Malaria Laboratory 1, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
PLoS One. 2010 Oct 27;5(10):e13653. doi: 10.1371/journal.pone.0013653.
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.
在感染疟原虫的蚊子吸血过程中，有 10 到 100 个寄生虫被接种到皮肤中，其中一部分通过血液迁移到肝脏，在那里它们感染肝细胞。尽管疟原虫肝期在临床上没有明显症状，但它是抗疟药物和疫苗方法的一个极具前景的靶点。成功入侵的寄生虫在肝细胞中大量增殖，产生数千个裂殖子，然后被输送到血管中感染红细胞。为了从肝期成功发育为感染性裂殖子，需要对基因表达进行严格的调控。尽管这是疟原虫生物学中非常有趣的一个方面，但人们对寄生虫一般和肝期特定的基因调控知之甚少。我们对啮齿动物寄生虫疟原虫伯氏疟原虫的一个新的启动子区域进行了功能分析，该区域在寄生虫的肝期是唯一活跃的。为了证明启动子的阶段特异性活性，已经成功建立了 GFP 和荧光素酶报告基因检测，允许进行定性和准确的定量分析。为了进一步表征启动子区域，通过快速扩增 cDNA 末端（5'-RACE）确定了转录起始位点。通过启动子截断实验和潜在转录因子结合位点的定点突变，我们认为最小启动子包含一个以上的结合位点，用于最近鉴定的寄生虫特异性 ApiAP2 转录因子。在 P. berghei 中鉴定出肝期特异性启动子证实了寄生虫在其生命周期中能够严格调控基因表达。鉴定出的启动子区域现在可用于研究疟原虫肝期的生物学，这在分子水平上一直是一个难题。表达显性负突变蛋白的阶段特异性表达和过表达通常在其他生命周期阶段活跃的蛋白将有助于了解所研究蛋白的功能。
