Expression and cellular localisation of Trypanosoma cruzi calpains

BACKGROUND Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.

研究背景：钙蛋白酶（calpains）广泛存在于几乎所有生物体内，是一类依赖钙离子的半胱氨酸蛋白酶家族，参与诸多关键细胞生理功能。克氏锥虫（Trypanosoma cruzi）是恰加斯病（Chagas disease）的病原体，其体内该基因家族发生了扩增，但其相关生物学特性尚未得到深入研究。
研究目的：本研究旨在从克氏锥虫基因组中筛选钙蛋白酶基因，检测其mRNA表达水平、钙蛋白酶活性与蛋白表达情况，并明确该蛋白在寄生虫三种生命周期阶段的细胞定位。
方法与结果：本研究在克氏锥虫CL Brener基因组中共鉴定出63条钙蛋白酶序列，涵盖14种结构域排列模式。通过实时定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）检测钙蛋白酶mRNA丰度，结果显示7条序列在无鞭毛体（amastigotes）和/或血流型锥鞭毛体（bloodstream trypomastigotes）中呈上调表达，另有5条序列在上鞭毛体（epimastigotes）中上调。蛋白质免疫印迹（Western Blotting）分析显示，在三种寄生虫生命周期阶段中共检测到7种不同的钙蛋白酶分子，其中1种为无鞭毛体特异性蛋白，但未检测到蛋白水解活性。流式细胞术检测结果表明，与上鞭毛体相比，无鞭毛体和/或锥鞭毛体的胞内钙蛋白酶含量更高。超微结构分析显示，三种寄生虫阶段的细胞质、囊泡膜与质膜中均存在钙蛋白酶，锥鞭毛体的鞭毛旁杆（paraflagellar rod）中也检测到该蛋白。
研究结论：钙蛋白酶在克氏锥虫的生命周期各阶段呈现差异化表达与定位模式。本研究为克氏锥虫钙蛋白酶的存在特征与表达模式提供了新的实验数据。