Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum
Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum
复制标题
疟原虫恶性疟原虫临床分离株和实验室适应克隆之间的裂殖体转录组变异
作者：
Sarah J Tarr;Ofelia Díaz;L. Stewart;S. Hocking;Lee Murray;Craig W. Duffy;T. Otto;L. Chappell;J. Rayner;G. Awandare;D. Conway
疟疾寄生虫具有遗传多态和表型可塑性。在研究来自不同感染的寄生虫之间的转录组差异时，克服样本之间潜在的混淆技术和生物差异是具有挑战性的。我们研究了人类主要寄生虫恶性疟原虫的变异，从一组临床分离株和长期实验室适应的克隆中产生含有裂殖子的红细胞内裂殖子的多个独立复制样本制备的RNA-SEQ数据，目的是强有力地识别差异表达的基因。对生物样本复制的分析表明，数量的增加提高了差异表达基因的真实发现率，每个寄生虫系的六个独立复制可以识别出可以用更大的数量检测到的大多数差异。对于高表达的基因，集中在裂殖体阶段的顶部四分位数，有更强的能力来检测差异。与培养的临床分离株和实验室适应的克隆相比，实验室适应的克隆中表达更高的基因包括编码AP2转录因子(PF3D7_0420300)、泛素结合蛋白和两个假定的甲基转移酶的基因。相比之下，裂殖子表面蛋白基因dblmsp2在临床分离株中的表达较高，该基因被认为是裂殖体形成致力于性别分化的裂殖子的标志。可变表达与已知的异染色质蛋白1靶标的基因有极强的相关性，但不是唯一的。临床分离株显示了几个已知的裂殖子入侵配体的可变表达，以及其他基因的可变表达，新的RT-qPCR方法验证了这些基因的定量，并允许在材料更有限的样本中进行表征。这些基因在不同临床分离株的裂殖体标本中的表达水平在患者红细胞中的第一个体外循环中有所不同，但平均水平与连续培养的临床分离株相似。对多个生物样本重复的分析极大地改进了对不同培养寄生虫品系之间可变表达基因的识别。最近在培养中建立的临床分离株在特定基因的转录水平上显示出与长期适应克隆的差异，适合于需要生物复制来了解寄生虫表型和可能在自然界中相关的可变表达的分析。
Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes. Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates. Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.
实验室繁殖期间的遗传变化：恶性疟原虫网织红细胞结合蛋白 1 位点的拷贝数。
影响因子：
1.5
作者：
Nair,Shalini;Nkhoma,Standwell;Nosten,François;Mayxay,Mayfong;French,Neil;Whitworth,Jim;Anderson,Tim
通讯作者：
Anderson,Tim