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Plasmodium falciparum transfected with ultra bright NanoLuc luciferase offers high sensitivity detection for the screening of growth and cellular trafficking inhibitors.

Azevedo MF, Nie CQ, Elsworth B, Charnaud SC, Sanders PR, Crabb BS, Gilson PR - PLoS ONE (2014)

Bottom Line: We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase.To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol.As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.

View Article: PubMed Central - PubMed

Affiliation: Macfarlane Burnet Institute of Medical Research and Public Health, Melbourne, Victoria, Australia.

ABSTRACT
Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.

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Detection of secretion and export inhibition.(A) Parasites expressing the secreted (SP) or the exported (PEXEL) Nluc were treated with Brefeldin A (BFA) for 6 hours and luciferase activity determined in each sub-cellular fraction. Results are the mean of 4 experiments and the error bars represent standard deviations. Statistical significance (* p<0.05) was determined by 2 way ANOVA test comparing the activity detected in the parasite fraction of treated and DMSO control parasites of each line and of PV and RBC fractions of DMSO against treated SP-Nluc and PEXEL-Nluc lines respectively. (B) The toxicity of each treatment was determined by measuring total luciferase activity as relative to the DMSO control.
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pone-0112571-g005: Detection of secretion and export inhibition.(A) Parasites expressing the secreted (SP) or the exported (PEXEL) Nluc were treated with Brefeldin A (BFA) for 6 hours and luciferase activity determined in each sub-cellular fraction. Results are the mean of 4 experiments and the error bars represent standard deviations. Statistical significance (* p<0.05) was determined by 2 way ANOVA test comparing the activity detected in the parasite fraction of treated and DMSO control parasites of each line and of PV and RBC fractions of DMSO against treated SP-Nluc and PEXEL-Nluc lines respectively. (B) The toxicity of each treatment was determined by measuring total luciferase activity as relative to the DMSO control.

Mentions: With the Nluc, SP-Nluc and PEXEL-Nluc parasites having such distinct and characteristic reporter fraction patterns, we decided to test known trafficking inhibitors on the lines to assess their potential usefulness for screening for novel trafficking inhibitor compounds. To our knowledge, however there are no known Plasmodium-specific protein export inhibitors, so we instead used the antibiotic brefeldin A (BFA), which blocks the translocation of proteins from the ER to the Golgi. Since this pathway is common for both protein secretion and export, SP-Nluc and PEXEL-Nluc lines were treated with BFA followed by RBC fractionation (Fig. 5A). Treatment of trophozoites with 5 mg/mL BFA for 6 hrs in both lines caused an increase in reporter activity in the parasite suggesting luciferase is accumulating in the parasite as expected. In the SP-Nluc line, activity decreased in the PV fraction but not in the RBC, since transit from the PV into the RBC was not expected to be affected by BFA. In the PEXEL-Nluc line, activity was reduced in the RBC, but no change was seen in the PV fraction suggesting that BFA does not block the PTEX protein transporter. The general BFA toxicity to the cells was evaluated by measuring total reporter activity and it dropped by 40 and 35% for the SP-Nluc and PEXEL-Nluc lines respectively, during the assay period (Fig. 5B).


Plasmodium falciparum transfected with ultra bright NanoLuc luciferase offers high sensitivity detection for the screening of growth and cellular trafficking inhibitors.

Azevedo MF, Nie CQ, Elsworth B, Charnaud SC, Sanders PR, Crabb BS, Gilson PR - PLoS ONE (2014)

Detection of secretion and export inhibition.(A) Parasites expressing the secreted (SP) or the exported (PEXEL) Nluc were treated with Brefeldin A (BFA) for 6 hours and luciferase activity determined in each sub-cellular fraction. Results are the mean of 4 experiments and the error bars represent standard deviations. Statistical significance (* p<0.05) was determined by 2 way ANOVA test comparing the activity detected in the parasite fraction of treated and DMSO control parasites of each line and of PV and RBC fractions of DMSO against treated SP-Nluc and PEXEL-Nluc lines respectively. (B) The toxicity of each treatment was determined by measuring total luciferase activity as relative to the DMSO control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231029&req=5

pone-0112571-g005: Detection of secretion and export inhibition.(A) Parasites expressing the secreted (SP) or the exported (PEXEL) Nluc were treated with Brefeldin A (BFA) for 6 hours and luciferase activity determined in each sub-cellular fraction. Results are the mean of 4 experiments and the error bars represent standard deviations. Statistical significance (* p<0.05) was determined by 2 way ANOVA test comparing the activity detected in the parasite fraction of treated and DMSO control parasites of each line and of PV and RBC fractions of DMSO against treated SP-Nluc and PEXEL-Nluc lines respectively. (B) The toxicity of each treatment was determined by measuring total luciferase activity as relative to the DMSO control.
Mentions: With the Nluc, SP-Nluc and PEXEL-Nluc parasites having such distinct and characteristic reporter fraction patterns, we decided to test known trafficking inhibitors on the lines to assess their potential usefulness for screening for novel trafficking inhibitor compounds. To our knowledge, however there are no known Plasmodium-specific protein export inhibitors, so we instead used the antibiotic brefeldin A (BFA), which blocks the translocation of proteins from the ER to the Golgi. Since this pathway is common for both protein secretion and export, SP-Nluc and PEXEL-Nluc lines were treated with BFA followed by RBC fractionation (Fig. 5A). Treatment of trophozoites with 5 mg/mL BFA for 6 hrs in both lines caused an increase in reporter activity in the parasite suggesting luciferase is accumulating in the parasite as expected. In the SP-Nluc line, activity decreased in the PV fraction but not in the RBC, since transit from the PV into the RBC was not expected to be affected by BFA. In the PEXEL-Nluc line, activity was reduced in the RBC, but no change was seen in the PV fraction suggesting that BFA does not block the PTEX protein transporter. The general BFA toxicity to the cells was evaluated by measuring total reporter activity and it dropped by 40 and 35% for the SP-Nluc and PEXEL-Nluc lines respectively, during the assay period (Fig. 5B).

Bottom Line: We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase.To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol.As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.

View Article: PubMed Central - PubMed

Affiliation: Macfarlane Burnet Institute of Medical Research and Public Health, Melbourne, Victoria, Australia.

ABSTRACT
Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.

Show MeSH
Related in: MedlinePlus