Transcriptome analysis of immature and mature male and female gametocytes of the malaria parasite Plasmodium falciparum

In the human host, Plasmodium falciparum parasites propagate asexually in erythrocytes causing the symptoms of malaria. However, in every asexual cycle, a small proportion of the parasites commits to sexual differentiation, which is critical for transmission. During a period of about 10 days, the sexually committed parasites either develop into male or female gametocytes. In this study, gametocytes carrying an endogenously GFP tagged version of the female specifically expressed ABCG2 gene were FACS sorted to separate male (GFP low) and female (GFP high) gametocyte populations at different days of development. RNA was subsequently extracted from these populations as well as from asexual parasites as controls and subjected to directional RNAseq. These data reveal the transcriptional differences between asexual parasite stages and developing gametocytes, but also between male and female gametocytes as well as for each sex during the course of differentiation. In particular, male gametocytes undergo distinct transcriptional transitions when comparing the transcriptome between day 4, 6 and 10 of gametocytogenesis. In contrast, female gametocyte transcriptomes are more static during the course of gametocytogenesis and only show minimal variation. This is the first study to longitudinally monitor gene expression in developing male and female gametocytes Overall design: RNA-Seq of Plasmodium falciparum parasites in differnet developmental stages: ring stage parasites, schizont stage parasites, day 4, 6, and 10 female gametocytes, and day 4, 6, and 10 male gametocytes

在人体宿主中，恶性疟原虫（Plasmodium falciparum）会在红细胞内进行无性增殖，进而引发疟疾症状。但在每一轮无性增殖周期中，仅有少量寄生虫会启动有性分化程序，这对于疟疾的传播至关重要。启动有性分化的寄生虫会在约10天的周期内发育为雄性或雌性配子体（gametocyte）。
本研究中，研究人员对携带雌性特异性表达的ABCG2基因的内源绿色荧光蛋白（GFP）标签版本的配子体，通过荧光激活细胞分选术（FACS）按照发育天数进行分选，分离出雄性（GFP低表达）与雌性（GFP高表达）配子体群体。随后，研究人员从这些配子体群体以及作为对照的无性增殖期寄生虫中提取RNA，并进行定向RNA测序（directional RNA-seq）。
这些数据不仅揭示了无性增殖期寄生虫与发育中配子体之间的转录组差异，同时也展现了雌雄配子体之间，以及不同分化阶段的同一性别配子体之间的转录差异。具体而言，在配子体发生（gametocytogenesis）的第4、6、10天进行转录组对比时，可观察到雄性配子体存在显著的转录组转变。与之相反，雌性配子体的转录组在配子体发生过程中更为稳定，仅存在极细微的表达变化。本研究是首个对发育中的雌雄配子体进行纵向基因表达监测的研究。
实验整体设计：对不同发育阶段的恶性疟原虫进行RNA测序，涵盖环状体期疟原虫、裂殖体期疟原虫，以及配子体发生第4、6、10天的雌性和雄性配子体。