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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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Plasmodium falciparum stably expressing Nluc.(A) The Nluc gene was cloned in the pEF vector for stable expression in P. falciparum. (B) Aliquots (100 µL) of cultures at 1% hematocrit and 5% parasitemia corresponding to 500,000 P. falciparum infected RBCs transfected with pEF-Nluc were mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and reporter activity measured (1∶1). Nano-Glo Luciferase Assay Reagent was further diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent and used to determine reporter activity of the same culture. As a negative control, wild type parasites (wt) were mixed 1∶1 with Nano-Glo Luciferase Assay Reagent. (C) Parasites stably transfected with pEF-Nluc were diluted in 2-fold increments in RPMI + RBC maintaining 0.5% hematocrit. For each sample, 10 µL of the culture dilutions were mixed with 40 µL of Nano-Glo diluted 1∶400 in water and measured in the luminometer for 2 s with the gain adjusted 10% below saturation for the brightest sample. The solid line represents the mean RLU after linear regression. The dashed line represents the background +3 standard deviations. (D) Nluc expressing parasites were cultured in varying concentrations of chloroquine (CQ) and their growth determined by the LDH standard method or by measuring reporter activity using Nano-Glo Luciferase Assay Reagent at its standard dilution (1∶1) or diluted 1∶1000 as described in (C). Similarly, wild type parasites were transiently transfected with pPfNluc and their growth determined using Nano-Glo Luciferase Assay Reagent (Transient). IC50 was calculated by non-linear regression and represents the mean of 3 experiments. (E) The IC50s determined in D were plotted with 95% confidence intervals (CI).
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pone-0112571-g002: Plasmodium falciparum stably expressing Nluc.(A) The Nluc gene was cloned in the pEF vector for stable expression in P. falciparum. (B) Aliquots (100 µL) of cultures at 1% hematocrit and 5% parasitemia corresponding to 500,000 P. falciparum infected RBCs transfected with pEF-Nluc were mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and reporter activity measured (1∶1). Nano-Glo Luciferase Assay Reagent was further diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent and used to determine reporter activity of the same culture. As a negative control, wild type parasites (wt) were mixed 1∶1 with Nano-Glo Luciferase Assay Reagent. (C) Parasites stably transfected with pEF-Nluc were diluted in 2-fold increments in RPMI + RBC maintaining 0.5% hematocrit. For each sample, 10 µL of the culture dilutions were mixed with 40 µL of Nano-Glo diluted 1∶400 in water and measured in the luminometer for 2 s with the gain adjusted 10% below saturation for the brightest sample. The solid line represents the mean RLU after linear regression. The dashed line represents the background +3 standard deviations. (D) Nluc expressing parasites were cultured in varying concentrations of chloroquine (CQ) and their growth determined by the LDH standard method or by measuring reporter activity using Nano-Glo Luciferase Assay Reagent at its standard dilution (1∶1) or diluted 1∶1000 as described in (C). Similarly, wild type parasites were transiently transfected with pPfNluc and their growth determined using Nano-Glo Luciferase Assay Reagent (Transient). IC50 was calculated by non-linear regression and represents the mean of 3 experiments. (E) The IC50s determined in D were plotted with 95% confidence intervals (CI).

Mentions: P. falciparum tolerance for Nluc expression in continuous culturing was next investigated. The stable transfection vector pEF-Nluc was made by replacing the firefly luciferase of pEF-Luc [30] with Nluc (Fig. 2A) and this vector was stably transfected in P. falciparum. To confirm that the transfected parasites were expressing Nluc, 100 µL of parasite culture at 1% hematocrit and 5% parasitemia, corresponding to 500,000 infected red blood cells (iRBC), were washed and resuspended in the original volume of PBS. The cells were then lysed in 100 µL of Nano-Glo Luciferase Assay Reagent (referred to as 1∶1 in Fig. 2B) and this, measured for 10 seconds in the luminometer with its gain adjusted to 10% below saturation, yielded almost 107 RLU, which is the value where the signal saturates. The high signal strength prompted us to determine if the Nano-Glo Luciferase Assay Reagent could be more economically used and so it was diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent, part of the Luciferase Assay System (Promega), and tested with the same number of Nluc expressing parasites in the same conditions. A 10-fold dilution of Nano-Glo decreased the signal by less than 10%, suggesting the substrate was in excess for the amount of enzyme present in the extract (1∶10, Fig. 2B). The 100 and 1,000-fold dilutions resulted in signal decreasing about 3 and 24-fold, respectively (1∶100 and 1∶1000, Fig. 2B). In spite of the lower signal detected, 1,000-fold dilution of Nano-Glo still permitted detection at a S/B ratio of approximately 125 (1∶1000 versus 1∶1 wt, Fig. 2B).


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)

Plasmodium falciparum stably expressing Nluc.(A) The Nluc gene was cloned in the pEF vector for stable expression in P. falciparum. (B) Aliquots (100 µL) of cultures at 1% hematocrit and 5% parasitemia corresponding to 500,000 P. falciparum infected RBCs transfected with pEF-Nluc were mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and reporter activity measured (1∶1). Nano-Glo Luciferase Assay Reagent was further diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent and used to determine reporter activity of the same culture. As a negative control, wild type parasites (wt) were mixed 1∶1 with Nano-Glo Luciferase Assay Reagent. (C) Parasites stably transfected with pEF-Nluc were diluted in 2-fold increments in RPMI + RBC maintaining 0.5% hematocrit. For each sample, 10 µL of the culture dilutions were mixed with 40 µL of Nano-Glo diluted 1∶400 in water and measured in the luminometer for 2 s with the gain adjusted 10% below saturation for the brightest sample. The solid line represents the mean RLU after linear regression. The dashed line represents the background +3 standard deviations. (D) Nluc expressing parasites were cultured in varying concentrations of chloroquine (CQ) and their growth determined by the LDH standard method or by measuring reporter activity using Nano-Glo Luciferase Assay Reagent at its standard dilution (1∶1) or diluted 1∶1000 as described in (C). Similarly, wild type parasites were transiently transfected with pPfNluc and their growth determined using Nano-Glo Luciferase Assay Reagent (Transient). IC50 was calculated by non-linear regression and represents the mean of 3 experiments. (E) The IC50s determined in D were plotted with 95% confidence intervals (CI).
© Copyright Policy
Related In: Results  -  Collection

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pone-0112571-g002: Plasmodium falciparum stably expressing Nluc.(A) The Nluc gene was cloned in the pEF vector for stable expression in P. falciparum. (B) Aliquots (100 µL) of cultures at 1% hematocrit and 5% parasitemia corresponding to 500,000 P. falciparum infected RBCs transfected with pEF-Nluc were mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and reporter activity measured (1∶1). Nano-Glo Luciferase Assay Reagent was further diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent and used to determine reporter activity of the same culture. As a negative control, wild type parasites (wt) were mixed 1∶1 with Nano-Glo Luciferase Assay Reagent. (C) Parasites stably transfected with pEF-Nluc were diluted in 2-fold increments in RPMI + RBC maintaining 0.5% hematocrit. For each sample, 10 µL of the culture dilutions were mixed with 40 µL of Nano-Glo diluted 1∶400 in water and measured in the luminometer for 2 s with the gain adjusted 10% below saturation for the brightest sample. The solid line represents the mean RLU after linear regression. The dashed line represents the background +3 standard deviations. (D) Nluc expressing parasites were cultured in varying concentrations of chloroquine (CQ) and their growth determined by the LDH standard method or by measuring reporter activity using Nano-Glo Luciferase Assay Reagent at its standard dilution (1∶1) or diluted 1∶1000 as described in (C). Similarly, wild type parasites were transiently transfected with pPfNluc and their growth determined using Nano-Glo Luciferase Assay Reagent (Transient). IC50 was calculated by non-linear regression and represents the mean of 3 experiments. (E) The IC50s determined in D were plotted with 95% confidence intervals (CI).
Mentions: P. falciparum tolerance for Nluc expression in continuous culturing was next investigated. The stable transfection vector pEF-Nluc was made by replacing the firefly luciferase of pEF-Luc [30] with Nluc (Fig. 2A) and this vector was stably transfected in P. falciparum. To confirm that the transfected parasites were expressing Nluc, 100 µL of parasite culture at 1% hematocrit and 5% parasitemia, corresponding to 500,000 infected red blood cells (iRBC), were washed and resuspended in the original volume of PBS. The cells were then lysed in 100 µL of Nano-Glo Luciferase Assay Reagent (referred to as 1∶1 in Fig. 2B) and this, measured for 10 seconds in the luminometer with its gain adjusted to 10% below saturation, yielded almost 107 RLU, which is the value where the signal saturates. The high signal strength prompted us to determine if the Nano-Glo Luciferase Assay Reagent could be more economically used and so it was diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent, part of the Luciferase Assay System (Promega), and tested with the same number of Nluc expressing parasites in the same conditions. A 10-fold dilution of Nano-Glo decreased the signal by less than 10%, suggesting the substrate was in excess for the amount of enzyme present in the extract (1∶10, Fig. 2B). The 100 and 1,000-fold dilutions resulted in signal decreasing about 3 and 24-fold, respectively (1∶100 and 1∶1000, Fig. 2B). In spite of the lower signal detected, 1,000-fold dilution of Nano-Glo still permitted detection at a S/B ratio of approximately 125 (1∶1000 versus 1∶1 wt, Fig. 2B).

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