Abstract
BackgroundBioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work.ResultsNanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo.ConclusionsPbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate requirements of NanoLuc are different from those of firefly luciferase, dual bioluminescence imaging for the simultaneous characterization of two lines, or two separate biological processes, is possible, as demonstrated in this work.
Highlights
Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development
PbmCherryHsp70 parasites [98] were transfected with the pNLuc construct, and this resulted in single cross-over integration events
The benefits and applications (See figure on page.) Fig. 7 Bioluminescence imaging of P. berghei blood stages. a Luminescence values from various concentrations of infected red blood cell (iRBC) from PbNLuc- and PbFlucinfected mice, ranging from 1 to 10,000 iRBCs. b Ratio of luminescence of PbNLuc iRBCs compared to PbFLuc iRBCs showing a 150-fold higher signal. 1000 parasites were isolated from a drop of blood of synchronously infected mice at 18 h post-infection
Summary
Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Rodent models, have proven to be extremely valuable tools for basic research on parasite and host biology, host-pathogen interactions, and anti-malarial drug activity [4,5,6]. Among the applications of bioluminescence in Plasmodium research, some of the most relevant studies include the in vivo and in vitro assessment of parasite growth in liver [8,9,10,11,12,13,14,15] and blood stages [14]; schizont sequestration in vivo ([16,17,18,19], De Niz et al, unpublished); analysing stage-specificity of promoters and other regulatory elements [20,21,22,23,24,25,26,27,28,29,30,31,32,33]; testing anti-malarial activity of drugs targeting liver [11, 34,35,36,37,38,39,40], and blood stage parasites [41,42,43,44,45,46]; screening for transmission-reducing activity of anti-malarials [47,48,49,50]; functional studies of parasite proteins and/or the adequacy of parasite attenuation by genetic manipulation ([16, 18, 51, 52]; De Niz et al, unpublished); evaluation of antimalarial immunity [53,54,55]; visualization of sporozoites in the skin [56], and specific differentiation of gametocyte stages (I-V) [47]
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