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Functional analysis and importance for host cell infection of the Ca²⁺-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi.

ABSTRACT: We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc and TcMCUd. The predicted structure of the proteins indicates that, as predicted for the TcMCU and TcMCUb 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 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 CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues 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 with those in the mammalian complex.

SUBMITTER: Chiurillo MA

PROVIDER: S-EPMC6727756 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The mitochondrial calcium uniporter complex (MCUC) is a highly selective channel that conducts calcium ions across the organelle inner membrane. We previously characterized Trypanosoma brucei's MCU (TbMCU) as an essential component of the MCUC required for parasite viability and infectivity. In this study, we characterize its paralog T. brucei MCUb (TbMCUb) and report the identification of two novel components of the complex that we named TbMCUc and TbMCUd. These new MCUC proteins are unique and conserved only in trypanosomatids. In situ tagging and immunofluorescence microscopy revealed that they colocalize with TbMCU and TbMCUb to the mitochondria of T. brucei Blue Native PAGE and immunodetection analyses indicated that the MCUC proteins exist in a large protein complex with a molecular weight of approximately 380 kDa. RNA interference (RNAi) or overexpression of the TbMCUc and TbMCUd genes significantly reduced or enhanced mitochondrial Ca2+ uptake in T. brucei, respectively, without affecting the mitochondrial membrane potential, indicating that they are essential components of the MCUC of this parasite. The specific interactions of TbMCU with TbMCUb, TbMCUc, or TbMCUd were confirmed by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Furthermore, combining mutagenesis analysis with MYTH assays revealed that transmembrane helices (TMHs) were determinant of the interactions between TbMCUC subunits. In summary, our study has identified two novel essential components of the MCUC of T. brucei and defined their direct physical interactions with the other subunits that result in a hetero-oligomeric MCUC.IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and nagana in animals. The finding of a mitochondrial calcium uniporter (MCU) conserved in this parasite was essential for the discovery of the gene encoding the pore subunit. Mitochondrial Ca2+ transport mediated by the MUC complex is critical in Trypanosoma brucei for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. We found that one component of the complex (MCUb) does not act as a dominant negative effector of the channel as in vertebrate cells and that the TbMCUC possesses two unique subunits (MCUc and MCUd) present only in trypanosomatids and required for Ca2+ transport. The study of the interactions between these four subunits (MCU, MCUb, MCUc, and MCUd) by a variety of techniques that include coimmunoprecipitation, split-ubiquitin membrane-based yeast two-hybrid assays, and site-directed mutagenesis suggests that they interact through their transmembrane helices to form hetero-oligomers.

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Functional analysis and importance for host cell infection of the Ca²⁺-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi.

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Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Functional analysis and importance for host cell infection of the Ca²⁺-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi.