MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.

The mitochondrial Ca 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 to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca uptake. 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 and knockout (-KO) cell lines. Ablation of either or showed a significantly reduced mitochondrial Ca 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 Ca concentrations ([Ca]), as occurs with their mammalian orthologs. -KO and -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. 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 Ca sensors and gatekeepers of the MCU, preventing Ca uptake under resting conditions and favoring it at high cytosolic Ca concentrations ([Ca]). Using the CRISPR/Cas9 technique, we generated and knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca] but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.