Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium.
Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium.
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DOI：
10.1371/journal.pone.0013653
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发表时间：
2010-10-27
期刊：
影响因子：
3.7
通讯作者：
Heussler VT
中科院分区：
文献类型：
--
作者：
Helm S;Lehmann C;Nagel A;Stanway RR;Horstmann S;Llinas M;Heussler VT
在被疟原虫感染的蚊子吸食血液的过程中，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。肝期疟原虫尽管临床沉默，但它是抗疟药物和疫苗方法的一个极有希望的靶点。成功入侵的寄生虫在肝细胞中大量增殖，产生数千个分裂子，这些分裂子被运送到血管中感染红细胞。要成功地从肝期发育为感染性的分裂子，需要对基因表达进行严格调控。虽然这是疟原虫生物学中一个非常有趣的方面，但人们对疟原虫的基因调控知之甚少，尤其是在肝脏阶段。我们从功能上分析了啮齿动物寄生虫伯氏疟原虫的一个新的启动子区域，该区域在寄生虫的肝脏阶段只活跃。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方法，可以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cDNA末端的快速扩增（5 ' -RACE）绘制了转录起始位点。通过启动子截断实验和潜在转录因子结合位点的定点诱变，我们发现最小启动子包含多个结合位点，用于最近鉴定的寄生虫特异性ApiAP2转录因子。在伯氏螺旋体中鉴定出肝脏阶段特异性启动子，证实该寄生虫能够在其生命周期中严格调节基因表达。鉴定的启动子区域现在可能用于研究疟原虫肝期的生物学，迄今为止在分子水平上证明存在问题。显性阴性突变蛋白的阶段特异性表达和在其他生命周期阶段正常活跃的蛋白的过表达将有助于了解所研究蛋白的功能。
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.
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