PfAP2-I-GFP-msp5MUT vs PfAP2-I-GFP 3D7 [timecourse 1]. Plasmodium falciparum

Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor belonging to the Apicomplexan Apetala 2 (ApiAP2) family that is responsible for regulating the expression of a subset of merozoite genes involved in RBC invasion (PfAP2-I). Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of these genes. msp5 transcription levels decrease when the PfAP2-I DNA-binding motif is mutated in PfAP2-I-GFP parasites, showing that PfAP2-I must bind the DNA motif in order for msp5 to be transcribed. Overall design: Plasmodium falciparum strains AP2-I-GFP and AP2-I-GFP were highly synchronized and two parallel timecourses resulting in a total of 8 samples (4 wildtype PfAP2-I-GFP, 4 msp5 mutant) were hybridized against a Cy3-labeled reference pool of 3D7 mixed stage parasites on a two-color array. Starting at 18 hours post-invasion, samples were harvested in Trizol for total RNA extraction every 6 hours until 36h post-invasion and then every 3h until the end of the IDC (48h post-invasion).

专性胞内寄生虫必须高效侵入宿主细胞，方能完成成熟与传播过程。对于恶性疟原虫（Plasmodium falciparum）而言，侵入宿主红细胞（RBCs）是其生存必需的关键步骤。本研究报道一种属于顶复门Apetala 2（ApiAP2）家族的寄生虫特异性转录因子，该因子负责调控参与红细胞侵入过程的部分裂殖子基因的表达，将其命名为PfAP2-I。我们通过染色质免疫沉淀测序（ChIP-seq）开展的全基因组分析显示，PfAP2-I可与上述基因启动子区域的特定DNA基序相结合。当在PfAP2-I-GFP寄生虫中对PfAP2-I的DNA结合基序进行突变后，msp5的转录水平显著下调，这证实PfAP2-I必须结合该DNA基序，才能启动msp5的转录。

实验总体设计：将恶性疟原虫AP2-I-GFP野生株与msp5突变株进行高度同步化处理，设置两组平行时间序列实验，共获得8份样本（4份野生型PfAP2-I-GFP样本、4份msp5突变样本）。将所有样本与经Cy3标记的3D7多阶段寄生虫混合参考池进行双色基因芯片杂交。本实验于红细胞入侵后18小时启动样本收集，每6小时收获一次样本直至入侵后36小时，之后改为每3小时收获一次，直至红细胞内期发育周期（IDC）结束（入侵后48小时），所有样本均采用Trizol试剂进行总RNA提取。