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Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites.

Yap A, Azevedo MF, Gilson PR, Weiss GE, O'Neill MT, Wilson DW, Crabb BS, Cowman AF - Cell. Microbiol. (2014)

Bottom Line: DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle.This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole.These results show that PfAMA1 is an essential protein for merozoite invasion in P.  falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., 3052, Australia.

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Rapamycin strongly and specifically depletes PfAMA1 expression in late stage parasites.A. Representative immunoblots show significantly decreased PfAMA1 expression following rapamycin treatment in B4 (top two panels) and 2F9 (bottom two panels) parasites over four time points. A monoclonal antibody specific for 3D7 AMA1 was used, which detected both the unprocessed 83 kDa and processed 66 kDa PfAMA1. Anti-HSP70 was used as loading control.B and C. By using densitometry quantification, PfAMA1 knockdown was estimated at an average of 81% for both parasite lines and in each of the four time points (n = 2). Error bars indicate SD.D. Immunoblot analysis indicates that expression of other rhoptry and micronemal proteins such as PfRh4, PfRON4 and PfEBA175 were not reduced by rapamycin treatment.E. Schizonts of rapamycin-treated parasites were dual labelled with anti-MSP1 (red) and anti-PfAMA1 (green) to show reduction in PfAMA1 expression. Schizonts displaying a gradient of high (arrow, top row), medium (bottom row) and low (arrowhead, top row) levels of PfAMA1 expression were observed. Scale bar is 5 μm.F. Histogram of pixel brightness of PfAMA1 antibody-labelled parasites, categorized into three groups: strong, medium and weak/none. Average pixel intensity for PfAMA1 was measured within a 70 pixel diameter surrounding the parasite and only MSP1-positive schizont stages were selected.
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fig04: Rapamycin strongly and specifically depletes PfAMA1 expression in late stage parasites.A. Representative immunoblots show significantly decreased PfAMA1 expression following rapamycin treatment in B4 (top two panels) and 2F9 (bottom two panels) parasites over four time points. A monoclonal antibody specific for 3D7 AMA1 was used, which detected both the unprocessed 83 kDa and processed 66 kDa PfAMA1. Anti-HSP70 was used as loading control.B and C. By using densitometry quantification, PfAMA1 knockdown was estimated at an average of 81% for both parasite lines and in each of the four time points (n = 2). Error bars indicate SD.D. Immunoblot analysis indicates that expression of other rhoptry and micronemal proteins such as PfRh4, PfRON4 and PfEBA175 were not reduced by rapamycin treatment.E. Schizonts of rapamycin-treated parasites were dual labelled with anti-MSP1 (red) and anti-PfAMA1 (green) to show reduction in PfAMA1 expression. Schizonts displaying a gradient of high (arrow, top row), medium (bottom row) and low (arrowhead, top row) levels of PfAMA1 expression were observed. Scale bar is 5 μm.F. Histogram of pixel brightness of PfAMA1 antibody-labelled parasites, categorized into three groups: strong, medium and weak/none. Average pixel intensity for PfAMA1 was measured within a 70 pixel diameter surrounding the parasite and only MSP1-positive schizont stages were selected.

Mentions: The amount of Pfama1 gene excision in the B4 and 2F9 populations was between 70–92% and we next determined the corresponding level of PfAMA1 protein expression when Cre recombinase activity was induced by rapamycin. Schizont-stage parasites were analysed by immunoblot using α-3D7 AMA1 antibody. There was approximately 81% knockdown of AMA1 protein levels across the four time points when compared with untreated parasites (Fig. 4A–C). Expression of other invasion proteins such as PfRh4, RON4 and EBA175 was unaffected (Fig. 4D). This level of PfAMA1 protein knockdown correlated with the level of Pfama1 gene excision mediated by DiCre.


Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites.

Yap A, Azevedo MF, Gilson PR, Weiss GE, O'Neill MT, Wilson DW, Crabb BS, Cowman AF - Cell. Microbiol. (2014)

Rapamycin strongly and specifically depletes PfAMA1 expression in late stage parasites.A. Representative immunoblots show significantly decreased PfAMA1 expression following rapamycin treatment in B4 (top two panels) and 2F9 (bottom two panels) parasites over four time points. A monoclonal antibody specific for 3D7 AMA1 was used, which detected both the unprocessed 83 kDa and processed 66 kDa PfAMA1. Anti-HSP70 was used as loading control.B and C. By using densitometry quantification, PfAMA1 knockdown was estimated at an average of 81% for both parasite lines and in each of the four time points (n = 2). Error bars indicate SD.D. Immunoblot analysis indicates that expression of other rhoptry and micronemal proteins such as PfRh4, PfRON4 and PfEBA175 were not reduced by rapamycin treatment.E. Schizonts of rapamycin-treated parasites were dual labelled with anti-MSP1 (red) and anti-PfAMA1 (green) to show reduction in PfAMA1 expression. Schizonts displaying a gradient of high (arrow, top row), medium (bottom row) and low (arrowhead, top row) levels of PfAMA1 expression were observed. Scale bar is 5 μm.F. Histogram of pixel brightness of PfAMA1 antibody-labelled parasites, categorized into three groups: strong, medium and weak/none. Average pixel intensity for PfAMA1 was measured within a 70 pixel diameter surrounding the parasite and only MSP1-positive schizont stages were selected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231980&req=5

fig04: Rapamycin strongly and specifically depletes PfAMA1 expression in late stage parasites.A. Representative immunoblots show significantly decreased PfAMA1 expression following rapamycin treatment in B4 (top two panels) and 2F9 (bottom two panels) parasites over four time points. A monoclonal antibody specific for 3D7 AMA1 was used, which detected both the unprocessed 83 kDa and processed 66 kDa PfAMA1. Anti-HSP70 was used as loading control.B and C. By using densitometry quantification, PfAMA1 knockdown was estimated at an average of 81% for both parasite lines and in each of the four time points (n = 2). Error bars indicate SD.D. Immunoblot analysis indicates that expression of other rhoptry and micronemal proteins such as PfRh4, PfRON4 and PfEBA175 were not reduced by rapamycin treatment.E. Schizonts of rapamycin-treated parasites were dual labelled with anti-MSP1 (red) and anti-PfAMA1 (green) to show reduction in PfAMA1 expression. Schizonts displaying a gradient of high (arrow, top row), medium (bottom row) and low (arrowhead, top row) levels of PfAMA1 expression were observed. Scale bar is 5 μm.F. Histogram of pixel brightness of PfAMA1 antibody-labelled parasites, categorized into three groups: strong, medium and weak/none. Average pixel intensity for PfAMA1 was measured within a 70 pixel diameter surrounding the parasite and only MSP1-positive schizont stages were selected.
Mentions: The amount of Pfama1 gene excision in the B4 and 2F9 populations was between 70–92% and we next determined the corresponding level of PfAMA1 protein expression when Cre recombinase activity was induced by rapamycin. Schizont-stage parasites were analysed by immunoblot using α-3D7 AMA1 antibody. There was approximately 81% knockdown of AMA1 protein levels across the four time points when compared with untreated parasites (Fig. 4A–C). Expression of other invasion proteins such as PfRh4, RON4 and EBA175 was unaffected (Fig. 4D). This level of PfAMA1 protein knockdown correlated with the level of Pfama1 gene excision mediated by DiCre.

Bottom Line: DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle.This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole.These results show that PfAMA1 is an essential protein for merozoite invasion in P.  falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., 3052, Australia.

Show MeSH
Related in: MedlinePlus