Let it glow: genetically encoded fluorescent reporters in Plasmodium

Abstract

The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed.

Keywords: Plasmodium; Apicomplexa; Fluorescent protein; GFP; Malaria; Parasite.

Fig. 1

Stage-specific and constitutive expression of FPs along the Plasmodium life cycle. Human and mosquito outlines displayed in the center illustrate the division of the parasite cycle in insect-specific and vertebrate-specific stages. Fluorescent P. berghei and P. falciparum reporter lines driving expression of the respective FP from classical genomic loci used for ectopic gene expression are shown. Reporter lines are classified in constitutive, oocyst- and sporozoite-specific, zoite-specific, sporozoite- and/or liver stage-specific as well as gametocyte-specific. The colour code highlighting the different expression groups matches with that of the life cycle sections the respective lines express the fluorescent reporter. Images of the P. berghei fluo line are shown as an example of a reporter line with both constitutive (GFP driven by the ef1a promoter) and stage-specific FP expression (mCherry driven by the CSP promoter in late oocysts, sporozoites and early liver stages). Merged images showing GFP and mCherry expression in combination with nuclear staining are shown for all stages. The scale bar for all images is 10 µm except for midgut and salivary gland where it represents 100 µm. Please note that P. falciparum reporter lines making use of the etramp10.3./peg4 promoter also display FP expression in gametocytes, although not shown here. Also, the P. berghei reporter line PbGFP103464 is fluorescent in late liver stages only, while the reporter line PbANKA-Cherry 2204cl5 is fluorescent in sporozoites and liver stages

Fig. 2

Fluorescent protein biosensors. A–C. The mode of action of three redox biosensors as well as their fluorescence spectra are shown as examples. A rxYFP is an intensiometric biosensor with a single excitation and emission peak. It was generated by introducing two cysteines in β-strands 7 and 11 (N149C, S202C) of YFP. rxYFP fluorescence decreases when a disulfide bridge forms between the two cysteines under oxidizing conditions. B roGFP2 is a ratiometric probe that was generated similarly as rxYFP. While the reduced form of roGFP2 has a single excitation peak at 488 nm, the oxidized form has two, at 405 and 488 nm. Ratio measurement provides a readout of the parameter of interest. C ECFP-RL7-EYFP is a ratiometric FRET sensor where ECFP and EYFP have been coupled by a small linker carrying 4 cysteines. Only ECFP is excited at 434 nm. Under reducing conditions, only ECFP emits fluorescence at 477 nm. Under oxidizing conditions, two disulfide bridges form in the linker, bringing both FPs in close proximity and allowing energy transfer from ECFP to EYFP, thus shifting the emission peak to 527 nm. D A non-exhaustive list of FP biosensors according to the physiological parameter they measure. The FPs constituting these sensors and the sensor type are indicated. Additional FP biosensors for small molecules and protein activity can be found at https://www.addgene.org/fluorescent-proteins/biosensors/. ^a already expressed in Plasmodium; ^b YFP-based sensors are especially sensitive to pH and must be used in parallel with a pH control probe sharing similar pKa and properties when employed to measure parameters other than pH