MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca ²⁺ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi

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 <i>Trypanosoma cruzi</i> to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca²⁺ uptake. <i>T. cruzi</i> 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 <i>TcMICU1</i> and <i>TcMICU2</i> knockout (-KO) cell lines. Ablation of either <i>TcMICU1</i> or <i>TcMICU2</i> 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. <i>TcMICU1</i>-KO and <i>TcMICU2</i>-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.<b>IMPORTANCE</b><i>Trypanosoma cruzi</i> 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 <i>TcMICU1</i> and <i>TcMICU2</i> 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.