An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle

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.

Spatial organization of protein export in malaria parasite blood stages

“…Here, we have utilized a trappable nanoluciferase (Nluc) reporter construct containing the murine dihydrofolate reductase (mDHFR) enzyme which is refractory to unfolding in the presence of WR99210 (WR) to further probe PTEX function . Nluc reporter proteins are highly quantifiable and have been used previously to examine the export of PEXEL proteins, resistance to sorbitol lysis, merozoite egress and invasion and other aspects of the parasite's life cycle . Here, we generated multiple exported constructs containing both Nluc and mDHFR, but with different additional epitope and affinity tags and followed their fates.…”

“…20 Nluc reporter proteins are highly quantifiable and have been used previously to examine the export of PEXEL proteins, resistance to sorbitol lysis, merozoite egress and invasion and other aspects of the parasite's life cycle. [21][22][23][24][25][26] Here, we generated multiple exported constructs containing both Nluc and mDHFR, but with different additional epitope and affinity tags and followed their fates. We show that different constructs do indeed behave differently upon trapping at the PVM with some constructs partially resuming export after WR removal, while others remain completely blocked.…”

In vivo imaging of pathogen homing to the host tissues

“…On the other hand, bioluminescence imaging can reveal the distribution of living luciferase-expressing cells in a non-invasive and quantitative manner in the whole body of small hosts, such as mice. This technique, which is based on an enzymatic reaction that generates photons following the oxidation of a substrate, has been frequently applied to study Plasmodium behaviour [10] in the blood [11], [12], liver [13], [14], [15] and skin of rodents [8], [16]. The firefly luciferase has been preferentially used for in vivo studies when compared to the NanoLuc and Gaussia luciferase, which despite emitting more light in vitro, seem to be less efficient in vivo [17].…”

Toolbox for In Vivo Imaging of Host–Parasite Interactions at Multiple Scales

“…In recent years, an important aim has been to achieve ultra-bright luminescence to detect signals with high sensitivity in deep tissues. This has been initially achieved by generating parasites expressing codon-optimized red-shifted firefly luciferases [83,84] and ultra-bright luminescent probes such as NanoLuc [85,86]. Although NanoLuc luciferase has been extremely useful in vitro due to the extremely bright signal it produces [87], its performance in vivo is very poor due to its short emission wavelength.…”