Abstract
Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70) family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein.
Highlights
Malaria is caused by blood infection of the obligate intracellular parasite Plasmodium, single cell eukaryotes that follow a complex developmental program during life cycle progression
We focused on members of the heat shock protein 70 (HSP70) family, because they are ubiquitous, typically abundant, and likely to perform important functions in Plasmodium parasites [13,14,15]
Profiling of steady-state transcript abundance by qRT PCR using gene-specific primer pairs and normalization to green fluorescent protein (GFP) expressed under the control of elongation factor 1 alpha (EF1a) revealed robust expression for all transcripts analyzed (Fig. 2)
Summary
Malaria is caused by blood infection of the obligate intracellular parasite Plasmodium, single cell eukaryotes that follow a complex developmental program during life cycle progression. Many aspects of the parasite life cycle are known since decades, previously unrecognized aspects of the clinically silent and diagnostically inaccessible pre-erythrocytic phase, such as cell traversal prior to productive invasion [1], intradermal migration of sporozoites [2], and formation of merosomes as the final step in liver stage maturation [3], have profoundly transformed our understanding of Plasmodium biology. These observations were possible because generation of a bright green fluorescent sporozoite line, CSP::GFP [4], permitted live imaging of sporozoite-infected animals. This limitation was partially overcome by generation of fluorescent parasites that express GFP under control of the elongation factor 1 alpha (EF1a) promoter, resulting in constitutive, but only moderate, fluorescence throughout the parasite life cycle [6]
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