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.
Highlights
Malaria remains one of the main health burdens in developing countries, especially in sub-Saharan Africa
During parasite development in hepatocytes, GFP expression under the PB103464.00.0 promoter was strongly upregulated at 48 hpi and continued until merosome formation, the end-point of liver stage development
Several liver stage- expressed genes have been described in the literature [11,12,13,14] but previous studies have not analyzed the promoters of these genes
Summary
Malaria remains one of the main health burdens in developing countries, especially in sub-Saharan Africa. There is a great discrepancy between the Plasmodium liver stage being the main target of vaccine development against malaria and our knowledge of the biology of the parasite at this particular stage. The ApiAP2 transcription factor family has been suggested to regulate stagespecific gene expression in the human parasite Plasmodium falciparum [3,4]. These in vitro investigations far have only been conducted on the blood stage of the parasite. ApiAP2 regulators for sporozoite and ookinete development in P. berghei have been characterized, showing for the first time that there are stage-specific transcription factors governing parasite development [5,6]. The aim of this study was to identify in the rodent model parasite P. berghei a liver stage-specific promoter and to verify its stage-specific expression using GFP and luciferase reporter assays
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