Identification and Characterization of a Liver Stage-Specific Promoter Region of the Malaria Parasite Plasmodium

Abstract

During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5′-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.

The s48/45 six-cysteine proteins: mediators of interaction throughout the Plasmodium life cycle

“…These studies also determined that LISP2, named after its expression pattern comparable with LISP1 (Tarun et al, 2008), is exclusively expressed in the mid-to-late liver stages stages. Another group has independently confirmed these results by characterizing stage-specific expression using the promoter region of P. berghei LISP2, in conjunction with a dual luminescence system (Helm et al, 2010; De Niz et al, 2015). Immunofluorescence assays of liver stages showed that LISP2 is transported to the vacuolar space and then, after processing, exported into the hepatocyte cytoplasm (Orito et al, 2013; Itani et al, 2014).…”

Imaging Plasmodium immunobiology in the liver, brain, and lung

“…Stable transgene integration into the dispensable S1 (sporozoite expressed gene 1) locus of P. yoelii , which enables fluorescence expression throughout the parasite life cycle, has overcome the possibility of reversion by plasmid excision and also eliminated survival disadvantages associated with transgene expression in the small ribosomal subunit locus, a strategy previously utilized for both P. berghei and P. yoelii [32, 78]. The recent construction of powerful P. berghei strains such as luciferase-GFP, mCherry, tdTomato as well as novel GFP-expressing strains with improved brightness [79-83], in particular if used in combination with the wide range of transgenic or full knockout C57Bl/6 mice, which are more susceptible to P. berghei , should compensate for the higher levels of liver inflammation and the lower rates of LS maturation previously associated with this parasite.…”

Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

“…The observed time course of PbMAPK1 subcellular localization was found to be independent of the integrity of the DFG motif putatively involved in ATP positioning (and thus kinase activity) (PbMAPK1(D178A)), integrity of the putative activation motif (PbMAPK1(T198A/Y200A)) and the presence of a putative coiled-coil domain in the kinase’s C-terminal domain (PbMAPK1-Δcc) ( Figure 2 and Figure S2 ). The observed localizations were identical for parasite lines expressing PbMAPK1 N-terminally and C-terminally fused to GFP and independent of the promoter (constitutive EEF1alpha promoter or liver stage-specific promoter [17]) used to control the expression of the fusion protein ( Figure 2 ). Expression of the full-length PbMAPK1-GFP fusion protein was confirmed by western blot analysis of mixed blood stage parasites ( Figure 3B ).…”

“…Waters). Constructs for liver stage-specific expression were produced using the plasmid pGFP 103464 [17], a pL0017-based vector in which the constitutive EEF1alpha promoter has been replaced by a liver stage-specific promoter. Plasmids were linearized with ApaI and SacII and used for transfection of P. berghei blood stage schizonts as described by Janse et al [54].…”