Santos Cyndia Mara Bezerra Dos, Ludwig Adriana, Kessler Rafael Luis, Rampazzo Rita de Cássia Pontello, Inoue Alexandre Haruo, Krieger Marco Aurélio, Pavoni Daniela Parada, Probst Christian Macagnan
Centro Politécnico, Universidade Federal do Paraná, Curitiba, PR, Brasil.
Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brasil.
Mem Inst Oswaldo Cruz. 2018;113(5):e170404. doi: 10.1590/0074-02760170404. Epub 2018 Apr 9.
BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.
克氏锥虫是一种重要的原生动物寄生虫，也是恰加斯病的病原体。了解克氏锥虫生物学特性的关键步骤之一是研究其生长曲线中呈现的细胞和分子特征。
我们旨在全面了解克氏锥虫在无鞭毛体生长过程中的基因表达谱。
除了细胞活力和细胞周期分析外，还对体外培养10天的克氏锥虫无鞭毛体生长曲线进行了总RNA和多核糖体/颗粒RNA组分的RNA测序分析。我们还分析了多核糖体谱，并通过荧光原位杂交和蛋白质印迹法研究了颗粒RNA的存在情况。
我们鉴定出1082个差异表达基因（DEG），其中220个在两个组分中均受到调控。根据调控模式，DEG被分为12个簇，并显示出重要基因本体（GO）术语的富集。此外，我们发现，在生长曲线的第6天，多核糖体含量大幅下降，而RNA颗粒含量似乎增加，这表明在生长后期从蔗糖梯度中分离出的一部分mRNA与RNA颗粒相关，而不仅仅与多核糖体相关。此外，我们还讨论了几个可能参与克氏锥虫生长的调控基因，主要是在稳定期，例如与细胞周期、发病机制、代谢过程和RNA结合蛋白相关的基因。
