putative phosphorylation sites to alanine within the cytoplasmic domain (PM, reducing the calculated MW by 0.6 kDa) leads to a reduced molecular weight of approximately 3 kDa of both fragments. (D) Western blot analysis of phosphatase-treated AMA-1-GFP. AMA-1-GFP was extracted and either treated with (+) or without (−) lambda-phosphatase followed by precipitation and then subjected to Western analysis. The pellet fraction (P) and the precipitated lysate supernatant (SN) without any treatment was loaded as a control. Treatment with lambda-phosphatase leads to a reduced molecular weight of approximately 3 kDa of both fragments, which is not seen in the control (−). (E) The treatment of the phosphorylation mutant (PM) with phosphatase does not affect the mobility of AMA-1-PM (with (+) and without (−) phosphatase treatment). Supernatant and pellet fraction served as controls. The size of all fragments is identical to that of the phosphatase-treated wild type AMA-1-GFP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654807&req=5

ppat-1000322-g006: Phosphorylation of the cytoplasmic domain.(A) After invasion, secondary processing of AMA-1-GFP N- and C-terminal of domain III results in two additional AMA-1 GFP fragments of 44 and 49 kDa. Both fragments are indicated by coloured asterisks (49 kDa blue; 44 kDa red). (B) Live images showing AMA-1-GFP distribution (green) in two young ring stage or reinvaded merozoites. Blue: DAPI-stained nucleus. (C) Western blot analysis of AMA-1-GFP wild type and AMA-1-PM. Mutation of all putative phosphorylation sites to alanine within the cytoplasmic domain (PM, reducing the calculated MW by 0.6 kDa) leads to a reduced molecular weight of approximately 3 kDa of both fragments. (D) Western blot analysis of phosphatase-treated AMA-1-GFP. AMA-1-GFP was extracted and either treated with (+) or without (−) lambda-phosphatase followed by precipitation and then subjected to Western analysis. The pellet fraction (P) and the precipitated lysate supernatant (SN) without any treatment was loaded as a control. Treatment with lambda-phosphatase leads to a reduced molecular weight of approximately 3 kDa of both fragments, which is not seen in the control (−). (E) The treatment of the phosphorylation mutant (PM) with phosphatase does not affect the mobility of AMA-1-PM (with (+) and without (−) phosphatase treatment). Supernatant and pellet fraction served as controls. The size of all fragments is identical to that of the phosphatase-treated wild type AMA-1-GFP.
Mentions: During the invasion process AMA-1 is shed by a protease from the merozoite surface releasing the extracellular domain into the supernatant (Figure 6A). The remaining small C-terminus of AMA-1 is carried into the host cell [24] (Figure 6B). A bioinformatics screen predicted six amino acids within the cytoplasmic domain to be phosphorylated (www.cbs.dtu.dk/services/NetPhos) (Figure 4B). To validate phosphorylation, all conserved putative phosphorylation sites (and those that might be phosphorylated due to the substitutions) were mutated to generate the parasite line AMA-1PM-GFP (Figure 4A). Taking advantage of the increased length of the processed C-terminal fragment due to the GFP tag, the released C-terminal fragments were easily detectable as 45 and 49 kDa fragments (Figure 6A and 6C). The introduced mutations (calculated to alter the MW by 0.6 kDa) led to a clearly reduced molecular weight (significantly more than 0.6 kDa) of both the 45 and the 49 kDa fragments (Figure 6C). In order to test if the observed shift in mobility was due to phosphorylation, AMA-1-GFP was treated with lambda-phosphatase (Figure 6D). This reduced the apparent molecular weight of wild type AMA-1-GFP, resulting in a size identical to that of the phosphorylation mutant AMA-1PM-GFP, whereas AMA-1PM-GFP itself showed no shift after the phosphatase treatment (Figure 6E). Thus the observed size difference between the mutated and wild type cytoplasmic domains of AMA1 is due to phosphorylation. Interestingly, this phosphorylation mutant (AMA-1PM) led to a functional inactivation of AMA-1 as shown in the complementation assays (Figures 4E and 5C).

Bottom Line: We identify several residues in the cytoplasmic tail that are essential for AMA-1 function.We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes.We show that the cytoplasmic domain of AMA-1 is phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Parasitology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.

ABSTRACT
A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus