Abstract
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.IMPORTANCETrypanosoma 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.
Highlights
The mitochondrial Ca2ϩ 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
We investigated whether T. cruzi mitochondrial calcium uptake 1 (MICU1) (TcMICU1) and TcMICU2 behave as the animal orthologs in modulating mitochondrial Ca2ϩ uptake
We included in this experiment TcMCU-KO cells, which exhibited an increased level of pyruvate dehydrogenase (PDH) phosphorylation [33]. These results suggest that in TcMICU1-KO and TcMICU2-KO cells, the reduced capacity of mitochondria to take up Ca2ϩ determines a low activity of Ca2ϩ-sensitive T. cruzi PDH (TcPDH) phosphatase (TcPDP) and in consequence an increase in the inactive form of TcPDH
Summary
The mitochondrial Ca2ϩ 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. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2ϩ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity None of these proteins had a gatekeeper function at low cytosolic Ca2ϩ concentrations ([Ca2ϩ]cyt), as occurs with their mammalian orthologs. Using the CRISPR/Cas technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2ϩ]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations. The mitochondrial calcium uniporter (MCU) complex mediates Ca2ϩ uptake from the cytosol into the mitochondrial matrix driven by the electrochemical gradient generated by the respiratory chain or ATP hydrolysis [1] and regulates mitochondrial metabolism [2], cytoplasmic Ca2ϩ signaling [3], and cell death [4]. Trypanosomatids possess, in addition, two extra MCU paralogs that were named MCUc, and MCUd, which are Ca2ϩ-conducting subunits and form hetero-oligomers with MCU and MCUb [32]
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