Evolution, dynamics and specialized functions of glycosomes in metabolism and development of trypanosomatids

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites' life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids' metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosome brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style

Enolase: A Key Player in the Metabolism and a Probable Virulence Factor of Trypanosomatid Parasites—Perspectives for Its Use as a Therapeutic Target

Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasite's invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination

Delineating transitions during the evolution of specialised peroxisomes: glycosome formation in kinetoplastid and diplonemid protists

One peculiarity of protists belonging to classes Kinetoplastea and Diplonemea within the phylum Euglenozoa is compartmentalisation of most glycolytic enzymes within peroxisomes that are hence called glycosomes. This pathway is not sequestered in peroxisomes of the third Euglenozoan class, Euglenida. Previous analysis of well-studied kinetoplastids, the ‘TriTryps’ parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., identified within glycosomes other metabolic processes usually not present in peroxisomes. In addition, trypanosomatid peroxins, i.e. proteins involved in biogenesis of these organelles, are divergent from human and yeast orthologues. In recent years, genomes, transcriptomes and proteomes for a variety of euglenozoans have become available. Here, we track the possible evolution of glycosomes by querying these databases, as well as the genome of Naegleria gruberi, a non-euglenozoan, which belongs to the same protist supergroup Discoba. We searched for orthologues of TriTryps proteins involved in glycosomal metabolism and biogenesis. Predicted cellular location(s) of each metabolic enzyme identified was inferred from presence or absence of peroxisomal-targeting signals. Combined with a survey of relevant literature, we refine extensively our previously postulated hypothesis about glycosome evolution. The data agree glycolysis was compartmentalised in a common ancestor of the kinetoplastids and diplonemids, yet additionally indicates most other processes found in glycosomes of extant trypanosomatids, but not in peroxisomes of other eukaryotes were either sequestered in this ancestor or shortly after separation of the two lineages. In contrast, peroxin divergence is evident in all euglenozoans. Following their gain of pathway complexity, subsequent evolution of peroxisome/glycosome function is complex. We hypothesize compartmentalisation in glycosomes of glycolytic enzymes, their cofactors and subsequently other metabolic enzymes provided selective advantage to kinetoplastids and diplonemids during their evolution in changing marine environments. We contend two specific properties derived from the ancestral peroxisomes were key: existence of nonselective pores for small solutes and the possibility of high turnover by pexophagy. Critically, such pores and pexophagy are characterised in extant trypanosomatids. Increasing amenability of free-living kinetoplastids and recently isolated diplonemids to experimental study means our hypothesis and interpretation of bioinformatic data are suited to experimental interrogation

Proteomic analysis of glycosomes from Trypanosoma cruzi epimastigotes

In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite’s glycosomes

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection

Genetic and Chemical Evaluation of Trypanosoma brucei Oleate Desaturase as a Candidate Drug Target

Background: Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. Methodology: Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. Principal Findings: Growth of PCF T. brucei was totally inhibited by 100 mM of 12-TS and 13-TS, with EC50 values of 4062 and 3062 mM, respectively. The BSF was more sensitive, with EC50 values of 763 and 261 mM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itsel

Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei.

Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M(r) of 100kDa and 72kDa. 5'-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72kDa. However, recombinant PEX5 migrates aberrantly in SDS-PAGE with an apparent M(r) of 100kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and (35)S-labelled PEX5 showed truncation of the 100kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed

Translocation of solutes and proteins across the glycosomal membrane of trypanosomes; possibilities and limitations for targeting with trypanocidal drugs.

SUMMARY Glycosomes are specialized peroxisomes found in all kinetoplastid organisms. The organelles are unique in harbouring most enzymes of the glycolytic pathway. Matrix proteins, synthesized in the cytosol, cofactors and metabolites have to be transported across the membrane. Recent research on Trypanosoma brucei has provided insight into how these translocations across the membrane occur, although many details remain to be elucidated. Proteins are imported by a cascade of reactions performed by specialized proteins, called peroxins, in which a cytosolic receptor with bound matrix protein inserts itself in the membrane to deliver its cargo into the organelle and is subsequently retrieved from the glycosome to perform further rounds of import. Bulky solutes, such as cofactors and acyl-CoAs, seem to be translocated by specific transporter molecules, whereas smaller solutes such as glycolytic intermediates probably cross the membrane through pore-forming channels. The presence of such channels is in apparent contradiction with previous results that suggested a low permeability of the glycosomal membrane. We propose 3 possible, not mutually exclusive, solutions for this paradox. Glycosomal glycolytic enzymes have been validated as drug targets against trypanosomatid-borne diseases. We discuss the possible implications of the new data for the design of drugs to be delivered into glycosomes