Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in .

is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect and a mammalian host, transitioning between developmental stages in response to microenvironmental changes. Among the second messengers driving differentiation in , cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite.

MICU1 and MICU2 potentiation of Ca uptake by the mitochondrial Ca uniporter of Trypanosoma cruzi and its inhibition by Mg.

The mitochondrial Ca uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca overload at low cytosolic Ca levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca uptake at low extramitochondrial Ca concentrations ([Ca]) and it was though that in the absence of one MICU the other would replace its role.

Different Sensitivity of Control and MICU1- and MICU2-Ablated Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors.

The mitochondrial Ca uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on , the agent of Chagas disease.

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.

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

The mitochondrial Ca 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 to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca uptake.

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of .

In vertebrate cells, mitochondrial Ca uptake by the mitochondrial calcium uniporter (MCU) leads to Ca-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca-mediated PDP activation has not been studied.

The mitochondrial calcium uniporter complex in trypanosomes.

The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components.

Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of .

is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of , or its paralog , by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca While knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of , in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU.