Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi
We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogs of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc, and TcMCUd. The predicted structure of the proteins indicates that, as that predicted for the TcMCU and TcMCUb paralogs, 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 Ca2+ uptake while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca2+ uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-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 knock-in approach we also studied the importance of critical amino acid residues of the four paralogs on mitochondrial Ca2+ uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared to those in the mammalian complex.