Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi

Abstract

Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of glycosomes is their harboring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle's membrane-homologs of the ABC and MCF transporter families-and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

Keywords: biogenesis; drug discovery; glycolysis; glycosomes; metabolic networks; metabolite transport; peroxisomes; trypanosomes.

Figure 1

(A–D) Ultrastructure of Trypanosoma cruzi epimastigote form showing glycosomes (G) and their proximity to other cell structures, especially the kinetoplast (K) and mitochondrial branches (M). Glycosomes are also seen close to reservosomes (R); N, nucleus. (B,C) Are insets that show areas of (A) with higher magnification. Cells were processed to transmission electron microscopy as follows. Protists were fixed for 1 h in 2.5% type II glutaraldehyde (Sigma, Missouri, USA) diluted in 0.1 M cacodylate buffer (pH 7.2). Then, they were washed twice in cacodylate buffer and post-fixed (1% osmium tetroxide, 0.8% potassium ferrocyanide, 5 mM calcium chloride diluted in 0.1 M cacodylate buffer) for 1 h. After fixation, samples were washed in cacodylate buffer, dehydrated in a graded series of acetone solutions (50%, 70%, 90%, and two exchanges of 100% acetone) for 10 min each step, and embedded in Polybed resin. Ultrathin sections were stained with 5% uranyl acetate for 45 min and lead citrate for 5 min before observation in a Jeol 1200ex operating at 80 kV. (E) T. cruzi contains multiple glycosomes distributed throughout the cell body as demonstrated by fluorescent puncta in transgenic epimastigotes and metacyclic trypomastigotes expressing green fluorescent protein (GFP) containing a C-terminal PTS1 (-SKL) to target it to glycosomes.

Figure 2

Diagrammatic representation of the glycosomal proteome of T. cruzi epimastigotes. Proteins involved in metabolic pathways, solute transport and glycosome biogenesis are represented in circles. Lines represent connections between different processes by exchange of metabolites. Glycolysis: HXK, hexokinase; GlcK, glucokinase; PGI, phosphoglucose isomerase; PFK, phosphofructokinase; ALD, aldolase; TIM, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase. Auxiliary to glycolysis and gluconeogenesis: G3PDH, glycerol-3-phosphate dehydrogenase; GK, glycerol kinase; PEPCK, phosphoenolpyruvate carboxy kinase; MDH, malate dehydrogenase; FH, fumarate hydratase; FRD, NADH-fumarate reductase; PPDK, pyruvate phosphate dikinase; ALDH, aldehyde dehydrogenase; HADH, D-isomer specific 2-hydroxyacid dehydrogenase-protein; ALADH, alanine dehydrogenase; ADH, alcohol dehydrogenase; AK, adenylate kinase; ArgK, arginine kinase. Other sugar metabolism: GALK, galactokinase; USPase, UDP-sugar pyrophosphorylase; GALE, UDP-galactose 4-epimerase; UGPUT, UTP-glucose-1-phosphate uridylyltransferase; PMM, phosphomannomutase-like protein; GalO, L-galactonolactone oxidase; PMI, phosphomannose isomerase; GNPI, glucosamine-6-phosphate isomerase; NagA, N-acetylglucosamine-6-phosphate deacetylase-like protein; GlyK, glycerate kinase; GarR, 2-hydroxy-3-oxopropionate reductase; DHAK, dihydroxyacetone kinase; DHAS, dihydroxyacetone synthase. Pentose-phosphate pathway: HPE, D-hexose-6-phosphate-1-epimerase; G6PDH, glucose-6-phosphate dehydrogenase; LAC, lactonase; 6PGD, 6-phosphogluconate dehydrogenase; RPI, ribulose-5-phosphate isomerase; R5PE, ribulose-5-phosphate epimerase; TKT, transketolase; SBTA, sedoheptulose-1,7-phosphate transaldolase; TAL, transaldolase; SBPase, sedoheptulose-1,7-bisphosphatase; RK, ribulokinase; RBK, ribokinase. Gluconeogenesis: FBPase, fructose-1,6-bisphosphatase. Ether-lipid synthesis: FAR, fatty-acyl-CoA reductase; ADHAPS, alkyl-DHAP synthase; DHAPAT, DHAP acyltransferase; AGPO, 1-alkyl G3P(NADP⁺)-oxidoreductase. β-oxidation: FACS, fatty-acyl CoA synthetase; ACAD, acyl-CoA dehydrogenase; ECH, enoyl-CoA hydratase; HADH, 3-hydroxyacyl-CoA dehydrogenase; 3-KAT, 3-ketoacyl-CoA thiolase; TECI, 3,2-trans-enoyl-CoA isomerase; ECI, enoyl-CoA isomerase; FAE, fatty acid elongase. Sterol and isoprenoid synthesis: HMCS, 3-hydroxy-3-methylglutaryl CoA synthase; HMCR, 3-hydroxy-3-methylglutaryl CoA reductase; MVK, mevalonate kinase; IDI, isopentenyl-diphosphate delta-isomerase; SQS, squalene synthase; SM, squalene monooxygenase; LDM, lanosterol 14-alpha-demethylase; SD, NAD(P)-dependent steroid dehydrogenase protein; SMT, sterol 24-C-methyltransferase; SI, C-8 sterol isomerase; 24-SR, sterol C-24 reductase; 3O5-SDH, 3-oxo-5-alpha-steroid 4-dehydrogenase. Purine salvage: NPase, nucleoside phosphorylase; APRT, adenine phosphoribosyltransferase; PRPPS, phosphoribosyl pyrophosphate synthetase; AMPD, AMP deaminase; IMPDH, inosine-5'-monophosphate dehydrogenase; GMPR, guanosine monophosphate reductase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; PPM, phosphopentomutase. Pyrimidine synthesis: OPRTase, orotate phosphoribosyltransferase; PRPPS, phosphoribosylpyrophosphate synthetase; OPDC, orotidine-5-phosphate decarboxylase. Amino acid metabolism: HAL, histidine ammonia-lyase; IPP, imidazolonepropionase; ProDH, proline dehydrogenase; P5CR, pyrroline-5-carboxylate reductase; T3DH, L-threonine-3-dehydrogenase; TDase, threonine dehydratase-like, OVDH, 2-oxoisovalerate dehydrogenase alpha subunit. Defense against oxidative stress: Argase, arginase; IDH, isocitrate dehydrogenase; GP, glutathione peroxidase-like protein; TR, trypanothione reductase; SOD, iron superoxide dismutase; AOD, acetylornithine deacetylase-like; Cyt B5 red, Cytochrome-B5 reductase; THR, thiol-dependent reductase 1. Other enzymes: AKR, aldo-keto reductase; NA, nicotinamidase. Membrane (associated) proteins: PEX, peroxin; GIM5A, glycosomal integral membrane protein 5. Pore-forming protein: PEX11. Solute transporters: MCF, mitochondrial carrier family protein; GAT, ABC transporter. Membrane-associated enzyme: CAT, carnitine/choline O-acyltransferase. For a detailed assessment of the results and a presentation of glycosomal metabolic pathways (see Acosta et al., 2019).