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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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Schematic for the sequential generation of W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites. In the first transfection, the gene for Pfama1 in W2mef was genetically disrupted and complemented with a codon-altered 3D7 Pfama1 gene using the pAMA1-loxP plasmid. The main features of pAMA1-loxP are a homologous target sequence for recombination followed by a W2mef promoter sequence and a ‘floxed’ 3D7 Pfama1 gene. A hdhfr selectable drug cassette, which confers resistance to the antifolate WR99210, was used to select for transfectants. Homologous integration of two tandemly arranged pAMA1-loxP plasmids into the W2mef AMA1 locus resulted in a duplication of the 3D7 Pfama1 gene. The architecture of the modified locus is shown along with AflIII and NdeI restriction sites used for Southern blot analysis. Sizes of the digested DNA fragments are shown in kilobase (kb). W2mef/AMA1-loxP parasites were cloned by limiting dilution. In the second transfection, DiCre was introduced into a W2mef/AMA1-loxP clone using the pDiCre plasmid. The expression casette of pDiCre is shown in the boxed insert (top right). The N- and C-terminus fragments of Cre recombinase are driven by the bi-directional P. berghei EF1α promoter and hsp86 promoter respectively with both Cre fusions placed in a head-to-tail orientation. Transcription termination is modulated by 3′ UTR sequences of P. berghei dihydrofolate reductase-thymidylate synthase (DT 3′) and P. falciparum calmodulin (CAM 3′) genes. In the DiCre system, Cre recombinase is fused to an F2 linker and either the FKBP12 or FRB sequence to induce dimerization in the presence of rapamycin. We have included a Gal4 nuclear localization signal to target the DiCre fusion proteins to the nucleus once expressed and a bsd resistance marker, which confers resistance to blasticidin. The predicted modification of the Pfama1 locus following DiCre-mediated Pfama1 excision is shown along with the restriction digest sizes.
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fig01: Schematic for the sequential generation of W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites. In the first transfection, the gene for Pfama1 in W2mef was genetically disrupted and complemented with a codon-altered 3D7 Pfama1 gene using the pAMA1-loxP plasmid. The main features of pAMA1-loxP are a homologous target sequence for recombination followed by a W2mef promoter sequence and a ‘floxed’ 3D7 Pfama1 gene. A hdhfr selectable drug cassette, which confers resistance to the antifolate WR99210, was used to select for transfectants. Homologous integration of two tandemly arranged pAMA1-loxP plasmids into the W2mef AMA1 locus resulted in a duplication of the 3D7 Pfama1 gene. The architecture of the modified locus is shown along with AflIII and NdeI restriction sites used for Southern blot analysis. Sizes of the digested DNA fragments are shown in kilobase (kb). W2mef/AMA1-loxP parasites were cloned by limiting dilution. In the second transfection, DiCre was introduced into a W2mef/AMA1-loxP clone using the pDiCre plasmid. The expression casette of pDiCre is shown in the boxed insert (top right). The N- and C-terminus fragments of Cre recombinase are driven by the bi-directional P. berghei EF1α promoter and hsp86 promoter respectively with both Cre fusions placed in a head-to-tail orientation. Transcription termination is modulated by 3′ UTR sequences of P. berghei dihydrofolate reductase-thymidylate synthase (DT 3′) and P. falciparum calmodulin (CAM 3′) genes. In the DiCre system, Cre recombinase is fused to an F2 linker and either the FKBP12 or FRB sequence to induce dimerization in the presence of rapamycin. We have included a Gal4 nuclear localization signal to target the DiCre fusion proteins to the nucleus once expressed and a bsd resistance marker, which confers resistance to blasticidin. The predicted modification of the Pfama1 locus following DiCre-mediated Pfama1 excision is shown along with the restriction digest sizes.

Mentions: The gene encoding PfAMA1 is refractory to genetic ablation via conventional knockout strategies (Triglia et al., 2000) and consequently we constructed a P. falciparum line in which it was placed under conditional regulation. To do this we simultaneously disrupted the endogenous Pfama1 gene, in the W2mef line by single recombination, with the plasmid pAMA1-loxP allowing complementation with a transfected gene (encodes the antigenically distinct 3D7 allele) flanked with loxP sites (Fig. 1). The resulting W2mef/AMA1-loxP parasites were cloned by limiting dilution and the clones derived had two copies of the plasmid inserted into the Pfama1 gene resulting in two functional copies of the 3D7 allele (Figs 1 and 2A, Fig. S1).


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)

Schematic for the sequential generation of W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites. In the first transfection, the gene for Pfama1 in W2mef was genetically disrupted and complemented with a codon-altered 3D7 Pfama1 gene using the pAMA1-loxP plasmid. The main features of pAMA1-loxP are a homologous target sequence for recombination followed by a W2mef promoter sequence and a ‘floxed’ 3D7 Pfama1 gene. A hdhfr selectable drug cassette, which confers resistance to the antifolate WR99210, was used to select for transfectants. Homologous integration of two tandemly arranged pAMA1-loxP plasmids into the W2mef AMA1 locus resulted in a duplication of the 3D7 Pfama1 gene. The architecture of the modified locus is shown along with AflIII and NdeI restriction sites used for Southern blot analysis. Sizes of the digested DNA fragments are shown in kilobase (kb). W2mef/AMA1-loxP parasites were cloned by limiting dilution. In the second transfection, DiCre was introduced into a W2mef/AMA1-loxP clone using the pDiCre plasmid. The expression casette of pDiCre is shown in the boxed insert (top right). The N- and C-terminus fragments of Cre recombinase are driven by the bi-directional P. berghei EF1α promoter and hsp86 promoter respectively with both Cre fusions placed in a head-to-tail orientation. Transcription termination is modulated by 3′ UTR sequences of P. berghei dihydrofolate reductase-thymidylate synthase (DT 3′) and P. falciparum calmodulin (CAM 3′) genes. In the DiCre system, Cre recombinase is fused to an F2 linker and either the FKBP12 or FRB sequence to induce dimerization in the presence of rapamycin. We have included a Gal4 nuclear localization signal to target the DiCre fusion proteins to the nucleus once expressed and a bsd resistance marker, which confers resistance to blasticidin. The predicted modification of the Pfama1 locus following DiCre-mediated Pfama1 excision is shown along with the restriction digest sizes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231980&req=5

fig01: Schematic for the sequential generation of W2mef/AMA1-loxP and W2mef/AMA1-loxP/DiCre parasites. In the first transfection, the gene for Pfama1 in W2mef was genetically disrupted and complemented with a codon-altered 3D7 Pfama1 gene using the pAMA1-loxP plasmid. The main features of pAMA1-loxP are a homologous target sequence for recombination followed by a W2mef promoter sequence and a ‘floxed’ 3D7 Pfama1 gene. A hdhfr selectable drug cassette, which confers resistance to the antifolate WR99210, was used to select for transfectants. Homologous integration of two tandemly arranged pAMA1-loxP plasmids into the W2mef AMA1 locus resulted in a duplication of the 3D7 Pfama1 gene. The architecture of the modified locus is shown along with AflIII and NdeI restriction sites used for Southern blot analysis. Sizes of the digested DNA fragments are shown in kilobase (kb). W2mef/AMA1-loxP parasites were cloned by limiting dilution. In the second transfection, DiCre was introduced into a W2mef/AMA1-loxP clone using the pDiCre plasmid. The expression casette of pDiCre is shown in the boxed insert (top right). The N- and C-terminus fragments of Cre recombinase are driven by the bi-directional P. berghei EF1α promoter and hsp86 promoter respectively with both Cre fusions placed in a head-to-tail orientation. Transcription termination is modulated by 3′ UTR sequences of P. berghei dihydrofolate reductase-thymidylate synthase (DT 3′) and P. falciparum calmodulin (CAM 3′) genes. In the DiCre system, Cre recombinase is fused to an F2 linker and either the FKBP12 or FRB sequence to induce dimerization in the presence of rapamycin. We have included a Gal4 nuclear localization signal to target the DiCre fusion proteins to the nucleus once expressed and a bsd resistance marker, which confers resistance to blasticidin. The predicted modification of the Pfama1 locus following DiCre-mediated Pfama1 excision is shown along with the restriction digest sizes.
Mentions: The gene encoding PfAMA1 is refractory to genetic ablation via conventional knockout strategies (Triglia et al., 2000) and consequently we constructed a P. falciparum line in which it was placed under conditional regulation. To do this we simultaneously disrupted the endogenous Pfama1 gene, in the W2mef line by single recombination, with the plasmid pAMA1-loxP allowing complementation with a transfected gene (encodes the antigenically distinct 3D7 allele) flanked with loxP sites (Fig. 1). The resulting W2mef/AMA1-loxP parasites were cloned by limiting dilution and the clones derived had two copies of the plasmid inserted into the Pfama1 gene resulting in two functional copies of the 3D7 allele (Figs 1 and 2A, Fig. S1).

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