MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi
MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi
复制标题
DOI：
10.1128/mbio.00348-19
复制
发表时间：
2019-05-01
期刊：
影响因子：
6.4
通讯作者：
Docampo, Roberto
中科院分区：
文献类型：
--
作者：
Bertolini, Mayara S.;Chiurillo, Miguel A.;Docampo, Roberto
属于真核生物超类群Excavata的锥虫的线粒体Ca 2+摄取与动物的线粒体Ca 2+摄取具有相同的生化特征，动物的线粒体Ca 2+摄取与真菌一起属于超类群Opisthokonta。然而，锥虫的线粒体钙单向转运体（MCU）复合物的组成是非常特殊的，这表明谱系特异性的适应。在这项工作中，我们使用克氏锥虫来研究线粒体钙摄取1（MICU 1）和MICU 2的直系同源物在线粒体钙摄取中的作用。T. cruzi MICU 1（TcMICU 1）和TcMICU 2具有线粒体靶向信号，两个典型的EF-手形钙结合结构域，并定位于线粒体。使用CRISPR/Cas9系统（i.例如，用Cas9成簇的规则间隔的短回文重复序列），我们产生了TcMICU 1和TcMICU 2敲除（-KO）细胞系。TcMICU 1或TcMICU 2的消融显示在透化的上鞭毛体中线粒体Ca 2+摄取显著降低，而不耗散线粒体膜电位或对AMP/ATP比率或柠檬酸合酶活性的影响。然而，这些蛋白质中没有一个在低胞质Ca 2+浓度（[Ca 2 +]（cyt））下具有看门人功能，如它们的哺乳动物直向同源物所发生的那样。TcMICU 1-KO和TcMICU 2-KO外鞭毛体具有较低的生长速率和受损的氧化代谢，而感染性锥鞭毛体具有降低的侵入宿主细胞和作为无鞭毛体在其中复制的能力。这项研究首次研究了MICU 1和MICU 2在进化上远离动物的生物体中的作用，其结果表明，尽管这些组分可能存在于最后一个真核生物共同祖先（LECA）中，但它们在不同真核生物超群的进化过程中发挥了不同的作用。这项工作也提供了新的见解锥虫适应其特定的生活方式。重要克氏锥虫是南美锥虫病的病原体，属于早期分支真核生物超组挖掘。它的线粒体钙单向转运体（MCU）亚基与发现其编码基因的重要动物直系同源物具有相似性。在动物细胞中，MICU 1和MICU 2蛋白充当MCU的Ca 2+传感器和看门人，在静息条件下阻止Ca 2+摄取，并在高胞质Ca 2+浓度（[Ca 2 +] cyt）下促进其摄取。使用CRISPR/Cas9技术，我们产生了TcMICU 1和TcMICU 2敲除细胞系，并表明MICU 1和-2在低[Ca 2 +] cyt下不充当看门人，但对于正常生长，宿主细胞侵袭和细胞内复制至关重要，揭示了谱系特异性适应。
The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i. e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+](cyt)), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+] cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+] cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.