Comprehensive analysis of transcripts and proteins relative abundance during Plasmodium falciparum blood stages

Plasmodium falciparum is the main causative agent of human malaria. During the intraerythrocytic development cycle, P. falciparum morphology changes dramatically from circulating young rings to sequestered mature trophozoites and schizonts. Sequestered forms contribute to the pathophysiology of severe malaria as the infected erythrocytes obstruct the microvascular flow in deep organs and induce local inflammation. However, the sequestration mechanism limits the access to the corresponding parasitic form in clinical samples from patients infected with P. falciparum. To complement this deficiency, we aimed to evaluate the relevance of mRNA study as a proxy of protein expression in sequestered parasites. To do so, we conducted a proteotranscriptomic analysis using five independent P. falciparum laboratory strains samples. RNA sequencing was performed on circulating ring stage parasites and LC-MS/MS on the corresponding sequestered mature forms. All analyzes were performed within the same development cycle for direct individual correlation. mRNA expression level was assessed at the circulating ring stage and the corresponding protein expression level was measured after 18h-24h of maturation to reach the mature trophozoite stage. Overall, our results showed a strong transcriptome/transcriptome and proteome/proteome correlation between samples. Moreover, strong correlations of mRNA and proteins expressions levels were found between ring stage transcriptomes and mature forms proteomes. However, twice more transcripts were identified at ring stage than proteins at mature trophozoite stage. A high level of transcript expression did not guarantee the detection of the corresponding protein. Finally, we pointed out discrepancies at the individual gene level. Taken together, our results show that transcripts and proteins expression are overall correlated. However, mRNA abundance is not a perfect proxy of protein expression at the individual level. Importantly, our study shows limitations of the “blind” use of RNA-seq and the importance of multi-omics approaches for P. falciparum blood stage study in clinical samples. Overall design: We used 3D7, HB3 and ITG laboratory strains, with specific binding background phenotypes (ICAM-1, Hbec-5i and CD36) for a total of five samples.

恶性疟原虫（Plasmodium falciparum）是引发人类疟疾的主要病原体。在红细胞内发育周期中，恶性疟原虫的形态会发生显著变化：从循环中的早期环状体，逐步转变为黏附滞留的成熟滋养体与裂殖体。黏附滞留的虫体可参与重症疟疾的病理生理过程：受感染的红细胞会阻塞深部器官的微血管血流，并诱发局部炎症。然而，这种黏附滞留机制使得临床样本中难以获取对应阶段的恶性疟原虫虫体。为弥补这一缺陷，本研究旨在评估信使RNA（mRNA）表达作为黏附滞留寄生虫蛋白质表达替代标志物的相关性。为此，我们针对5株独立的恶性疟原虫实验室毒株开展了蛋白质组-转录组联合分析：对循环中的环状体阶段虫体进行RNA测序（RNA-seq），对其对应的黏附滞留成熟阶段虫体进行液相色谱-串联质谱（LC-MS/MS）检测。所有分析均在同一发育周期内完成，以实现直接的个体间相关性比对。
研究中，我们在循环环状体阶段检测了mRNA表达水平，并在经过18~24小时成熟至滋养体阶段后，测定了对应的蛋白质表达水平。整体结果显示，样本间的转录组（transcriptome）与转录组、蛋白质组（proteome）与蛋白质组均呈现强相关性。此外，环状体阶段的转录组与成熟阶段蛋白质组之间，mRNA与蛋白质的表达水平也存在显著相关性。然而，环状体阶段鉴定到的转录本数量是成熟滋养体阶段鉴定到的蛋白质数量的两倍以上。高转录本表达水平并不一定能保证对应蛋白质被检测到。最后，我们还发现了单个基因水平上的表达差异。
综上，本研究结果表明，转录本与蛋白质的整体表达水平存在相关性，但mRNA丰度并不能完美作为单个基因水平上蛋白质表达的替代标志物。重要的是，本研究揭示了盲目使用RNA-seq的局限性，以及多组学方法在临床样本中恶性疟原虫红内期研究中的重要性。
整体实验设计：我们选用了3D7、HB3及ITG这3株实验室毒株，其分别具有特定的黏附背景表型（ICAM-1、Hbec-5i及CD36），共计5个样本。