Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.
Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.
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DOI：
10.1128/mbio.00540-21
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发表时间：
2021-04-06
期刊：
影响因子：
6.4
通讯作者：
Docampo R
中科院分区：
文献类型：
--
作者：
Negreiros RS;Lander N;Chiurillo MA;Vercesi AE;Docampo R
克氏锥虫是恰加斯病的病原体。丙酮酸是糖酵解的最终产物，其转运到线粒体是由线粒体丙酮酸载体(MPC)亚单位介导的。丙酮酸是糖酵解的最终代谢物，可在线粒体中转化为乙酰辅酶A(乙酰辅酶A)，在线粒体中用作三羧酸循环的底物。丙酮酸在线粒体中的有效性取决于其通过线粒体丙酮酸载体1和2形成的异质复合体(MPC1/MPC2)的主动转运。本文报道了查加斯病的病原体克氏锥虫MPC1/MPC2的研究。克氏锥虫MPC1(TcMPC1)和TcMPC2与3×c-Myc的内源标记表明，这两种编码蛋白都与MitoTracker共定位于上胚体的线粒体。利用CRISPR/Cas9对TcMPC1和TcMPC2基因进行了单独敲除(KO)，经PCR和Southern印迹分析证实。当丙酮酸而不是琥珀酸作为线粒体底物时，洋地黄素通透性的TcMPC1-KO和TcMPC2-KO表基的耗氧率降低，而α-酮戊二酸由于α-酮戊二酸脱氢酶活性的增加而增加了它们的耗氧率。线粒体丙酮酸输入缺陷导致钙摄取减少。抑制剂UK5099和丙二酸削弱了通透性对照细胞中丙酮酸驱动的氧耗。丙二酸对琥珀酸脱氢酶的抑制表明丙酮酸需要转化为琥珀酸以增加呼吸。TcMPC1-KO和TcMPC2-KO上胚体在标准或低糖培养基中的生长差异不大。然而，在TcMPC-KOS中，类鞭毛虫感染组织培养细胞并作为细胞内无鞭毛体复制的能力降低。总体而言，克鲁氏毛滴虫MPC1和MPC2对于丙酮酸存在时的细胞呼吸、宿主细胞的入侵和无鞭毛体的复制是必不可少的。
Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruzi MPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.