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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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NanoLuc is targeted to the PV and to the RBC.Diagrams of gene constructs and the IFA images are shown on the left and right respectively. Nluc fused at its N-terminus to (A) the N-terminal region of an exported protein (PEXEL), (B) to a secretion signal peptide (SP) or (C) original (cytosolic). The gene encoding each fusion protein was cloned in the pEF vector. Transfected parasites were analysed by IFA using antibodies to detect Nluc and the PVM marker Exp2. DAPI was used for nuclear staining. Size bar = 5 µm.
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pone-0112571-g003: NanoLuc is targeted to the PV and to the RBC.Diagrams of gene constructs and the IFA images are shown on the left and right respectively. Nluc fused at its N-terminus to (A) the N-terminal region of an exported protein (PEXEL), (B) to a secretion signal peptide (SP) or (C) original (cytosolic). The gene encoding each fusion protein was cloned in the pEF vector. Transfected parasites were analysed by IFA using antibodies to detect Nluc and the PVM marker Exp2. DAPI was used for nuclear staining. Size bar = 5 µm.

Mentions: Much of the virulence of Plasmodium parasites can be attributed to the export of effector proteins into the host compartment, which extensively modifies the iRBC [10], [17], [39]–[43]. Consequently, there has been much interest in understanding how parasites export proteins beyond the PVM that envelops them within the RBC cytoplasm and whether the export system can be targeted with drug inhibitors [6], [12], [39], [44]. Methods such as tagging exported proteins with fluorescent markers like green fluorescent protein or by immuno-labeling with protein-specific antibodies have been extensively applied for detection of exported proteins in Plasmodium spp[17], [41], [45]–[47]. Microscopy of individual parasites has then been used to follow the tagged proteins but this is time consuming and not amenable to quantification. In order to create a more quantitative approach we attempted to export Nluc into the RBC compartment so we could quantitatively follow its passage. To do this the first 113 residues of the P. falciparum exported protein Hyp1 (PF3D7_0113300), containing the PEXEL cleavage site RLLTE, was fused to Nluc at its N-terminus (Fig. 3A). PEXEL-Nluc parasites were analyzed by immunofluorescence (IFA) with antibodies for Nluc and Exp2, a PVM marker used to delimit the parasite boundaries. PEXEL-Nluc did not concentrate and co-localize with Exp2 rather occupying the entire RBC cytosolic region that lies beyond the PV, suggesting it is correctly exported (Fig. 3A). There was also an increased Nluc signal surrounding the parasites’ nuclei that could represent newly synthesized Nluc transiting through the endoplasmic reticulum (ER).


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)

NanoLuc is targeted to the PV and to the RBC.Diagrams of gene constructs and the IFA images are shown on the left and right respectively. Nluc fused at its N-terminus to (A) the N-terminal region of an exported protein (PEXEL), (B) to a secretion signal peptide (SP) or (C) original (cytosolic). The gene encoding each fusion protein was cloned in the pEF vector. Transfected parasites were analysed by IFA using antibodies to detect Nluc and the PVM marker Exp2. DAPI was used for nuclear staining. Size bar = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112571-g003: NanoLuc is targeted to the PV and to the RBC.Diagrams of gene constructs and the IFA images are shown on the left and right respectively. Nluc fused at its N-terminus to (A) the N-terminal region of an exported protein (PEXEL), (B) to a secretion signal peptide (SP) or (C) original (cytosolic). The gene encoding each fusion protein was cloned in the pEF vector. Transfected parasites were analysed by IFA using antibodies to detect Nluc and the PVM marker Exp2. DAPI was used for nuclear staining. Size bar = 5 µm.
Mentions: Much of the virulence of Plasmodium parasites can be attributed to the export of effector proteins into the host compartment, which extensively modifies the iRBC [10], [17], [39]–[43]. Consequently, there has been much interest in understanding how parasites export proteins beyond the PVM that envelops them within the RBC cytoplasm and whether the export system can be targeted with drug inhibitors [6], [12], [39], [44]. Methods such as tagging exported proteins with fluorescent markers like green fluorescent protein or by immuno-labeling with protein-specific antibodies have been extensively applied for detection of exported proteins in Plasmodium spp[17], [41], [45]–[47]. Microscopy of individual parasites has then been used to follow the tagged proteins but this is time consuming and not amenable to quantification. In order to create a more quantitative approach we attempted to export Nluc into the RBC compartment so we could quantitatively follow its passage. To do this the first 113 residues of the P. falciparum exported protein Hyp1 (PF3D7_0113300), containing the PEXEL cleavage site RLLTE, was fused to Nluc at its N-terminus (Fig. 3A). PEXEL-Nluc parasites were analyzed by immunofluorescence (IFA) with antibodies for Nluc and Exp2, a PVM marker used to delimit the parasite boundaries. PEXEL-Nluc did not concentrate and co-localize with Exp2 rather occupying the entire RBC cytosolic region that lies beyond the PV, suggesting it is correctly exported (Fig. 3A). There was also an increased Nluc signal surrounding the parasites’ nuclei that could represent newly synthesized Nluc transiting through the endoplasmic reticulum (ER).

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