Search

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.)

Membrane proteins in trypanosomatids are, in general, weakly expressed and confirmation of their subcellular localization frequently requires their overexpression with epitope tags. However, overexpression can lead to mislocalization of the probes. Viswanathan et al. (Nat. Methods, 2015, 12:568) described high performance tags for localization of weakly expressed proteins. We report here the use of these protein tags, named "spaghetti monster," for CRISPR/Cas9-mediated C-terminal endogenous tagging of Trypanosoma cruzi to localize two weakly expressed transient receptor potential channels to acidic compartments. The results indicate that this method will improve the detection of membrane proteins in T. cruzi., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Generation of conditional mutants in Trypanosoma brucei can be done by the use of RNA interference (RNAi). However, RNAi frequently produces off target effects. Here, we present an alternative strategy in which the glmS ribozyme is inserted in the C-terminal region of one allele of a GOI and effectively knocks it down in response to the presence of glucosamine in the culture medium. Using several endogenous genes, we show that the glmS ribozyme cleaves the mRNA in vivo leading to reduction in mRNA and protein expression following glucosamine treatment in both T. brucei procyclic and bloodstream forms. Glucosamine-induced ribozyme activation can be rapidly reversed by removing the inducer. In summary, the glmS ribozyme could be used as a tool to study essential genes in T. brucei., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno-electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites., (© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.)

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites., (© 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.)

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy., (© 2011 The Author(s). Journal of Eukaryotic Microbiology© 2011 International Society of Protistologists.)

Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PP(i)) and polyphosphate (poly P) complexed with calcium and other cations. The acidocalcisome membrane contains a number of pumps (Ca(2+)-ATPase, V-H(+)-ATPase, H(+)-PPase), exchangers (Na(+)/H(+), Ca(2+)/H(+)), and channels (aquaporins), while its matrix contains enzymes related to PP(i) and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non-pathogenic prokaryotes and eukaryotes, e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PP(i) and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PP(i), poly P, and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.

The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca(2+) compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca(2+)-ATPase activity was found to be responsible for intracellular Ca(2+) transport in these cells, with no evidence of a mitochondrial Ca(2+) transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A(1), and stimulated by Na(+) ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca(2+) and H(+) transport in promastigotes of P. francai.

The plasma membrane potential (deltapsi) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of deltapsi in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H(+)-ATPase inhibitors (e.g. dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A1). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium-rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the deltapsi of the procyclic stages in a sodium-rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H(+)-ATPase inhibitor dicyclohexylcarbo-diimide, indicating the presence of an ouabain-sensitive sodium pump whose activity is masked by the H(+)-ATPase. Unlike procyclic forms, the deltapsi of bloodstream-stage trypomastigotes was markedly sensitive to extracellular Na+ and K+ concentrations. Thus, there are significant differences between procyclic and bloodstream forms in the maintenance of the deltapsi and in their permeability to cations.

Respiration, oxidative phosphorylation. and the corresponding changes in membrane potential (deltapsi) of Trypanosoma cruzi epimastigotes grown either in liver infusion-tryptose (LIT) or brain heart infusion (BHI) culture medium were assayed in situ using digitonin to render their plasma membrane permeable to succinate, ADP, safranine O, and other small molecules. When the cells were permeabilized with 64 microM digitonin, a concentration previously used with epimastigotes, the ability of the cells grown in LIT medium to sustain oxidative phosphorylation was demonstrated by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. In contrast, the cells grown in BHI medium were not able to sustain a stable membrane potential and did not respond to ADP addition. Analyses of oxygen consumption by these permeabilized cells indicated that the rate of basal respiration, which was similar in both cell types, was significantly decreased by 64 microM digitonin. Addition of ADP to the permeabilized cells grown in LIT medium promoted an oligomycin-sensitive transition from resting to phosphorylating respiration in contrast to the cells grown in BHI medium, whose respiration decreased steadily and did not respond either to ADP or CCCP. Titration of the cells grown in BHI medium with different digitonin concentrations indicated that their mitochondria have higher sensitivity to digitonin than those grown in LIT medium. Analysis of the sterol composition of epimastigotes grown in the two different media showed a higher percentage of cholesterol in total and mitochondrial extracts of epimastigotes grown in BHI medium as compared to those grown in LIT medium, suggesting the involvement of this sterol in their increased sensitivity to digitonin-permeabilization.

[35S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 microM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [35S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [3H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.

We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.