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

Trypanosoma cruzi 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 T. cruzi, cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite. Adenylate cyclases (ACs) catalyze the conversion of ATP to cAMP. They comprise a multigene family encoding putative receptor-type ACs in T. cruzi. Using protein sequence alignment, we classified them into five groups and chose a representative member from each group to study their localization (TcAC1-TcAC5). We expressed an HA-tagged version of each protein in T. cruzi and performed immunofluorescence analysis. A peculiar dual localization of TcAC1 and TcAC2 was observed in the flagellar distal domain and in the contractile vacuole complex (CVC), and their enzymatic activity was confirmed by gene complementation in yeast. Furthermore, TcAC1 overexpressing parasites showed an increased metacyclogenesis, a defect in host cell invasion, and a reduced intracellular replication, highlighting the importance of this protein throughout T. cruzi life cycle. These mutants were more tolerant to hypoosmotic stress and showed a higher adhesion capacity during in vitro metacyclogenesis, whereas the wild-type phenotype was restored after disrupting TcAC1 localization. Finally, TcAC1 was found to interact with cAMP response protein 3 (TcCARP3), co-localizing with this protein in the flagellar tip and CVC. IMPORTANCE We identified three components of the cAMP signaling pathway (TcAC1, TcAC2, and TcCARP3) with dual localization in Trypanosoma cruzi: the flagellar distal domain and the CVC, structures involved in cell adhesion and osmoregulation, respectively. We found evidence on the role of TcAC1 in both cellular processes, as well as in metacyclogenesis. Our data suggest that TcACs act as signal sensors and transducers through cAMP synthesis in membrane microdomains. We propose a model in which TcACs sense the harsh conditions in the triatomine hindgut (nutrient deprivation, acidic pH, osmotic stress, ionic composition, hydrophobic interactions) and become active. Synthesis of cAMP then triggers cell adhesion prior completion of metacyclogenesis, while mediating a response to osmotic stress in the parasite. These results shed light into the mechanisms driving cAMP-mediated cell differentiation in T. cruzi, while raising new questions on the activation of TcACs and the role of downstream components of this pathway.

MICU1 and MICU2 potentiation of Ca2+ uptake by the mitochondrial Ca2+ uniporter of Trypanosoma cruzi and its inhibition by Mg2.

The mitochondrial Ca2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca2+ 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 Ca2+ overload at low cytosolic Ca2+ levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca2+ uptake at low extramitochondrial Ca2+ concentrations ([Ca2+]ext) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca2+ uptake at low [Ca2+]ext, significantly decreased the mitochondrial Ca2+ uptake at different [Ca2+]ext, without dissipation of the mitochondrial membrane potential, and increased the [Ca2+]ext set point needed for Ca2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca2+ uptake at different [Ca2+]ext. Occlusion of the MCUC pore by Mg2+ could partially explain the lack of mitochondrial Ca2+ uptake at low [Ca2+]ext in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.

Mitochondrial Ca2+ homeostasis in trypanosomes.

Mitochondrial calcium ion (Ca2+) uptake is important for buffering cytosolic Ca2+ levels, for regulating cell bioenergetics, and for cell death and autophagy. Ca2+ uptake is mediated by a mitochondrial Ca2+ uniporter (MCU) and the discovery of this channel in trypanosomes has been critical for the identification of the molecular nature of the channel in all eukaryotes. However, the trypanosome uniporter, which has been studied in detail in Trypanosoma cruzi, the agent of Chagas disease, and T. brucei, the agent of human and animal African trypanosomiasis, has lineage-specific adaptations which include the lack of some homologues to mammalian subunits, and the presence of unique subunits. Here, we review newly emerging insights into the role of mitochondrial Ca2+ homeostasis in trypanosomes, the composition of the uniporter, its functional characterization, and its role in general physiology.

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

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ 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 Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

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 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.

MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.

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.IMPORTANCETrypanosoma 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.

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

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-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 Ca2+-mediated PDP activation has not been studied. In this work, we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.

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. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.

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

Trypanosoma cruzi 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 TcMCU, or its paralog TcMCUb, by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca2+ 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 Ca2+ While TcMCU-knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb 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 TcMCUb, in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU.IMPORTANCE The finding of a mitochondrial calcium uniporter (MCU) in Trypanosoma cruzi was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for T. cruzi to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.