TcCARP3 modulates compartmentalized cAMP signals involved in osmoregulation, infection of mammalian cells, and colonization of the triatomine vector in the human pathogen .

is the causative agent of Chagas disease, a zoonotic infectious disease considered a leading cause of cardiomyopathy, disability, and premature death in the Americas. This parasite spends its life between a mammalian host and an arthropod vector, undergoing essential transitions among different developmental forms. How senses microenvironmental changes that trigger cellular responses necessary for parasite survival has remained largely unknown.

A key regulator of male gametogenesis in Cryptosporidium.

Gamete development is a precisely programmed process in Cryptosporidium parvum, a leading cause of diarrheal disease worldwide. Nava et al. recently described the developmentally regulated expression of CDPK5 during male gametogenesis.

Gene editing of putative cAMP and Ca -regulated proteins using an efficient cloning-free CRISPR/Cas9 system in Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca ) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics.

Gene editing of putative cAMP and Ca -regulated proteins using an efficient cloning-free CRISPR/Cas9 system in .

, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca ) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics.

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.

Ablation of the Gene of Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication.

P21 is an immunomodulatory protein expressed throughout the life cycle of , the etiologic agent of Chagas disease. and studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization.

Drug Target Validation of the Protein Kinase Essential for Proliferation, Host Cell Invasion, and Intracellular Replication of the Human Pathogen Trypanosoma cruzi.

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 () 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 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs).

The long and winding road of reverse genetics in .

Trypanosomes are early divergent protists with distinctive features among eukaryotic cells. Together with and spp., has been one of the most studied members of the group.

Mitochondrial Ca homeostasis in trypanosomes.

Mitochondrial calcium ion (Ca) uptake is important for buffering cytosolic Ca levels, for regulating cell bioenergetics, and for cell death and autophagy. Ca uptake is mediated by a mitochondrial Ca 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.

Trypanosoma cruzi Letm1 is involved in mitochondrial Ca transport, and is essential for replication, differentiation, and host cell invasion.

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca 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 uptake and release. The results show that both mitochondrial Ca influx and efflux are reduced in TcLetm1 knockdown (TcLetm1-KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential.

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.

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 , the etiologic agent of Chagas disease.

Signaling pathways involved in environmental sensing in Trypanosoma cruzi.

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.

IP receptor-mediated Ca release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi.

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIPR) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIPR is a Ca release channel gated by IP when expressed in DT40 cells knockout for all vertebrate IP receptors, and is required for Ca uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O consumption, and preventing autophagy.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

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

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

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.

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi.

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.

State-of-the-art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids.

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida.

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.

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation.

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.

Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in .

chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences.