Functional analysis and importance for host cell infection of the Ca-conducting subunits of the mitochondrial calcium uniporter of .

We report here that the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex subunit that we named and . The predicted structure of the proteins indicates that, as predicted for the and paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca uptake, while knockout (KO) of either or led to a loss of mitochondrial Ca uptake, without affecting the mitochondrial membrane potential. -KO and -KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca uptake. In conclusion, the results predict a hetero-oligomeric structure for the MCU complex, with structural and functional differences, as compared with those in the mammalian complex.