Proteomic analysis of acidocalcisomes of Trypanosoma brucei uncovers their role in phosphate metabolism, cation homeostasis, and calcium signaling.
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ABSTRACT:
SUBMITTER: Huang G
PROVIDER: S-EPMC4594416 | biostudies-other | 2015 Mar-Apr
REPOSITORIES: biostudies-other
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Project description:Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi 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+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, 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.
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Project description:Potassium channels from prokaryotes and eukaryotes are usually recognized by a typical amino acid sequence TXTGY(F)G representing the ionic selectivity filter. Using a screening approach with ion channel family profiles but without the above motif, we identified a gene in Trypanosoma brucei that exhibits homology to inward rectifying potassium channels. We report here cloning of this ion channel named TbIRK. The protein is localized to acidocalcisomes in procyclic and in bloodstream form parasites. Functional properties of this channel were established after expression in Xenopus oocytes. Currents recorded in potassium medium show inward rectification and little time dependence. Surprisingly, this channel retains selectivity for potassium ions over sodium ions >7, in spite of the lack of the classical selectivity filter. The sequence GGYVG was predicted in silico to replace this filter motif. Point mutations of the corresponding glycine residues confirmed this at the functional level. The channel is inhibited by caesium ions but remains unaffected by barium ions up to 10 mM. TbIRK is to our knowledge the first potassium channel in T. brucei that localizes to the acidocalcisomes, organelles involved in the storage of phosphates and the response to osmotic stress that occurs during the life cycle of trypanosomes.
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Project description:Trypanosoma brucei membranes consist of all major eukaryotic glycerophospholipid and sphingolipid classes. These are de novo synthesized from precursors obtained either from the host or from catabolised endocytosed lipids. In recent years, substantial progress has been made in the molecular and biochemical characterisation of several of these lipid biosynthetic pathways, using gene knockout or RNA interference strategies or by enzymatic characterization of individual reactions. Together with the completed genome, these studies have highlighted several possible differences between mammalian and trypanosome lipid biosynthesis that could be exploited for the development of drugs against the diseases caused by these parasites.
| S-EPMC3744938 | biostudies-literature
Project description:Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery.
| S-EPMC5630508 | biostudies-literature
Project description:Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+ [Vercesi, Moreno and Docampo (1994) Biochem. J. 304, 227-233; Scott, Moreno and Docampo (1995) Biochem. J. 310, 789-794; Docampo, Scott, Vercesi and Moreno (1995) Biochem. J. 310, 1005-1012]. The data presented here indicate that Na+ stimulates Ca2+ release from the acidocalcisomes of digitonin-permeabilized Trypanosoma brucei procyclic trypomastigotes in a dose-dependent fashion, this effect being enhanced by increasing pH of the medium from 7.0 to 7.8. The hypothesis that this Na+ effect was mediated by alkalinization of the acidocalcisomes via a Na+/H+ antiporter was supported by experiments showing that Na+ promotes release of Acridine Orange previously accumulated in these vacuoles. This putative antiporter did not transport Li+ and was not sensitive to the amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride. Addition of the Na+/H+ ionophore monensin to intact cells loaded with fura 2, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, was followed by an increase in cytosolic Ca2+ concentration ([Ca2+]i), which was more accentuated in the presence of extracellular Na+. An increase in intracellular pH (pHi) of BCECF-loaded cells was detected after addition of monensin in the presence of extracellular Na+, whereas a dramatic decrease in pHi was detected in its absence, thus indicating the presence of a significant amount of releasable protons in the acidic compartments. These results are consistent with the presence of a Na+/H+ antiporter in the acidocalcisomes that could be involved in the regulation of pH1 and [Ca2+]1 in these parasites.
| S-EPMC1217181 | biostudies-other
Project description:Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca(2+) release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP(3)R) for Ca(2+) release. Localization studies in cell lines expressing TbIP(3)R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP(3) was able to stimulate Ca(2+) release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP(3)Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP(3)R, providing evidence of its function. IP(3) was also able to release Ca(2+) from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP(3) in intact trypanosomes loaded with Fluo-4 elicited a transient Ca(2+) increase in their cytosol. Ablation of TbIP(3)R by RNA interference caused a significant reduction of IP(3)-mediated Ca(2+) release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP(3)R as a Ca(2+) release channel in acidocalcisomes of trypanosomes and suggest that a Ca(2+) signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.
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Project description:The Earth's rotation forced life to evolve under cyclic day and night environmental changes. To anticipate such daily cycles, prokaryote and eukaryote free-living organisms evolved intrinsic clocks that regulate physiological and behavioural processes. Daily rhythms have been observed in organisms living within hosts, such as parasites. Whether parasites have intrinsic molecular clocks or whether they simply respond to host rhythmic physiological cues remains unknown. Here, we show that Trypanosoma brucei, the causative agent of human sleeping sickness, has an intrinsic circadian clock that regulates its metabolism in two different stages of the life cycle. We found that, in vitro, ∼10% of genes in T. brucei are expressed with a circadian rhythm. The maximum expression of these genes occurs at two different phases of the day and may depend on a post-transcriptional mechanism. Circadian genes are enriched in cellular metabolic pathways and coincide with two peaks of intracellular adenosine triphosphate concentration. Moreover, daily changes in the parasite population lead to differences in suramin sensitivity, a drug commonly used to treat this infection. These results demonstrate that parasites have an intrinsic circadian clock that is independent of the host, and which regulates parasite biology throughout the day.
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Project description:Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes.
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Project description:Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.
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Project description:Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation.
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