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.

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.

Edited by Dennis VoelkerMethods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP 3 R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was colocalized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP 3 R, endogenously tagged TcIP 3 R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.The application of the CRISPR/Cas9 technology to the study of protist parasites has dramatically increased the tools available for their genetic manipulation (1). Trypanosoma cruzi, the etiologic agent of Chagas disease, which is a significant cause of morbidity and mortality from the South of the United States to the South of Argentina and Chile, has been particularly refractory to genetic manipulation. However, the recent use of the CRISPR/Cas9 technology to knock down or knock out genes (2, 3) has revolutionized their study.The localization of proteins is important to determine their cellular function, and previous studies in T. cruzi have used either antibodies or gene tagging methods with vectors that overexpressed the proteins (4). Although specific antibodies are useful to detect the endogenous proteins, it is not always possible to obtain them because either the proteins have low antigenicity or the antibodies cross-react with other proteins. Plasmids that enable the tagging of genes at their endogenous loci are not available for T. cruzi, and a major drawback of the overexpression of tagged proteins is that the proteins of interest sometimes are retained in the endoplasmic reticulum (ER) 4 or localize to other compartments.Here we have adapted the CRISPR/Cas9 system to tag genes of T. cruzi at their endogenous loci and tested this system with two genes encoding proteins of well recognized localization (flagellar calcium binding protein or TcFCaBP and the acidocalcisome vacuolar proton pyrophosphatase or TcVP1) and two encoding proteins of undefined or disputed localizati...

In vertebrate cells, mitochondrial Ca 2؉ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca 2؉-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1␣ subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1␣ subunit of PDH from nonvertebrate organisms has been described, the Ca 2؉-mediated PDP activation has not been studied. In this work, we investigated the Ca 2؉ 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 Ca 2؉-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 Ca 2؉-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.

Edited by Ursula Jakob Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (P i) transport are unclear. We found here that Tbpho91 ؊/؊ trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased P i content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and P i conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na ؉ / P i-loaded giant vacuoles prepared from WT or from TbPHO91expressing pho91⌬ strains but not from the pho91⌬ yeast strains or from the pho91⌬ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar P i and Na ؉ efflux and that myo-inositol polyphosphates stimulate the Na ؉ /P i symporter activities through their SPX domains.