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 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. IMPORTANCE Trypanosoma 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.

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…Important findings have been reported on the structural and biochemical characterization of proteins involved in different metabolic pathways and signaling cascades in T. cruzi, T. brucei, and Leishmania spp. Bertolini et al 2019;Chiurillo et al 2017Chiurillo et al , 2019Costa et al 2018;Cruz-Bustos et al 2018b;Lander et al 2017Lander et al , 2018Martel et al 2017;Vasquez et al 2018; Table 1). Several approaches have been successfully used to adapt the CRISPR/Cas9 system and modify these parasites' genomes.…”

“…Selection-free methods are useful when there is an expected phenotype associated to the deletion of a gene, as for example drug resistance (Rico et al 2018;Soares Medeiros et al 2017;Zhang and Matlashewski 2015) or flagellar detachment (Burle-Caldas et al 2018). However, for genes encoding proteins of unknown function, the inclusion of a resistance marker or reporter gene in the DNA donor template is the best option to efficiently achieve genome editing in trypanosomatids Bertolini et al 2019;Chiurillo et al 2017Chiurillo et al , 2019Costa et al 2018;Cruz-Bustos et al 2018a,b;Lander et al 2015Lander et al , 2016bLander et al , 2018Liu et al 2019;Martel et al 2017). Figure 1 describes the two strategies that have been most widely used for genome editing in kinetoplastids Lander et al 2015).…”

“…, ), mitochondrial calcium uptake proteins TcMICU1 and TcMICU2 (Bertolini et al. ), TcAMT (Cruz‐Bustos et al. 2018b) and TcPDP (Lander et al.…”

“…In general, there were few molecular tools available for the genetic manipulation of T. cruzi and Leishmania spp., compared to T. brucei, thus limiting the study of biological processes in the first two parasites. Fortunately, genome editing by CRISPR/Cas9 has been recently achieved in T. cruzi (Lander et al 2015;, L. major (Sollelis et al 2015), L. donovani (Zhang and Matlashewski 2015), L. mexicana , T. brucei Rico et al 2018;Vasquez et al 2018) and the honey bee trypanosomatid Lotmaria passim (Liu et al 2019) and the system has been successfully used to investigate the role of several proteins in trypanosomatids, but mainly in T. cruzi Bertolini et al 2019;Chiurillo et al 2017Chiurillo et al , 2019Cruz-Bustos et al 2018b;Lander et al 2015Lander et al , 2018Martel et al 2017;Takagi et al 2019), as shown in Table 1. In this review we described the main strategies that have been developed to perform genome editing by CRISPR/Cas9 in trypanosomatids, presenting examples of their usefulness for solving biological questions.…”

The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans

“…The MCUb subunit is a Ca 2ϩ -conducting subunit and does not have a dominant negative effect on the channel like its mammalian ortholog (17). The mitochondrial calcium uptake 1 (MICU1) and MICU2 proteins do not have a gatekeeper function at low Ca 2ϩ concentrations (18), as occurs with the mammalian orthologs (19)(20)(21)(22)(23)(24)(25). Finally, the trypanosomatid genomes lack orthologs encoding subunits present in the mammalian MCU complex, like MCU regulator 1 (MCUR1) (26) and essential MCU regulator (EMRE) (27).…”