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Molecular & Biochemical Parasitology 168 (2009) 102–108

Contents lists available at ScienceDirect

Molecular & Biochemical Parasitology

lucose uptake in the mammalian stages of Trypanosoma cruzi

riel M. Silber a,1,2, Renata R. Tonelli a,2, Camila G. Lopes a, Narcisa Cunha-e-Silva b,na Cláudia T. Torrecilhas a, Robert I. Schumacher a, Walter Colli a, Maria Júlia M. Alves a,∗

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof Lineu Prestes 748, CEP 05508-900, São Paulo, SP, BrazilInstituto de Biofisica Carlos Chagas Filho, CCS, Bloco G, subsolo, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21949-900, Rio de Janeiro, RJ, Brazil

r t i c l e i n f o

rticle history:eceived 3 September 2008eceived in revised form 14 July 2009ccepted 15 July 2009vailable online 23 July 2009

eywords:rypanosoma cruziexose transporterlucose uptake

a b s t r a c t

Trypanosoma cruzi, the agent of Chagas’ disease, alternates between different morphogenetic stages thatface distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availabilityof glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is knownabout how the mammalian stages acquire this molecule. Herein glucose transport activity and expressionwere analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infectivetrypomastigotes showed the highest transport activity (Vmax = 5.34 ± 0.54 nmol/min per mg of protein;Km = 0.38 ± 0.01 mM) when compared to intracellular epimastigotes (Vmax = 2.18 ± 0.20 nmol/min per mgof protein; Km = 0.39 ± 0.01 mM). Under the conditions employed no transport activity could be detected inamastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in

ntracellular stages intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotesand intracellular epimastigotes protein expression could be detected by Western blot with an antibodyraised against the glucose transporter correlating well with the transport activity measured experimen-tally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome andreservosome organelles. A comparison between proline and glucose transport among the intracellular

resenon re

differentiation forms is pdifferent energy and carb

. Introduction

Trypanosoma cruzi, the etiological agent of Chagas’ disease,s a kinetoplastid parasite with a complex life cycle sharedy invertebrate and vertebrate hosts. During the life cycle thearasite alternates between glucose-rich and glucose-poor envi-onments having to adapt to different sources of energy andarbon. Epimastigotes, the non-infective dividing forms living in theigestive tract of the invertebrate host, differentiate to metacyclic

rypomastigotes (metacyclogenesis) in an amino-acid-rich andarbohydrate-poor medium. After vertebrate invasion, the trypo-astigotes differentiate into the dividing forms called amastigotes

nd establish the infection in the host cell cytoplasm, a medium

Abbreviations: FCS, fetal calf serum; CHO-K1, Chinese hamster ovary cells; LIT,iver-infusion tryptose; BSA, bovine serum albumin; TBS-T, Tris-buffered saline,ontaining 0.1% Tween 20; PCR, polymerase chain reaction; RT-PCR, reverseranscription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TcHT, Try-anosoma cruzi hexose transporter; PBS, phosphate-buffered saline.∗ Corresponding author. Tel.: +55 11 3091 3810x233/234; fax: +55 11 3815 5579.

E-mail address: mjmalves@iq.usp.br (M.J.M. Alves).1 Present address: Departamento de Parasitologia, Instituto de Ciências Biomédi-

as, USP, Av. Lineu Prestes 1374, 05508-000, São Paulo, SP, Brazil.2 Both the authors contributed equally to this work.

166-6851/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2009.07.006

ted. The data suggest that the regulation of glucose transporter reflectsquirements along the intracellular life cycle of T. cruzi.

© 2009 Elsevier B.V. All rights reserved.

poor in free glucose. Finally, after some rounds of replication,the parasites differentiate to trypomastigotes passing through anintermediate stage, the intracellular epimastigotes [1,2]. Trypo-mastigotes are liberated to the extracellular medium after ruptureof the host cells, being able to invade other cells or be ingested byan insect vector during the blood meal.

It is well established that trypanosomatids can use either glu-cose or amino acids as main carbon and energy source, although onecannot rule out the participation of fatty acids as demonstrated foramastigotes of Leishmania [3]. For instance, trypomastigote forms ofT. cruzi and T. brucei use glucose, abundant in vertebrate fluids. How-ever, the main source of carbon and energy in the insect stages areamino acids, especially l-proline and l-glutamine, which are abun-dant in the hemolymph and tissue fluids of the hematophagousvector [4,5]. Notably, l-proline is important for metacyclogenesis[6], for the differentiation of intracellular epimastigotes to try-pomastigotes in T. cruzi [7], and seems to be involved in severalmechanisms of resistance to oxidative, nutritional and thermalstress [8]. As expected, several amino acids including proline, aspar-

tate and glutamate are actively transported and oxidized by T.cruzi [9–11]. In particular, the catabolism of aromatic amino acidsappears to be related to the cytosolic NADH+ reoxidation [12]. Inaddition, the presence in the T. cruzi genome of at least 60 genesbelonging to a single family of amino acid transporters reinforces

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he relevance of amino acids in the biology of these organisms [13].he majority of trypanosomatids preferentially metabolize glucosehen grown in a glucose-rich medium, even if amino acids are

resent, as described for proline and glucose metabolism in T. brucei14]. Notwithstanding, some trypanosomatids as Crithidia deanei, a

onoxenic and non-pathogenic trypanosomatid living in the insectut, metabolize amino acids independently of the glucose contentf the culture medium [15].

In trypanosomatids the glycolytic pathway is compartmental-zed in peroxysome-derived organelles called glycosomes and theytoplasm, and in the particular case of T. cruzi glucose is metabo-ized further in mitochondria [16,17]. In T. cruzi it was describedp to now a single gene coding for a protein to which the glu-ose transport activity was attributed. This gene, present in theenome as a cluster of multiple tandem reiterated copies [18],ives rise to at least two mRNAs diverging in the 3′ untranslatedegion [19]. The ORF, which has been functionally characterized,odes for a protein with 12 transmembrane spanners belonging tohe sugar transporter superfamily that passively transports glucoseith high affinity and other hexoses, such as fructose, with lower

ffinities [20]. A similar Km was described for the glucose transportn epimastigotes and trypomastigotes, although a higher Vmax wasalculated for epimastigotes [19,21]. It was recently demonstratedhat glucose transporters in trypanosomatids are not only involvedn the sugar uptake to feed glycolysis, but are also involved in theegulation of the glycolytic flux [22,23]. While the glucose transportctivity is well studied in epimastigotes and trypomastigotes of T.ruzi, no data are available for the intracellular mammalian stages.n addition to shed light in the metabolism of the mammalian stagesn trypanosomatids, the study of nutrient transporters may con-ribute to the understanding of drug transport in these parasites24,25].

The glucose transport activity was measured in the mammalianntracellular stages and compared with previous data on the trans-ort of l-proline [7]. It is shown, herein, that the glucose transport isown regulated in the intracellular stages (amastigote and intracel-

ular epimastigote) when compared to trypomastigotes. Finally, aelationship among glucose and l-proline uptake, free proline con-ent and the intracellular life cycle progression of the parasite isroposed.

. Materials and methods

.1. Reagents

[14C-U]-d-glucose, [3H]-2-deoxyglucose, and l-[2,3,4,5-3H] pro-ine were purchased from NEN Life Science Products (Boston, MA,SA). All other reagents were from Sigma (Saint Louis, MO, USA).

.2. Cell culture

The Chinese Hamster Ovary cell line CHO-K1 was purchasedrom the ATCC (CCL 6.1) and routinely cultivated in RPMI mediumGibco BRL) supplemented with 10% heat-inactivated fetal calferum (FCS), 0.15% (w/v) NaHCO3, 100 units/ml penicillin and00 mg/ml streptomycin at 37 ◦C in a humid atmosphere contain-ng 5% CO2. Parasite viability was evaluated by direct microscopicbservation, or by using calcein AM (Molecular Probes) as viabilityrobe, following the manufacturer’s instructions.

.3. Parasite strain and growth conditions

Epimastigote forms of T. cruzi, CL-14, a clone derived from theL strain [26] were cultured in liver infusion-tryptose medium (LIT)upplemented with 10% FCS at 28 ◦C [27]. The parasites were kept inxponential growth by subculturing every other day. Intracellular

l Parasitology 168 (2009) 102–108 103

forms and trypomastigotes were obtained by infection of CHO-K1cells with trypomastigotes, as described [1]. Amastigotes and intra-cellular epimastigotes were purified from CHO-K1 on day 2 andaround days 4–5 post-infection, respectively. When necessary, theintracellular forms were purified after disrupting the infected cellswith a rubber policeman. After centrifugation, the pellet was sus-pended in RPMI medium and purified by centrifugation on 5 mlLymphoprep (Nycomed Pharma AS) for 10 min at 4300 × g. Trypo-mastigotes were collected from the extracellular medium on the6–7 days post-infection.

2.4. Antisera

Eight- and 11-amino acid length synthetic peptides, correspond-ing to an extracellular loop of the T. cruzi glucose transporter (serum#1: ACNRVLNAECS; and serum #2: ATEGISGG) were independentlycoupled to Keyhole limpet hemocyanin (KLH, Pierce) followingthe manufacturer’s instructions. Eight-week-old wild-type BALB/Cfemale mice were intraperitoneally inoculated (every 15 days) with30 �g of the KLH-coupled synthetic peptide in Freund’s adjuvant.After 3 weeks, the blood was collected and the serum separated.Before immunization, a blood sample was collected for the pre-immune control. Animals were housed under barrier conditions atthe animal care facility at the Instituto de Química, Universidade deSão Paulo (Brazil). Formal animal ethics approval was given for thiswork. Animals were housed and maintained according to institu-tion guidelines and in accordance with international guidelines forthe use of animals. Antibodies were affinity purified by adsorptionto the KLH-coupled peptide immobilized on nitrocellulose mem-branes as previously described [28].

All experiments were made using both sera, with the exceptionof TEM, which was made with serum #2 only.

2.5. Western blot

For Western blots, total extracts of different stages of the lifecycle of T. cruzi (50 �g/lane) were subjected to 12% SDS-PAGEand electroblotted to a nitrocellulose membrane. Membranes wereblocked with 5% non-fat milk in Tris-buffered saline, containing0.1% Tween 20 (TBS-T) for 1 h at 23 ◦C followed by incubation withprimary antibodies overnight at 4 ◦C. The following conditions wereused: (i) anti-TcHT diluted 1:50 in TBS-T containing 1% bovineserum albumin (BSA); (ii) anti-GAPDH diluted 1:1000 in TBS-T. Afterwashes with TBS-T, membranes were incubated with horseradishperoxidase (HRP)-protein A (GE Healthcare) diluted 1:2000 in TBS-. The bound antibodies were detected by using Immobilon western

chemiluminescence HRP substrate (Millipore). When necessary,the antibodies were stripped off the membranes by incubationwith 100 mM �-mercaptoethanol, 2% SDS, and 62.5 mM Tris–HCl(pH 6.7) at 50 ◦C for 30 min. Competition assays were performedby incubation of the membranes with 10 �g of the synthetic pep-tide diluted in TBS before incubation with the anti-TcHT antibodyas described above.

2.6. Transport assays

Transport assays were performed essentially as previouslydescribed [10]. Different stages of T. cruzi were washed three timesby centrifugation and resuspended in phosphate-buffered saline(PBS), pH 7.4 (intracellular amastigotes and epimastigote forms) orRPMI buffer solution (trypomastigotes) and distributed in aliquots

of 100 �l (2 × 107 parasites). Transport assays and V0 determinationwas performed at 28 ◦C for 30 s with the addition to the experimen-tal tube of 100 �l of the desired dilution of d-glucose in PBS in thepresence of 0.5 �Ci of [14C-U]-d-glucose. In all cases, glucose trans-port was stopped by adding 800 �l of cold 50 mM d-glucose in PBS,

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showed a higher transport activity in trypomastigotes (Vmax =5.34 ± 0.54 nmol/min per mg of protein; Km = 0.38 ± 0.01 mM),being approximately 2.5-fold higher than the activity found in theintracellular epimastigotes (Vmax = 2.18 ± 0.20 nmol/min per mg ofprotein; Km = 0.39 ± 0.01 mM), but 12-fold lower than the activ-

04 A.M. Silber et al. / Molecular & Bioch

ollowed by centrifugation at 10,000 × g for 60 s. The results areepresentative of triplicates from three independent experiments.hese measurements were also repeated by tracing glucose with

3H]-2-deoxyglucose. As no transport was detected in amastigotesnder the experimental conditions employed, a control experi-ent was performed by measuring proline transport as previously

escribed [7].

.7. Protein determination

The amount of protein was determined according to the methodf Bradford [29] using bovine serum albumin (BSA) as standard.

.8. Reverse transcription PCR (RT-PCR) and real-timeolymerase chain reaction

RT-PCR was performed with ImProm-II. Reverse Transcriptionystem (Promega) as described by the manufacturer’s instructions.omplementary DNA (cDNA) of each stage of T. cruzi or CHO-K1ells (used as control) was synthesized using 5 �g of RNA throughreverse transcription reaction (Superscript IITM, Invitrogen, USA).eal-time PCR quantitative mRNA analyses were performed in aastercyler® ep realplex (Eppendorf, Germany) using the SYBR-

reen fluorescence quantification system (Biotools QauntimixasySyg, Spain) for amplicon quantification. The standard PCR con-itions were 95 ◦C (10 min), and then 40 cycles of 94 ◦C (1 min),0 ◦C (1 min), and 72 ◦C (2 min), followed by the standard denatura-ion curve. The primers were designed using the software providedy Eppendorf (RealPlex v.1.5) based on nucleotide sequencesf T. cruzi glyceraldehyde-3-phosphate dehydrogenase (GAPDH)nd the hexose transporter (TcHT) (GeneBank accession num-ers AI007393 and U05588.1, respectively). The sequences of therimers used were: GAPDH forward: (5′-GTGGCAGCACCGGTAACG-′) and reverse: (5′-CAGGTCTTTCTTTTGCGAATAGG-3′); and TcHTorward: (5′-TGATGTACCATGTGTCCTCGGCAACG-3′) and reverse:5′-ATGGCACTGCGCTGGACCCGAA-3′). The reaction mixture con-ained 500 nM of each specific primer and 5 �g of cDNA samples.YBR Green PCR Mix (Biotools, Spain) was used as recommended byhe manufacturer. Data obtained were analyzed using the softwareealPlex v1.5. The relative levels of gene expression were obtainedy calculating the �CT for each sample.

.9. Indirect immunofluorescence and transmission electronicroscopy

For immunofluorescence experiments approximately 1 × 106

arasites were washed two times with PBS and resuspended inml of the same buffer. Twenty microlitres from the parasite sus-ension were added to the wells of the immunofluorescence slidesnd than incubated for 5 min at room temperature. After remov-ng excesses of the cell suspension, parasites were fixed with 2%araformaldehyde in PBS (pH 7.4) for 20 min at room temperaturend then washed three times with PBS. Fixed cells were permeabi-ized with 0.1% Triton X-100 for 5 min, washed three times with PBSnd than incubated with mouse anti-TcHT antibody diluted 1:30or 1 h at room temperature. For co-localization assays, at the sameime, cells were stained with rabbit anti-aldolase antibody diluted:4000 to detect glycosome or with rabbit anti-cruzipain anti-ody diluted 1:500 to detect the reservosome. Appropriate Alexaluor 488 and Alexa Fluor 555-conjugated goat anti-IgG (Molec-lar Probes) were used as secondary antibodies. Cells were also

tained with 500 ng of DAPI (4′,6′-diamidino-2-phenylindole)/ml.erial image stacks (0.2-�m Z-increment) were collected at 100×UPlanApo oil immersion 1.35 NA) on a motorized inverted micro-cope Olympus IX81 equipped with a rear-mounted excitation filterheel, a triple-pass (DAPI-fluorescein isothiocyanate-Tritc) emis-

l Parasitology 168 (2009) 102–108

sion cube, differential interference contrast optics, and a Orca ER/AGcharge-coupled device camera (Hamamatsu). All images were col-lected with Cell® Imaging software (Olympus), and fluorescenceimages were deconvoluted by using AutoQuant X version 2 software(Media Cybernetics).

For electron microscopy, epimastigote cells were fixed in 2.5%glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h atroom temperature, post-fixed in 1% OsO4 + 0.8% potassium ferri-cyanide + 5 mM calcium chloride in 0.1 M cacodylate buffer, pH 7.2,for 40 min. For the immunolabeling experiments, parasites werefixed in a solution containing 4% freshly prepared formaldehyde,0.1% glutaraldehyde, 0.1% picric acid and 0.1 M sodium cacody-late buffer, pH 7.2, washed in the same buffer, embedded in 10%gelatin cut into 1-mm2 pieces, and maintained overnight at 48 ◦Cin 2.3 M sucrose. After freezing in liquid nitrogen, the sampleswere dehydrated in methanol by cryosubstitution at −90 ◦C for48 h and embedded in Lowicryl HM20 resin at −45 ◦C. Ultrathinsections were collected on 300-mesh nickel grids, incubated for30 min with 0.05 M glycine in PBS, followed by 30 min in PBS con-taining 5% bovine serum albumin (BSA), 0.1% cold water fish skin,and 5% normal serum. The material was incubated overnight withthe primary antibody (10 �g/ml) in PBS-BSA, washed with PBS-BSA, and incubated for 60 min at room temperature with anti-IgGmouse antibody (1:20) coupled to colloidal gold particles (10 nm).After this step, the grids were washed with PBS and distilled water,stained with uranyl acetate, and observed in a Zeiss EM 900 trans-mission electron microscopy.

3. Results

3.1. Kinetics of d-glucose uptake in T. cruzi vertebrate stages

Glucose uptake was measured in the three developmental stagespresent in the vertebrate cycle of T. cruzi, CL-14 clone: amastigotes,intracellular epimastigotes and trypomastigotes. Comparativeanalysis of the glucose uptake in the mammalian stages

Fig. 1. Kinetics of initial d-glucose uptake in mammalian stages of Trypanosomacruzi. The rate of d-glucose uptake was measured over the concentration range of0.02–1 mM at 28 ◦C in the three developmental stages found in cultured cells infectedby T. cruzi: trypomastigotes (�), intracellular epimastigotes (�) and amastigotes (�).Values are expressed as means ± S.D. of triplicates from, at least, three experiments.The results plotted for amastigotes were not significant (p = 0.07).

A.M. Silber et al. / Molecular & Biochemical Parasitology 168 (2009) 102–108 105

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ig. 2. Quantitation of protein expression and glucose transporter mRNA. (A) Quanttages of T. cruzi and in CHO-K1 host cell. (B) Quantitation of T. cruzi glucose transpy real-time PCR. (C) Specificity of antibodies. Lane 1, incubation of antibody AS#2xtracellular epimastigotes; C, CHO-K1 cells; Ty, trypomastigotes; IE, intracellular e

ty measured in axenic-culture derived epimastigotes (Vmax =0.57 ± 2.56 nmol/min per mg protein; Km = 0.18 ± 0.02 mM [cf.18]]). Under the conditions employed no transport of glucose coulde found in amastigotes (Fig. 1).

.2. Expression of the glucose transporter is regulated along T.ruzi life cycle

In order to know at which level the glucose transport activity isegulated, specific mRNA levels and glucose transporter expressionere analyzed in T. cruzi stages by real-time PCR and Western blot.

or PCR analysis, total RNA purified from all stages was submitted to

ig. 3. Subcellular localization of T. cruzi glucose transporter in extracellular epimastigotesith anti-TcHT antibody (AS#2) and processed for indirect immunofluorescence microsc

hown (third column).

was evaluated by immunoblot with anti-TcHT antibody in different developmentalRNA and protein expression. Transcripts for d-glucose transporter were analyzed

peptide 2; lane 2, control substituting AS#2 for by PBS; lane 3, AS#2. Legends: E,tigotes; A, amastigotes.

reverse transcriptase and the resulting cDNA was used for ampli-fication. cDNAs from epimastigotes and CHO-K1 cells, were usedas controls. Quantitation of the amplicon by real-time PCR showedthat trypomastigotes contain approximately 4.2 times more specificmRNA than intracellular epimastigotes (Fig. 2B). Accordingly to thelack of transport activity observed, no specific mRNA was found inamastigotes. The reactions were shown to be parasite-specific, as

no amplification was observed when total RNA isolated from thehost cell was employed.

To analyze the presence of the glucose transporter in the intra-cellular stages of T. cruzi, two antisera (AS1 and AS2) againstdifferent synthetic peptides corresponding to specific regions of

(A) and tissue-cultured derived trypomastigotes (B). Parasites were fixed, incubatedopy (first column), stained with DAPI (second column). The merged image is also

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he protein (anti-TcHT) were raised. The expression of the hexoseransporter in amastigotes, intracellular epimastigotes and try-omastigotes was then analyzed by Western blot by using AS1.

56-kDa band corresponding to the molecular mass expectedor the glucose transporter was detected in the intracellular epi-

astigotes and in trypomastigotes (Fig. 2A), while no band wasetected in amastigotes. The hexose transporter/GAPDH ratio inhe trypomastigote stage was approximately 2.5-fold higher whenompared to the intracellular epimastigotes ratio. These data wereonfirmed by repeating the experiment with AS2. In addition, toonfirm the specificity of the obtained sera, the Western blots were

lso developed with both antibodies incubated in the presence ofhe corresponding peptide in competition assays. No bands wereetected under these conditions, confirming the specificity of bothntibodies, as shown with AS-2 (Fig. 2C).

ig. 4. Subcellular localization of the T. cruzi glucose transporter. Extracellular epimastignd DAPI (C), and processed for indirect immunofluorescence microscopy. The merged imlectron microscopy (E: bar = 0.5 �m; F and G: bar = 0.1 �m). Arrows, glycosomes; small d

l Parasitology 168 (2009) 102–108

3.3. Glycosomes and reservosomes are enriched in the glucosetransporter

The subcellular localization of the hexose transporter in T. cruziwas performed by immunofluorescence in the epimastigote andtrypomastigote stages due to the higher transport activity in theseforms. The anti-TcHT antibody labeled slightly the plasma mem-brane of the parasite but showed a consistently strong reactivitywith some internal compartments varying in size and localizationwithin the cell (Fig. 3). Some of these compartments consistedof large spherical organelles present at the post-nuclear region

of the parasite, resembling the reservosomes [30]. Others aresmall vesicle-like organelles distributed all over the cell body andseem to correspond to glycosomes. Co-localization by transmis-sion electronic microscopy experiments were then performed in

otes were fixed and incubated with �-TcHT antibody (A), �-cruzipain antibody (B)age is also shown (D). The fixed epimastigotes were also submitted to transmissionots, GAPDH; large dots, glucose transporter.

emical Parasitology 168 (2009) 102–108 107

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Fig. 5. Variations of the relative activities of thed-glucose and l-proline transportersand l-proline accumulation during the intracellular life cycle of T. cruzi. Data on intra-

A.M. Silber et al. / Molecular & Bioch

rder to characterize further these compartments using antibodiesgainst the hexose transporter, cruzipain (a reservosome marker),nd glycerol-3-phosphate dehydrogenase (GAPDH) (a glycosomearker). As shown in Fig. 4, the small organelles recognized by

nti-TcHT antibody, were compatible in size and reactive with anti-APDH antibody, showing to be glycosomes. It was also observed

hat in most of the cases, the hexose transporter seems to beresent in clusters in the parasite glycosomes (Fig. 4B). A strongeactivity was also observed co-localizing with cruzipain, con-rming the presence of TcHT in a reservosome-like organelle

Fig. 4A).

. Discussion

Most of the literature on T. cruzi glucose transport andetabolism relies on studies made in the extracellular stages.erein, it is shown that glucose transport activity is higher in try-omastigotes than in intracellular stages, an expected result sincetages living in glucose-rich and glucose-poor environments areeing compared. In fact, [14C-U]-d-glucose transport activity is 2.5-

old lower in intracellular epimastigotes than in trypomastigotesnd, interestingly, no transport, could be measured in amastigotes,nder the conditions employed (Fig. 1). The data were confirmedy real-time PCR and Western blot analysis (Fig. 2), suggestinghat glucose uptake is, at least in part, controlled at the mRNAevel among the stages of T. cruzi. Amastigotes did not express glu-ose transporter mRNA confirming previous observations on thebsence of transport activity (Fig. 1). In order to be metabolizedy T. cruzi and other trypanosomatids, glucose must reach the gly-osomes, a single membrane and rather impermeable organelle,here most of the glycolysis occurs. The exchange of substrates

nd other metabolites between the glycosome and the cytoplasmight occur through specific transporter proteins as the three ABC

ransporters associated with the glycosome membrane of T. brucei31] or MCF proteins [32] but this is, as yet, a matter of discussion33]. The data herein presented showed a strong reactivity of anti-lucose transporter antibody (anti-TcHT) with internal organelles.very weak reactivity with the plasma membrane of the epimastig-

tes and trypomastigotes could be observed in some experimentsFig. 3). However, further experiments to confirm the membraneocation of TcHT were not conclusive. As the used antibodies wereirected towards peptide sequences present in all fully sequencedenes coding for possible isoforms of the TcHT, it could be spec-lated that other proteins could contribute to the whole glucoseptake activity. However, no other protein with this putative func-ion was as yet described. Part of the labeled organelles is in factlycosomes, as confirmed by co-localization with an anti-GAPDHntibody, and part is reservosomes, as shown by their size, subcel-ular localization at the post-nuclear region of epimastigotes ando-localization with cruzipain, a reservosome marker (Fig. 4). Theresence of the glucose transporter in the reservosomes was rein-

orced by a proteomic analysis of a purified fraction of this organelle34].

As pointed out, glycosomes contain most of the glycolyticnzymes, corresponding to 90% of their protein content in T. bru-ei [33]. Reservosomes are late endosomes that stock endocytosedacromolecules and lysosomal enzymes and have distinct mor-

hologies varying with the nutritional conditions. The reservosomeontent is massively degraded during the differentiation of epi-astigotes to metacyclic trypomastigotes and, as a consequence,

he organelle disappears [30]. Reservosomes are also rich in cruzi-

ain, the main cysteine protease produced and secreted by thearasite [35]. How the hexose transporter is addressed to the gly-osomes or reservosomes is unclear. One possibility is that thelucose transporter located at the plasma membrane is rapidlynternalized by endocytosis and undergoes sorting steps en route

cellular l-proline concentration and transport activity described in [7]. For clarity,the data on trypomastigotes were repeated in the invading and evading representa-

tions: ( ) trypomastigote; ( ) amastigote; ( ) intracellular epimastigote.

to the glycosomes. During its recycling, it can be concentratedin reservosome/lysosome organelles for degradation. Alternatively,the glucose transporter can be internalized by different transportroutes, described for the transport of heme and hemoglobin inepimastigotes [36]. It should be mentioned that autophagy, a mech-anism for degradation and recycling of proteins and organelles, wasrecently described in T. cruzi and is involved in nutritional stressresponse and differentiation of the parasite [37], a mechanism thatcontributes to explain the vesicle size heterogeneity observed, asshown by antibody labeling.

Although the turnover of the glucose transporter in the plasmamembrane of T. cruzi was not addressed here, it should be remindedthat in the basal state, only 5% of the mammalian GLUT-4 trans-porter is present in the plasma membrane and that a slow andcontinuous recycling between the plasma membrane and severalintracellular compartments occurs [38].

Since the intracellular content and the transport of proline havebeen previously determined in the mammalian stages under thesame experimental conditions, a comparative picture is shown sug-gesting a metabolic link between the two main energy sources(Fig. 5).

Trypomastigotes have the highest glucose and the minimumproline transport activities in comparison with the other mam-malian stages. As relatively high quantities of intracellular freeproline were measured (3.8 mM) it is likely that trypomastigotesobtain this amino acid from other internal sources [7] but relymainly on glucose metabolism for energy requirements. Amastig-otes have the highest intracellular free proline concentration(8.09 mM) [7]. However, the proline transport is low and thetransport of glucose is undetectable. Amastigotes are then moredependent on endogenous amino acids either from biosynthesisor from protein hydrolysis [37]. Apparently, the amino acid poolof the amastigotes is consumed during replication and/or differ-entiation to the intracellular epimastigote stage. The intracellularepimastigote has the lowest levels of intracellular free proline con-centration (0.45 mM), and depends upon an external supplementof this metabolite to go ahead with the differentiation to the trypo-mastigote stage. Accordingly, this stage shows the highest prolinetransport activity and the amino acid is likely to be provided bythe proline pool of the host cell, which is approximately 0.3 mM,

thus, near the Km of the T. cruzi high affinity proline transporter[10]. Glucose then plays a less important role in the metabolismof the intracellular epimastigotes, since glucose transport activity,glucose transporter amounts, and its specific mRNA were detected

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08 A.M. Silber et al. / Molecular & Bioch

t this stage in very low amounts. The low level of the hexose trans-orter detected may be attributed to an early expression pattern ofrypomastigote-specific proteins, as was described for Tc-85 [39].t is worth stressing that the glucose concentration in the host–cellytoplasm is below the TcHT Km (for an example see Ref. [40]).nder such conditions, the hexose transporter may not be func-

ional or relevant. In that case, although the participation of fattycids cannot be ruled out, amino acids appear to have an importantole as an energy source for the intracellular stages of T. cruzi.

cknowledgements

The authors are indebted to Fundacão de Amparo à Pesquisao Estado de São Paulo (FAPESP) and to Conselho Nacional deesenvolvimento Científico e Tecnológico (CNPq) for financial help

projects #04/03303-5 to MJMA and WC, projects #08-57596-4nd CNPq 473906/2008-2 to AMS, and fellowship support to RRTnd ACTT). AMS is a member of the Instituto Nacional de Biotec-ologia Estrutural e Química Medicinal em Doencas InfecciosasINBEQMeDI). The help of Roberto Zangrandi with host cell andrypomastigote cultivation is greatly acknowledged.

eferences

[1] Almeida-de-Faria M, Freymuller E, Colli W, Alves MJ. Trypanosoma cruzi:characterization of an intracellular epimastigote-like form. Exp Parasitol1999;92:263–74.

[2] Tyler KM, Engman DM. The life cycle of Trypanosoma cruzi revisited. Int J Para-sitol 2001;31:472–81.

[3] Rosenzweig D, Smith D, Opperdoes F, Stern S, Olafson RW, Zilberstein D. Retool-ing Leishmania metabolism: from sand fly gut to human macrophage. FASEB J2008;22:590–602.

[4] Besteiro S, Barrett MP, Riviere L, Bringaud F. Energy generation in insect stagesof Trypanosoma brucei: metabolism in flux. Trends Parasitol 2005;21:185–91.

[5] Bringaud F, Riviere L, Coustou V. Energy metabolism of trypanosomatids: adap-tation to available carbon sources. Mol Biochem Parasitol 2006;149:1–9.

[6] Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S. In vitro differenti-ation of Trypanosoma cruzi under chemically defined conditions. Mol BiochemParasitol 1985;16:315–27.

[7] Tonelli RR, Silber AM, Almeida-de-Faria M, Hirata IY, Colli W, Alves MJ. l-Prolineis essential for the intracellular differentiation of Trypanosoma cruzi. Cell Micro-biol 2004;6:733–41.

[8] Magdaleno A, Ahn IY, Paes LS, Silber AM. Actions of a proline analogue,l-thiazolidine-4-carboxylic acid (T4C), on Trypanosoma cruzi. PLoS ONE2009;4:e4534.

[9] Canepa GE, Bouvier LA, Urias U, et al. Aspartate transport and metabolism in theprotozoan parasite Trypanosoma cruzi. FEMS Microbiol Lett 2005;247:65–71.

10] Silber A, Tonelli R, Martinelli M, Colli W, Alves M. Active transport of l-prolinein Trypanosoma cruzi. J Eukaryot Microbiol 2002;49:441–6.

11] Silber AM, Rojas RL, Urias U, Colli W, Alves MJ. Biochemical characterization ofthe glutamate transport in Trypanosoma cruzi. Int J Parasitol 2006;36:157–63.

12] Nowicki C, Cazzulo JJ. Aromatic amino acid catabolism in trypanosomatids.Comp Biochem Physiol A: Mol Integr Physiol 2008;151:381–90.

13] Bouvier LA, Silber AM, Galvao Lopes C, et al. Post genomic analysis of permeasesfrom the amino acid/auxin family in protozoan parasites. Biochem Biophys ResCommun 2004;321:547–56.

14] Lamour N, Riviere L, Coustou V, Coombs GH, Barrett MP, Bringaud F. Prolinemetabolism in procyclic Trypanosoma brucei is down-regulated in the presenceof glucose. J Biol Chem 2005;280:11902–10.

15] Galvez Rojas RL, Frossard ML, Machado Motta MC, Silber AM. l-Proline uptakein Crithidia deanei is influenced by its endosymbiont bacterium. FEMS MicrobiolLett 2008;283:15–22.

[

l Parasitology 168 (2009) 102–108

16] Cazzulo JJ. Aerobic fermentation of glucose by trypanosomatids. FASEB J1992;6:3153–61.

[17] Cazzulo JJ. Intermediate metabolism in Trypanosoma cruzi. J Bioenerg Biomembr1994;26:157–65.

18] Barrett MP, Tetaud E, Seyfang A, Bringaud F, Baltz T. Trypanosome glucose trans-porters. Mol Biochem Parasitol 1998;91:195–205.

19] Tetaud E, Bringaud F, Chabas S, Barrett MP, Baltz T. Characterization of glucosetransport and cloning of a hexose transporter gene in Trypanosoma cruzi. ProcNatl Acad Sci USA 1994;91:8278–82.

20] Barrett MP, Walmsley AR, Gould GW. Structure and function of facilitative sugartransporters. Curr Opin Cell Biol 1999;11:496–502.

21] Tetaud E, Chabas S, Giroud C, Barrett MP, Baltz T. Hexose uptake in Trypanosomacruzi: structure–activity relationship between substrate and transporter.Biochem J 1996;317(Pt 2):353–9.

22] Bakker BM, Walsh MC, ter Kuile BH, et al. Contribution of glucose transport tothe control of the glycolytic flux in Trypanosoma brucei. Proc Natl Acad Sci USA1999;96:10098–103.

23] Bakker BM, Westerhoff HV, Opperdoes FR, Michels PA. Metabolic control anal-ysis of glycolysis in trypanosomes as an approach to improve selectivity andeffectiveness of drugs. Mol Biochem Parasitol 2000;106:1–10.

24] Landfear SM. Drugs and transporters in kinetoplastid protozoa. Adv Exp MedBiol 2008;625:22–32.

25] Silber AM, Colli W, Ulrich H, Alves MJ, Pereira CA. Amino acid metabolic routesin Trypanosoma cruzi: possible therapeutic targets against Chagas’ disease. CurrDrug Targets Infect Disord 2005;5:53–64.

26] Brener Z, Chiari E. Morphological variations observed in different strains ofTrypanosoma cruzi. Rev Inst Med Trop Sao Paulo 1963;19:220–4.

27] Camargo EP. Growth and differentiation in Trypanosoma cruzi. I. Origin ofmetacyclic trypanosomes in liquid media. Rev Inst Med Trop Sao Paulo1964;12:93–100.

28] Pereira CM, Sattlegger E, Jiang HY, et al. IMPACT, a protein preferentiallyexpressed in the mouse brain, binds GCN1 and inhibits GCN2 activation. J BiolChem 2005;280:28316–23.

29] Bradford MM. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. AnalBiochem 1976;72:248–54.

30] Cunha-e-Silva N, Sant’Anna C, Pereira MG, Porto-Carreiro I, JeovanioAL, de Souza W. Reservosomes: multipurpose organelles? Parasitol Res2006;99:325–7.

31] Yernaux C, Fransen M, Brees C, Lorenzen S, Michels PA. Trypanosoma brucei gly-cosomal ABC transporters: identification and membrane targeting. Mol MembrBiol 2006;23:157–72.

32] Colasante C, Alibu VP, Kirchberger S, Tjaden J, Clayton C, Voncken F. Charac-terization and developmentally regulated localization of the mitochondrialcarrier protein homologue MCP6 from Trypanosoma brucei. Eukaryot Cell2006;5:1194–205.

33] Michels PA, Bringaud F, Herman M, Hannaert V. Metabolic functionsof glycosomes in trypanosomatids. Biochim Biophys Acta 2006;1763:1463–77.

34] Sant’Anna C, Nakayasu ES, Pereira MG, et al. Subcellular proteomics of Try-panosoma cruzi reservosomes. Proteomics 2009;9:1782–94.

35] Souto-Padron T, Campetella OE, Cazzulo JJ, de Souza W. Cysteine proteinasein Trypanosoma cruzi: immunocytochemical localization and involvement inparasite–host cell interaction. J Cell Sci 1990;96(Pt 3):485–90.

36] Lara FA, Sant’Anna C, Lemos D, et al. Heme requirement and intracellular traf-ficking in Trypanosoma cruzi epimastigotes. Biochem Biophys Res Commun2007;355:16–22.

37] Alvarez VE, Kosec G, Sant’Anna C, Turk V, Cazzulo JJ, Turk B. Autophagy isinvolved in nutritional stress response and differentiation in Trypanosoma cruzi.J Biol Chem 2008;283:3454–64.

38] Hou JC, Pessin JE. Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking.Curr Opin Cell Biol 2007;19:466–73.

39] Abuin G, Freitas-Junior LH, Colli W, Alves MJ, Schenkman S. Expression of trans-

sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentiallyregulated at the post-transcriptional level by labile protein factors. J Biol Chem1999;274:13041–7.

40] Malliopoulou V, Xinaris C, Mourouzis I, et al. High glucose protects embry-onic cardiac cells against simulated ischemia. Mol Cell Biochem 2006;284:87–93.