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Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins., Importance: Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism., Competing Interests: The authors declare no conflict of interest.

Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi . PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi . We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.

SUMMARYAcidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca 2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels., Competing Interests: The author declares no conflict of interest.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease., Competing Interests: Declaration of interests A.D.G. is a scientific founder of Maze Therapeutics. A.S.H. is a scientific consultant for Dewpoint Therapeutics and on the Scientific Advisory Board for Prose Foods., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of Competing Interest None.

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1 .

Acidocalcisomes are electron-dense organelles rich in polyphosphate and inorganic and organic cations that are acidified by proton pumps, and possess several channels, pumps, and transporters. They are present in bacteria and eukaryotes and have been studied in greater detail in trypanosomatids. Biogenesis studies of trypanosomatid acidocalcisomes found that they share properties with lysosome-related organelles of animal cells. In addition to their described roles in autophagy, cation and phosphorus storage, osmoregulation, pH homeostasis, and pathogenesis, recent studies have defined the role of these organelles in phosphate utilization, calcium ion (Ca 2+ ) signaling, and bioenergetics, and will be the main subject of this review., (© 2022 International Society of Protistologists.)

The mitochondrial Ca 2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca 2+ 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 2+ overload at low cytosolic Ca 2+ 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 2+ uptake at low extramitochondrial Ca 2+ concentrations ([Ca 2+ ] 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 Ca 2+ uptake at low [Ca 2+ ] ext , significantly decreased the mitochondrial Ca 2+ uptake at different [Ca 2+ ] ext , without dissipation of the mitochondrial membrane potential, and increased the [Ca 2+ ] ext set point needed for Ca 2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg 2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca 2+ uptake at different [Ca 2+ ] ext . Occlusion of the MCUC pore by Mg 2+ could partially explain the lack of mitochondrial Ca 2+ uptake at low [Ca 2+ ] 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., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2022. Published by Elsevier Ltd.)

Developing transfection protocols for marine protists is an emerging field that will allow the functional characterization of protist genes and their roles in organism responses to the environment. We developed a CRISPR/Cas9 editing protocol for Bodo saltans, a free-living kinetoplastid with tolerance to both marine and freshwater conditions and a close non-parasitic relative of trypanosomatids. Our results show that SaCas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex-mediated disruption of the paraflagellar rod 2 gene (BsPFR2) was achieved using electroporation-mediated transfection. The use of CRISPR/Cas9 genome editing can increase the efficiency of targeted homologous recombination when a repair DNA template is provided. Our sequence analysis suggests two mechanisms for repairing double-strand breaks in B. saltans are active; homologous-directed repair (HDR) utilizing an exogenous DNA template that carries an antibiotic resistance gene and likley non-homologous end joining (NHEJ). However, HDR was only achieved when a single (vs. multiple) SaCas9 RNP complex was provided. Furthermore, the biallelic knockout of BsPFR2 was detrimental for the cell, highlighting its essential role for cell survival because it facilitates the movement of food particles into the cytostome. Our Cas9/sgRNA RNP complex protocol provides a new tool for assessing gene functions in B. saltans and perhaps similar protists with polycistronic transcription., (© 2022 Woods Hole Oceanographic Institution. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named Tc BDF1 to Tc BDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid p Tc INDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of Tc BDF2 in T. cruzi . This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. Tc BDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of Tc BDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, Tc BDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized Tc BDF3. These results point to Tc BDF2 as a druggable target against T. cruzi .

Significance: Millions of people are infected with trypanosomatids and new therapeutic approaches are needed. Trypanosomatids possess one mitochondrion per cell and its study has led to discoveries of general biological interest. These mitochondria, as in their animal counterparts, generate reactive oxygen species (ROS) and have evolved enzymatic and nonenzymatic defenses against them. Mitochondrial calcium ion (Ca 2+ ) overload leads to generation of ROS and its study could lead to relevant information on the biology of trypanosomatids and to novel drug targets. Recent Advances: Mitochondrial Ca 2+ is normally involved in maintaining the bioenergetics of trypanosomes, but when Ca 2+ overload occurs, it is associated with cell death. Trypanosomes lack key players in the mechanism of cell death described in mammalian cells, although mitochondrial Ca 2+ overload results in collapse of their membrane potential, production of ROS, and cytochrome c release. They are also very resistant to mitochondrial permeability transition, and cell death after mitochondrial Ca 2+ overload depends on generation of ROS. Critical Issues: In this review, we consider the mechanisms of mitochondrial oxidant generation and removal and the involvement of Ca 2+ in trypanosome cell death. Future Directions: More studies are required to determine the reactions involved in generation of ROS by the mitochondria of trypanosomatids, their enzymatic and nonenzymatic defenses against ROS, and the occurrence and composition of a mitochondrial permeability transition pore. Antioxid. Redox Signal . 36, 969-983.

Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca 2+ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca 2+ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH 3 ) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.

Calcium ion (Ca 2+ ) signaling is one of the most frequently employed mechanisms of signal transduction by eukaryotic cells, and starts with either Ca 2+ release from intracellular stores or Ca 2+ entry through the plasma membrane. In intracellular protist parasites Ca 2+ signaling initiates a sequence of events that may facilitate their invasion of host cells, respond to environmental changes within the host, or regulate the function of their intracellular organelles. In this review we examine recent findings in Ca 2+ signaling in two groups of intracellular protist parasites that have been studied in more detail, the apicomplexan and the trypanosomatid parasites., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 ( TcAEK1 ) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line ( TcAEK1- SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca 2+ and K + homeostasis in mammalian cells. Here, we demonstrate that the Letm1 orthologue of Trypanosoma cruzi, the etiologic agent of Chagas disease, is important for mitochondrial Ca 2+ uptake and release. The results show that both mitochondrial Ca 2+ influx and efflux are reduced in TcLetm1 knockdown (TcLetm1-KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential. Remarkably, TcLetm1 knockdown or overexpression increases or does not affect mitochondrial Ca 2+ levels in epimastigotes, respectively. TcLetm1-KD epimastigotes have reduced growth, and both overexpression and knockdown of TcLetm1 cause a defect in metacyclogenesis. TcLetm1-KD also affected mitochondrial bioenergetics. Invasion of host cells by TcLetm1-KD trypomastigotes and their intracellular replication is greatly impaired. Taken together, our findings indicate that TcLetm1 is important for Ca 2+ homeostasis and cell viability in T cruzi., (© 2021 Federation of American Societies for Experimental Biology.)

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket ("big eye" phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target., (© 2021 Federation of American Societies for Experimental Biology.)

Trypanosoma cruzi is a unicellular parasite and the etiologic agent of Chagas disease. The parasite has a digenetic life cycle alternating between mammalian and insect hosts, where it faces a variety of environmental conditions to which it must adapt in order to survive. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Major environmental changes include temperature, nutrient availability, ionic composition, pH, osmolarity, oxidative stress, contact with host cells and tissues, host immune response, and intracellular life. Some of the signaling pathways and second messengers potentially involved in the response to these changes have been elucidated in recent years and will be the subject of this review., (© 2020 John Wiley & Sons Ltd.)

Diphosphoinositol-5-pentakisphosphate (5-PP-IP 5 ), also known as inositol heptakisphosphate (5-IP 7 ), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a posttranslational modification involving a β-phosphoryl transfer. In this study, utilizing an immobilized 5-IP 7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP 7 -regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP 7 -driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP 7 -mediated phosphorylation events at a polyserine tract (Ser 578-580 ). We also identified a novel SPX domain-containing phosphoribosyltransferase [EC 2.7.6.1] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP 7 in this divergent eukaryote, and provided potential new targets for chemotherapy., (© 2020 John Wiley & Sons Ltd.)

Ca 2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca 2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca 2+ homeostasis and the initiation of Ca 2+ signaling. We used a T. gondii mutant of the V-H + -ATPase, a pump previously shown to transport protons to the extracellular medium, and to control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca 2+ at physiological levels and for Ca 2+ influx. Proton transport was also important for Ca 2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca 2+ and concentrates in acidocalcisomes. This was supported by the co-localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V-ATPase in acidocalcisomes. Our work shows that proton transport regulates plasma membrane Ca 2+ transport and control acidocalcisome polyP and Ca 2+ content, impacting Ca 2+ signaling and downstream stimulation of motility and egress in T. gondii., (© 2021 John Wiley & Sons Ltd.)

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi , the etiologic agent of Chagas disease. Endogenous tagging of T. cruzi MPC1 ( TcMPC1 ) and TcMPC2 with 3× c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1 -KO and TcMPC2 -KO epimastigotes showed reduced O 2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O 2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca 2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1 -KO and TcMPC2 -KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC -KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruzi MPC1 ( TcMPC1 ) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication., (Copyright © 2021 Negreiros et al.)

Trypanosoma cruzi, and the T. brucei group of parasites cause neglected diseases that affect millions of people around the world. These unicellular microorganisms have complex life cycles involving an insect vector and a mammalian host. Both groups of pathogens possess an inositol 1,4,5-trisphosphate (IP 3 )/diacylglycerol (DAG) signaling pathway, and an IP 3 receptor, but with lineage-specific adaptations that make them different from their mammalian counterparts. The phospholipase C (PLC), which hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP 2 ) to IP 3 is N-terminally myristoylated and palmitoylated. Acidocalcisomes, which are lysosome-related organelles rich in polyphosphate, are the main intracellular Ca 2+ stores. The inositol 1,4,5-trisphosphate receptor (IP 3 R) localizes to acidocalcisomes instead of the endoplasmic reticulum. The trypanosome IP 3 R is stimulated by luminal phosphate and pyrophosphate, which are hydrolysis products of polyphosphate (polyP), and inhibited by tripolyphosphate (polyP 3 ), which is the most abundant polyP in acidocalcisomes. Ca 2+ signaling is important for host cell invasion and differentiation and to maintain cellular bioenergetics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins., Competing Interests: The authors have declared that no competing interests exist.

Inositol phosphates (IPs) are phosphorylated derivatives of myo-inositol involved in the regulation of several cellular processes through their interaction with specific proteins. Their synthesis relies on the activity of specific kinases that use ATP as phosphate donor. Here, we combined reverse genetics and liquid chromatography coupled to mass spectrometry (LC-MS) to dissect the inositol phosphate biosynthetic pathway and its metabolic intermediates in the main life cycle stages (epimastigotes, cell-derived trypomastigotes, and amastigotes) of Trypanosoma cruzi , the etiologic agent of Chagas disease. We found evidence of the presence of highly phosphorylated IPs, like inositol hexakisphosphate (IP 6 ), inositol heptakisphosphate (IP 7 ), and inositol octakisphosphate (IP 8 ), that were not detected before by HPLC analyses of the products of radiolabeled exogenous inositol. The kinases involved in their synthesis (inositol polyphosphate multikinase (TcIPMK), inositol 5-phosphate kinase (TcIP5K), and inositol 6-phosphate kinase (TcIP6K)) were also identified. TcIPMK is dispensable in epimastigotes, important for the synthesis of polyphosphate, and critical for the virulence of the infective stages. TcIP5K is essential for normal epimastigote growth, while TcIP6K mutants displayed defects in epimastigote motility and growth. Our results demonstrate the relevance of highly phosphorylated IPs in the life cycle of T. cruzi .

Mitochondrial calcium ion (Ca 2+ ) uptake is important for buffering cytosolic Ca 2+ levels, for regulating cell bioenergetics, and for cell death and autophagy. Ca 2+ uptake is mediated by a mitochondrial Ca 2+ 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 Ca 2+ homeostasis in trypanosomes, the composition of the uniporter, its functional characterization, and its role in general physiology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mitochondrial Ca 2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP 3 R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP 3 R is a Ca 2+ release channel gated by IP 3 when expressed in DT40 cells knockout for all vertebrate IP 3 receptors, and is required for Ca 2+ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O 2 consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP 3 R by CRISPR/Cas9 genome editing caused: a) a reduction in O 2 consumption rate and citrate synthase activity; b) decreased mitochondrial Ca 2+ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP 3 R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP 3 R-mediated acidocalcisome Ca 2+ release on cell bioenergetics in T. cruzi., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Lathosterol oxidase (LSO) catalyzes the formation of the C-5-C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5-C-6 desaturation, we generated an LSO -null mutant line ( lso - ) in Leishmania major , the causative agent for cutaneous leishmaniasis. lso - parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5-C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso - mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania All these defects in lso - were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5-C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5-C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5-C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development., (Copyright © 2020 Ning et al.)

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Bisphosphonates are the most commonly prescribed drugs for the treatment of osteoporosis and other bone illnesses. Some of them have also shown antiparasitic activity. In search of improving the pharmacological profile of commercial bisphosphonates, our group had previously developed first row transition metal complexes with N-containing bisphosphonates (NBPs). In this work, we extended our studies to heteroleptic palladium-NBP complexes including DNA intercalating polypyridyl co-ligands (NN) with the aim of obtaining potential multi-target species. Complexes of the formula [Pd(NBP) 2 (NN)]·2NaCl·xH 2 O with NBP = alendronate (ale) or pamidronate (pam) and NN = 1,10 phenanthroline (phen) or 2,2'-bipyridine (bpy) were synthesized and fully characterized. All the obtained compounds were much more active in vitro against T. cruzi (amastigote form) than the corresponding NBP ligands. In addition, complexes were nontoxic to mammalian cells up to 50-100 µM. Compounds with phen as ligand were 15 times more active than their bpy analogous. Related to the potential mechanism of action, all complexes were potent inhibitors of two parasitic enzymes of the isoprenoid biosynthetic pathway. No correlation between the anti-T. cruzi activity and the enzymatic inhibition results was observed. On the contrary, the high antiparasitic activity of phen-containing complexes could be related to their ability to interact with DNA in an intercalative-like mode. These rationally designed compounds are good candidates for further studies and good leaders for future drug developments. Four new palladium heteroleptic complexes with N-containing commercial bisphosphonates and DNA intercalating polypyridyl co-ligands were synthesized and fully characterized. All complexes displayed high anti-T. cruzi activity which could be related to the inhibition of the parasitic farnesyl diphosphate synthase enzyme but mainly to their ability to interact DNA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In continuation of our pharmacomodulation work on the nitroimidazooxazole series, we report the synthesis of new 5-substituted 6-nitroimidazooxazole derivatives. Our aim was to evaluate how functionalization of the 5-position of the 6-nitroimidazooxazole scaffold affects antileishmanial and antitrypanosomal in vitro activities. Twenty-one original compounds were synthesized and evaluated for their in vitro antileishmanial (L. donovani) and antitrypanosomal (T. cruzi) properties. Pallado-catalyzed cross-coupling reactions were used to introduce an aryl or ethynyl aryl substituent in 5-position from a 5-brominated-6-nitroimidazooxazole starting product. Unfortunately, the first series of compounds bearing an aryl group in 5-position presented limited in vitro activities against L. donovani and T. cruzi, with IC 50  > 10 μM (vs 0.18 μM and 2.31 μM for the reference drugs amphotericin B and benznidazole respectively). Interestingly, the second series of compounds bearing an ethynyl aryl substituent in 5-position showed more promising, particularly against T. cruzi. Compounds 6a, 6b, 6c, 6g and 6h had better activity than the reference drug benznidazole (0.92 μM ≤ IC 50  ≤ 2.18 μM vs IC 50  = 2.31 μM), whereas the non-functionalized 2-methyl-6-nitro-2,3-dihydroimidazo [2,1-b]oxazole 2 was not active against T. cruzi (IC 50  > 10 μM)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Mitochondrial Ca 2+ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and P i transport with ATP synthesis, a process that is stimulated by Ca 2+ in trypanosomes and human cells. IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei , the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells., (Copyright © 2020 Huang and Docampo.)

Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis , which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei . In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 ( TcGP72 ) and vacuolar proton pyrophosphatase ( TcVP1 ) with the active ( glmS ) or inactive ( M9 ) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro . Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite., (Copyright © 2020 Lander, Cruz-Bustos and Docampo.)

Lysine polyphosphorylation (K-PPn) is a relatively new post-translational modification, the full targets and functional consequences of which are unknown. A critical problem in the study of endogenous K-PPn of proteins in the yeast model system is that its nonenzymatic nature and its susceptibility to polyphosphatases make it potentially susceptible to artifacts during extraction. A new study confirms that K-PPn modifications can be altered during sample handling, provides new insights into the mechanism of K-PPn, and develops a yeast model strain, devoid of both vacuolar polyP and polyphosphatases, that allows detection of authentic endogenous K-PPn., (© 2020 Docampo.)

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PP i ) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca 2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca 2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP 3 R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

As an extension of our project aimed at the search for new chemotherapeutic agents against Chagas disease and toxoplasmosis, several 1,1-bisphosphonates were designed, synthesized and biologically evaluated against Trypanosoma cruzi and Toxoplasma gondii, the etiologic agents of these diseases, respectively. In particular, and based on the antiparasitic activity exhibited by 2-alkylaminoethyl-1,1-bisphosphonates targeting farnesyl diphosphate synthase, a series of linear 2-alkylaminomethyl-1,1-bisphosphonic acids (compounds 21-33), that is, the position of the amino group was one carbon closer to the gem-phosphonate moiety, were evaluated as growth inhibitors against the clinically more relevant dividing form (amastigotes) of T. cruzi. Although all of these compounds resulted to be devoid of antiparasitic activity, these results were valuable for a rigorous SAR study. In addition, unexpectedly, the synthetic designed 2-cycloalkylaminoethyl-1,1-bisphosphonic acids 47-49 were free of antiparasitic activity. Moreover, long chain sulfur-containing 1,1-bisphosphonic acids, such as compounds 54-56, 59, turned out to be nanomolar growth inhibitors of tachyzoites of T. gondii. As many bisphosphonate-containing molecules are FDA-approved drugs for the treatment of bone resorption disorders, their potential nontoxicity makes them good candidates to control American trypanosomiasis and toxoplasmosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca 2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP 3 R), which is essential for growth and infectivity of the parasite Trypanosoma brucei However, the mechanism by which TbIP 3 R controls Ca 2+ release is unclear. In this work, we expressed TbIP 3 R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP 3 Rs were stably ablated (DT40-3KO). We show that IP 3 -mediated Ca 2+ release depends on Ca 2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP 3 R disclosed that luminal inorganic orthophosphate (P i ) or pyrophosphate (PP i ), and neutral or alkaline pH can stimulate IP 3 -generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP 3 R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within acidocalcisomes or alkalinization of their luminal pH activate TbIP 3 R and Ca 2+ release. We conclude that TbIP 3 R is well-adapted to its role as the major Ca 2+ release channel of acidocalcisomes in T. brucei., (© 2019 Potapenko et al.)

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 Ca 2+ uptake, while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca 2+ 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 Ca 2+ 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.

Trypanosoma brucei , a protist parasite that causes African trypanosomiasis or sleeping sickness, relies mainly on glycolysis for ATP production when in its mammalian host. Glycolysis occurs within a peroxisome-like organelle named the glycosome. Previous work from our laboratory reported the presence of significant amounts of inorganic polyphosphate (polyP), a polymer of three to hundreds of orthophosphate units, in the glycosomes and nucleoli of T. brucei In this work, we identified and characterized the activity of two Nudix hydrolases (NHs), T. brucei Nudix hydrolase (TbNH) 2 and TbNH4, one located in the glycosomes and the other in the cytosol and nucleus, respectively, which can degrade polyP. We found that TbNH2 is an exopolyphosphatase with higher activity on short chain polyP, while TbNH4 is an endo- and exopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. We also found that only TbNH2 can dephosphorylate ATP and ADP but with lower affinity than for polyP. Our results suggest that NHs can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes, cytosol and nuclei of T. brucei ., (© 2019 The Author(s).)

The mitochondrial Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ concentrations ([Ca 2+ ] 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. IMPORTANCE Trypanosoma 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 Ca 2+ sensors and gatekeepers of the MCU, preventing Ca 2+ uptake under resting conditions and favoring it at high cytosolic Ca 2+ concentrations ([Ca 2+ ] 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 [Ca 2+ ] cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations., (Copyright © 2019 Bertolini et al.)

Inorganic pyrophosphate (PP i ) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PP i and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and P i conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na + /P i symporter. PP i is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PP i generated outward currents in Na + /P i -loaded giant vacuoles prepared from wild-type or pho91 Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PP i , at physiological concentrations, can function as a signaling molecule releasing P i from S. cerevisiae vacuoles and T. brucei acidocalcisomes. IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na + /P i symporter involved in P i release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PP i , at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule., (Copyright © 2019 Potapenko et al.)

As a continuation of our project aimed at searching for new chemotherapeutic agents against American trypanosomiasis (Chagas disease), new selenocyanate derivatives were designed, synthesized and biologically evaluated against the clinically more relevant dividing form of Trypanosoma cruzi, the etiologic agent of this illness. In addition, in order to establish the role of each part of the selenocyanate moiety, different derivatives, in which the selenium atom or the cyano group were absent, were conceived, synthesized and biologically evaluated. In addition, in order to study the optimal position of the terminal phenoxy group, new regioisomers of WC-9 were synthesized and evaluated against T. cruzi. Finally, the resolution of a racemic mixture of a very potent conformationally rigid analogue of WC-9 was accomplished and further tested as growth inhibitors of T. cruzi proliferation. The results provide further insight into the role of the selenocyanate group in its antiparasitic activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)