Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi
Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi
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DOI：
10.1091/mbc.e19-03-0152
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发表时间：
2019-07-01
影响因子：
3.3
通讯作者：
Docampo, Roberto
中科院分区：
文献类型：
--
作者：
Chiurillo, Miguel A.;Lander, Noelia;Docampo, Roberto
我们在这里报告，锥虫克氏锥虫，南美锥虫病的病原体，具有两个独特的旁系同源的线粒体钙单向转运体复合物TcMCU亚基，我们命名为TcMCUC和TcMCUD。蛋白质的预测结构表明，如预测的TcMCU和TcMCUB旁系同源物，它们由两个螺旋跨膜结构域组成，并含有WDXXEPXXY基序。每个基因的过表达导致线粒体Ca 2+摄取的显著增加，而敲除（KO）TcMCUC或TcMCUD导致线粒体Ca 2+摄取的损失，而不影响线粒体膜电位。TcMCUc-KO和TcMCUd-KO外鞭毛体在低葡萄糖培养基中表现出生长速率降低，呼吸速率、柠檬酸合酶活性和AMP/ATP比值改变，而锥虫鞭毛体有效感染宿主细胞和在细胞内作为无鞭毛体复制的能力降低。通过KO细胞系的基因互补或通过新开发的CRISPR/Cas9介导的敲入方法，我们还研究了四种旁系同源物的关键氨基酸残基对线粒体Ca 2+摄取的重要性。总之，结果预测了T. cruzi MCU复合体，与哺乳动物复合体相比，具有结构和功能上的差异。
We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc and TcMCUd. The predicted structure of the proteins indicates that, as predicted for the TcMCU and TcMCUb paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca2+ uptake, while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca2+ uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca2+ uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared with those in the mammalian complex.