Experimental Parasitology 111 (2005) 198–206

www.elsevier.com/locate/yexpr

Research brief

Trypanosoma cruzi: Sequence polymorphism of the gene encoding the Tc52 immunoregulatory-released factor in relation

to the phylogenetic diversity of the species �

Bruno Oury b,¤, Frédérique Tarrieu b, Adriano Monte-Alegre a, Ali Ouaissi a

a Institut de Recherche pour le Développement (IRD), Unité de Recherche no 8 “Pathogénie des Trypanosomatidae,” Montpellier, Franceb Institut de Recherche pour le Développement, Unité de Recherche IRD no 165 “Génétique et Evolution des Maladies Infectieuses,”

Unité Mixte de Recherche CNRS (Centre National de la Recherche ScientiWque)-IRD no 2724, Montpellier, France

Received 6 August 2004; received in revised form 4 July 2005; accepted 15 July 2005

Abstract

We have previously identiWed a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional prop-erties with the thioredoxin and glutaredoxin family involved in thiol-disulWde redox reactions. Gene targeting strategy and immuno-logical studies allowed showing that Tc52 is among T. cruzi virulence factors. Taking into account that T. cruzi has a geneticvariability that might be important determinant that governs the diVerent behaviour of T. cruzi clones in vitro and in vivo, wethought it was of interest to analyse the sequence polymorphism of Tc52 gene in several reference clones. The DNA sequences of 12clones which represent the whole genetic diversity of T. cruzi allowed showing that 40 amino-acid positions over 400 analysed aretargets for mutations. A number of residues corresponding to putative amino-acids playing a role in GSH binding and/or enzymaticfunction and others located nearby are subject to mutations. Although the immunological analysis showed that Tc52 is present inparasite extracts from diVerent clones, it is possible that the amino-acid diVerences could aVect the enzymatic and/or the immuno-modulatory function of Tc52 variants and therefore the parasite phenotype. 2005 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Trypanosoma cruzi; Tc52; Immunomodulatory factor; Sequence polymorphism; aa, amino-acid(s); Ala, ala-nine; Arg, arginine; bp, base pair(s); kbp, kilobase pair(s); cDNA, DNA complementary to RNA; dNTP, dinucleotide tri-phosphate; DTU, discretetyping unit; FCS, foetal calf serum; Gln, glutamine; Glu, glutamic acid; Gly, glycine; GSH, reduced tripeptide glutathione; GST, glutathione-S-trans-ferase; Ile, isoleucine; kDa, kilodalton(s); L. infantum, Leishmania infantum; L. lainsoni, Leishmania lainsoni; L. major, Leishmania major; LIT, liverinfusion tryptose; Lys, lysine; Met, methionine; PBS, phosphate buVer saline; PCR, polymerase chain reaction; P. discolor, Phyllostomum discolor;Pro, proline; RPMI 1640, culture medium no 1640 developed at Roswell Park Memorial Institute; SDS, sodium dodecyl sulphate; Ser, serine;T. infestans, Triatoma infestans; T. brucei gambiense, Trypanosoma brucei gambiense; T. cruzi, Trypanosoma cruzi; T. marinkellei, Trypanosoma marinkellei;T. rangeli, Trypanosoma rangeli

Trypanosoma cruzi, the etiological agent of Cha-gas’disease, is an obligate intracellular parasite causingchronic infections in humans and a large number of

� Accession numbers: AY195575; AY238345; AY238346;AY238347; AY238348; AY238349; L07510; AY238350; AY238351;AY238352; AY238353; AY238354; AY238355; AY238356;AY238357; AJ582069.

* Corresponding author. Fax: +33 4 67 41 61 47.E-mail address: Bruno.Oury@mpl.ird.fr (B. Oury).

0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2005.07.001

other mammalian species. This protozoan parasite istransmitted to man in the faeces of hematophagous bugsof the Reduviidae family. The complex lifecycle ofT. cruzi includes diVerent stages in the insect vector andthe vertebrate host. There are two parasite stages in thevector: epimastigotes and metacyclic trypomastigotes,whereas the vertebrate stages are bloodstream trypom-astigotes and intracellular amastigotes. These series oftransitions involve major modiWcations in morphology,

B. Oury et al. / Experimental Parasitology 111 (2005) 198–206 199

gene expression, and cell cycle (Bonaldo et al., 1988;Gonzales-Perdomo et al., 1988).

In previous studies, we reported the identiWcation of acDNA encoding a T. cruzi Tc52 protein sharing motifssimilar to those found in glutathione-S-transferase(GSTs) sequences and a set of small proteins mostlyinduced by diVerent stress conditions (Schöneck et al.,1994). The Tc52 protein was found by gene knockoutapproach to be among factors crucial for parasite sur-vival and virulence (Allaoui et al., 1999).

GSTs are ubiquitous housekeeping enzymes foundin nearly all animals and some parasites. They belongto a super family of isoenzymes catalysing the conjuga-tion of the reduced tripeptide glutathione (GSH) witha large variety of xenobiotics as well as endogenoussubstances like bilirubin, steroids, carcinogens, andsome organic compounds. Some GSH-conjugatesare known to be directly implicated in carcinogenesisbut also in detoxiWcation of those substances (Kettereret al., 1988). Drug resistance mediated by GSTshas been reported in insects, some plants, andperhaps also in tumours and parasites (Hayes andWolf, 1988).

Cytosolic GSTs are homo- or heterodimers composedof two subunits of 24–28 kDa, linked by non-covalentinteractions. They are shared out among Wve classes, �,�, �, �, and � (Landi, 2000). Identity between classes didnot exceed 30% and is found mainly within their N-ter-minal region also named glutathione binding site (G-site). The non-speciWc hydrophobic C-terminal region(H-site) has a much more variability than the G-site.

Evolutionary relationships allowed drawing thehypothesis that an ancestor gene product used GSH as acofactor to exert the reducer function, which could be itsmain role. The GSH binding does appear in the N-termi-nal region (G-region), whereas the individual function isrelated to the C-terminal hydrophobic region (H-region). From a progenitor protein all the others couldhave evolved by gene duplication and further divergenceleading to the large diversity in function. Another com-mon feature is that GSTs act as dimers, the only excep-tion being the T. cruzi Tc52 protein, which seems to beancestral. Indeed it is believed to be a result of geneduplication without further separation. The Tc52protein is composed of two homologous domains com-prising a GSH binding site (G-site) and a hydrophobicC-terminal region (H-site) (Ouaissi et al., 2002b). Themolecule may act as a dimeric-like complex where thetwo “pseudo-subunits” areas are arranged in an antipar-allel fashion separated by a strong �-turn motif (Ala225-Pro-Gly-Tyr228).

Biochemical studies have shown that the puriWedTc52 protein could function in vitro as a thioltransferase(Moutiez et al., 1995). Finally, immune protection exper-iments in murine model suggest that Tc52 is among can-didate molecules that may be used to design an optimal

vaccine to control T. cruzi infection (Ouaissi et al.,2002a).

It is well known that T. cruzi isolates show high levelsof genotypic diversity (Barnabé et al., 2000). Geneticsstudies have revealed that the population structure ofT. cruzi is predominantly clonal and genetic exchangescould occur but were too infrequent to inXuence on thegenetic structure and evolution of the species (Tibayrencet al., 1986). Natural clones appear to be identical for agiven set of genetic loci. Genetic diversity is caused byclonal divergence and rare genetic exchanges (Brisseet al., 1998; Gaunt et al., 2003; Machado and Ayala,2001). Phylogenetic approaches have evidenced that thespecies is distributed into two main lineages which werereferred to as discrete typing units (DTUs) I and II(Tibayrenc, 1998) or T. cruzi I and T. cruzi II (Momen,1999). DTU II or T. cruzi II is subdivided into Wve dis-crete sub-lineages referred to as IIa–IIe (Barnabé et al.,2000).

Various biological parameters including growthkinetics, virulence, and tissue tropism in mice, in vitrodrug sensitivity and vectorial transmissibility are highlylinked to phylogenetic divergence between variants ofthe parasite (De Lana et al., 1998, 2000; Dvorak, 1984;Laurent et al., 1997; Pinto Da Silva et al., 1998; Revolloet al., 1998). Therefore, in the light of these observations,we thought it was of interest to examine the presenceand the possible sequence variation of Tc52 gene in sev-eral reference variants of T. cruzi.

All parasite strains surveyed in this present work werecloned. T. cruzi, Trypanosoma marinkellei, and Trypano-soma rangeli epimastigotes were grown at high cell den-sity at 27 °C in LIT medium supplemented with 10%heat-inactivated foetal calf serum (FCS) and 50�g/mlgentamycin. Trypanosoma brucei gambiense procyclicforms were cultivated at 27 °C, in the Cunninghammedium supplemented with 10% heat-inactivated FCSand 50 �g/ml gentamycin. Leishmania infantum, Leish-mania lainsoni, and Endotrypanum clones were main-tained at 26 °C in RPMI medium supplemented with10% heat-inactivated FCS and 50 �g/ml gentamycin.When reaching the stationary phase, cultures were har-vested by centrifugation at 750g for 10 min at 4 °C. Para-site pellets were then washed in phosphate buVer saline(PBS) by three successive centrifugations and then fre-ezed at ¡80 °C until used.

To identify the Tc52 gene, we have used the polymer-ase chain reaction (PCR) to amplify a fragment encod-ing the domains of Tc52 protein containing the putativeamino-acids (aa) involved in GSH binding (aa 20–418),followed by direct sequencing of individual PCR prod-ucts. DNA was puriWed by phenol/chloroform extrac-tion as described by Oury et al. (1997). AmpliWcations ofthe Tc52 gene were performed in a volume of 50�l usingthe Tc52A sense primer (5�-ATGAAGGCTTTGAAACTTTTTAAAG-3�) and the Tc52B antisense primer

200 B. Oury et al. / Experimental Parasitology 111 (2005) 198–206

(5�-TCAAGACGATGGACGCAAAAACGTC-3�).BrieXy, genomic DNA samples (100 ng) were used astemplate for PCR in the speciWc buVer (10 mM Tris–HCl, pH 8, 50 mM KCl, and 1.5 mM MgCl2) containing2 £ 0.5�M primers, 4 £ 200 �M dNTP, and 2.5 U of TaqDNA polymerase (Roche Diagnostics, Mannheim, Ger-many). The ampliWcation program was achieved on aPTC-100-60 thermocycler equipped with a heating lid(MJ Research, Watertown, Massachusetts, USA) andconsisted of 40 cycles at 94 °C for 1 min, 59 °C for 1 min,and 72 °C for 1 min 30 s. For the last cycle, the elonga-tion step was extended to 5 min at 72 °C. AmpliWedDNA was kept at 4 °C until use.

PCR products were puriWed using the “Wizzard PCRPreps DNA PuriWcation System” (Promega), accordingto the protocol provided by the manufacturer. The qual-ity of puriWed fragments were controlled by scanning theUV absorption of DNA from 210 to 310 nm and afterelectrophoresis on 1.6% agarose horizontal gel. ThenpuriWed fragments were sequenced from each extremity.The sequencing was repeated twice at minimum to ascer-tain the sequence variability detected.

The Tc52 gene was ampliWed in a set of 12 clones,which represent the whole genetic diversity of T. cruzi(two clones for each DTU). The speciWc ampliWcationwas also performed in representative clones of other Try-panosomatidae: T. marinkellei, a species closely relatedto T. cruzi, isolated from chiropters in South Americawhich is not pathogenic for humans; T. rangeli, found inprimates in South America; T. brucei gambiense (group1), responsible for the chronic form of human sleepingsickness in Africa; L. infantum, responsible for visceralleishmaniasis in man and dogs; L. lainsoni, and Endotry-panum. The origins of clones are given in Table 1.

For each clone, the non-coding strand was convertedin coding strand using the program DNA StriderTM 1.0(Marck, C.). Both sequences were joined end to end toobtain the full ampliWed sequence. Then nucleotidesequences were translated in protein sequence using theprogram Multiple Translation available on the websiteof Infobiogen (France). Protein sequences were trun-cated to a minimal 400 aa region common to all clones.They were aligned using the program CLUSTAL X, ver-sion 1.8 for analysis. A neighbor-joining tree was built byusing Neighbor program and drawn with Treeview pro-gram, version 1.4. The analysis was carried out with theprotein sequence of the thiol-dependant reductase,TDR1, from Leishmania major, as outgroup (Dentonet al., 2004).

To analyse the Tc52 expression, we conducted West-ern blotting experiments using parasite extracts accord-ing to the procedures reported elsewhere (Ouaissi et al.,1990). Antibody probe consisted of a mouse immuneserum to the native Tc52 protein obtained as describedin previous reports (Ouaissi et al., 1995).

As shown in Fig. 1, selected number of DNA sam-ples from T. cruzi representative clones as well asT. marinkellei ones ampliWed using the primer pairTc52A/Tc52B in repeated experiments revealed a bandof molecular size mean value of 1.3 kbp. Although twofaint bands of 800 and 1000 bp could be distinguishedin the case of L. lainsoni, no ampliWcation product wasdetected when using DNA samples from T. rangeli,L. infantum, L. lainsoni, Endotrypanum, and T. bruceigambiense. The signiWcance of this observation isunknown. It might be that some homology sequencesare shared between Tc52 primers and sequencesin part of the L. lainsoni genome. Moreover, it is

Table 1Host and geographical origins of the 20 Trypanosomatidae clones used in the present study

n.k., not known.

Species/DTU Code Host Geographical origin (country, region, and/or locality)

T. cruzi/DTU I OPS21 cl11 Man Venezuela, Cojedes, MacuayasT. cruzi/DTU I P209 cl1 Man Bolivie, SucreT. cruzi/DTU IIa CanIII cl1 Man Brazil, BélemT. cruzi/DTU IIa Dog Theis Man USA, n.k.T. cruzi/DTU IIb CBB cl3 Man Chile, Coquimbo, TulahuènT. cruzi/DTU IIb Y cl7 Man Brazil, São PauloT. cruzi/DTU IIc M6241 cl6 Man Brazil, BélemT. cruzi/DTU IIc X110/8 Dog Paraguay, MakthlawaiyaT. cruzi/DTU IId Bug2148 cl1 T. infestans Brazil, Rio Grande do SulT. cruzi/DTU IId Mn cl2 Man Chile, Coquimbo, TulahuènT. cruzi/DTU IIe Cl Brener T. infestans Brazil, Rio Grande do SulT. cruzi/DTU IIe Tula cl2 Man Brazil, Rio Grande do SulT. marinkellei B3 (or 593) P. discolor Brazil, Bahía, São FelipeT. marinkellei B7 cl2 P. discolor Brazil, Bahía, São FelipeT. rangeli RGB Dog Venezuela, CaracasT. brucei gambiense Jua Man Cameroon, FontemL. infantum ITMAP263 Man Morocco, n.k.L. lainsoni M6426 Man Brazil, n.k.Endotrypanum LEM 2954 n.k. French Guiana, Petit Saut

B. Oury et al. / Experimental Parasitology 111 (2005) 198–206 201

noteworthy that a recent study has reported themolecular and functional characterization of aL. major enzyme, termed TDR1, able to reduce antile-ishmanial pentavalent antimonial drugs and whoseprimary sequence showed 45.7% identity overall withTc52 (Denton et al., 2004). Therefore, these new dataclearly demonstrated the existence of Tc52 homo-logues in Leishmania genus.

Analysis of Tc52 nucleotide sequences from theT. cruzi clones evidenced that 72 positions of the 1201ones taken into account are targets for mutations. Thirtysynonymous substitutions were counted (42%). Forty-two non-synonymous substitutions were identiWed on 40mutated codons.

Sequences analysis revealed ORF that share 93.75–99% homology with the Tc52 sequence P209 cl1, for T.cruzi and 90–91% for T. marinkellei (Fig. 2). Indeed, 62aa positions over 400 aa are targets for mutations in allsequences surveyed. In the 12 T. cruzi sequences, 40 sub-stitutions could be detected whereas there were 36 and42 in both T. marinkellei clones B3 and B7 cl2, respec-tively. Although this might appear as an unusual feature,similar heterogeneity in T. cruzi gene sequences havebeen already reported in previous studies (Machado andAyala, 2001; Gaunt et al. (2003)).

DTUs IId and IIe gather heterozygous genotypesresulting from hybridisation events between homozy-

gous genotypes pertaining to parental DTUs IIb andIIc. None determined residues X were detected only insequences of clones belonging to hybrid DTUs IId andIIe. They corresponded to irresolutions between 2 aafound in both alleles (Fig. 2). Thus sequences identi-Wed in the diVerent genetic variants correspond todiVerent alleles speciWc to DTU I, IIa, IIb, and IIc.

A certain degree of sequence variation could be seenwhen comparing T. cruzi clones belonging to DTU1 orDTU2: only Wve mutations were speciWc to DTUs I andII (K/T(28), V/I(201), (L/S202), (R/S213), and (S/R320)). When considering the existence of variationsfor certain biological properties between DTUs, espe-cially between DTUs I and II, the existence of asequence polymorphism associated with members ofthose two major subdivisions might suggest that itcould have an impact on some functions of Tc52 andsubsequently on biological properties in which this fac-tor of virulence is involved (De Lana et al., 1998, 2000;Pinto Da Silva et al., 1998; Revollo et al., 1998). Theexact signiWcance of this sequence polymorphismawaits further investigations.

Otherwise Tc52 sequences from T. marinkellei clonesdiVered from those of T. cruzi at 24 over 38–42 positionsidentiWed. Recent phylogenetic studies drew the hypoth-esis that the adaptation of T. cruzi to a wide variety ofhosts was a derived genetic character that was acquired

Fig. 1. PCR analysis of total DNA from diVerent T. cruzi genetic variants and other protozoan parasites using the Tc52 primers pair, Tc52A andTc52B. AmpliWcations showed in this experiment were performed on DNA from a reference clone of each DTU for T. cruzi (OPS21 cl11 for theDTU I, CanIII cl1 for the DTU IIa, Y cl7 for the DTU IIb, M6241 cl6 for the DTU IIc, Bug 2148 cl1 for the DTU IId, and Cl Brener for the DTUIIe), and T. marinkellei (B3), T. rangeli, L. infantum, L. lainsoni, Endotrypanum, and T. brucei gambiense. Molecular weight marker is a 100 bp DNAladder from Fermentas (Lithuania).

IIeIIcI IIa IIb IId

500 bp

1 000 bp1 300 bp

T. brucei g

ambiense

Negative co

ntrol

Endotrypanum

L. lainsoni

L. infantum

T.rangeli

T.marin

kelle

i

Marker

Marker T. cruzi

202 B. Oury et al. / Experimental Parasitology 111 (2005) 198–206

from a bat-restricted ancestor (Barnabé et al., 2003). Yetthe close phylogenetic relationship between T. cruzi andT. marinkellei suggest that these two species have a puta-

tive common ancestor, a parasite restricted to bats. Andthe pathogenicity is a property acquired by T. cruzi atthe time of its adaptation to man, whereas T. marinkellei

20 30 40 50 60 70 80 90P209 cl1 (DTU I) ITAKEKRVKLEEVEVPLGDDMPQWYKELNPRETVPTLQVDGKKCMIESDLISRYIDRISSPANALIGSSPYQRHRVEFFLOPS21 cl11 (DTU I) ................................................................................Can III cl1 (DTU IIa) ..E.....T.......................................................................Dog Theis (DTU IIa) ........T..........................................A............................CBB cl3 (DTU IIb) ........T........................................................M..............Y cl7 (DTU IIb) ........T........................................................M..............M6241 cl6 (DTU IIc) ........T..........................M.........................E..................X110/8 (DTU IIc) ........T..........................M.........................E..................Bug2148 cl1 (DTU IId) ........T..........................M.........................E..................MN cl2 (DTU IId) ........T....................................................X...X..............Cl Brener (DTU IIe) ........T..........................X.............................X..............Tula cl2 (DTU IIe) ........T....................................................X...X..............B3 (T. marinkellei) ...............A......................L.....L...........S......I.M.............MB7 cl2 (T. marinkellei) ...............A................R.....L.....L...........S......I.M.............M

**.*****.******.****************.**.**.*****.******.****.****.*.*.*************.

100 110 120 130 140 150 160 170 P209 cl1 (DTU I) SEIGDLIKAYFGLLRDPFNEEKRKSVDHNTAYIEGIIAEHQGDGPYFLDDTFSMAEVMVVPFLACFRPVLSYYCGYDIFHOPS21 cl11 (DTU I) ................................................................................Can III cl1 (DTU IIa) ......V......V..................................................................Dog Theis (DTU IIa) ......V......V..................................................................CBB cl3 (DTU IIb) G.....V......V.............N......D......................................W......Y cl7 (DTU IIb) G.....V......V.............N......D.............................................M6241 cl6 (DTU IIc) ......V......V..................................................................X110/8 (DTU IIc) ......V......V..................................................................Bug2148 cl1 (DTU IId) ......V......V..................................................................MN Cl2 (DTU IId) ......V......V.............X....................................................Cl Brener (DTU IIe) X.....V......V.............X......X.............................................Tula cl2 (DTU IIe) X.....V......V.............X....................................................B3 (T. marinkellei) ..V...V......X....................D......................L.L....................B7 cl2 (T. marinkellei) ..V...V...........................D......................L.L....................

.*.***.******.*************.******.**********************.*.*************.******

180 190 200 210 220 230 240 250P209 cl1 (DTU I) EAPRLKKMYVTSMQRTTVKETVLKPEEYIIGFKRKVPTSHVTWSLAPGYVLFVNKYSPFSDRPRLACALKNIDLPMLEIDOPS21 cl11 (DTU I) ................................................................................Can III cl1 (DTU IIa) ..........A....A.....ISR.........S...K..........................................Dog Theis (DTU IIa) .....................IS..........S...K..........................................CBB cl3 (DTU IIb) N....................IS..........S...K..........................................Y cl7 (DTU IIb) N....................IS..........S...K..........................................M6241 cl6 (DTU IIc) .....................IS..........S...K..........................................X110/8 (DTU IIc) .....................IS..........S...K..........................................Bug2148 cl1 (DTU IId) .....................IS..........S...K..........................................MN cl2 (DTU IId) D....................IS..........S...K..........................................Cl Brener (DTU IIe) X....................IS..........S...K..........................................Tula cl2 (DTU IIe) D....................IS..........S...K..........................................B3 (T. marinkellei) K.........A....A.............T...N...K..................C...................I...B7 cl2 (T. marinkellei) K.........A....A.............T...N...K..................C...................I...

+*********.****.*****...*****.***:***.******************.*****************.*.***

260 270 280 290 300 310 320 330 P209 cl1 (DTU I) LKQLPSWFRWFNQRETVPALLTPQGTHVHESQLIVHYLDDGFPEHGPALLPKDADGLYHVSFVESNVDYFMDAMFSLIKDOPS21 cl11 (DTU I) ..........L....L..............................R.................................Can III cl1 (DTU IIa) ......G...........T......AY.............................S...R.............Y.....Dog Theis (DTU IIa) ..................T.......Y.............................S...R.............Y.....CBB cl3 (DTU IIb) .....P............T.......Y.............................S...R.............Y.F...Y cl7 (DTU IIb) .....P............T.......Y.............................S...R.............Y.F...M6241 cl6 (DTU IIc) ..................T....R..Y.............................S...R.............Y.....X110/8 (DTU IIc) ..................T....R..Y.............................S...R.............Y.....Bug2148 cl1 (DTU IId) ..................T....R..Y.............................S...R.............Y.....MN cl2 (DTU IId) .....P............T.......Y.............................S...R.............Y.F...Cl Brener (DTU IIe) .....X............T....X..Y.............................S...R.............Y.X...Tula cl2 (DTU IIe) ..................T.......Y.............................S...R.............Y.F...B3 (T. marinkellei) .....T............T.......Y.............................S....L.....N.L.G..Y.....B7 cl2 (T. marinkellei) .....T............T.......Y.............V...............S....L.....N.L.G..Y.....

*****:.***.****.**.****.*..*************.*****.*********.***..*****.*.*.**.*.***

340 350 360 370 380 390 400 410P209 cl1 (DTU I) PKNMNAKEEFDWAAGELEKLLAEHQFGEGPFFGGASMNAADVSLVPMLVHLKACTPDLTEGQDLLANYKLLTAAAEAGLTOPS21 cl11 (DTU I) ...R............................................................................Can III cl1 (DTU IIa) ...................................T...................................A........Dog Theis (DTU IIa) ..................................TT...................................A........CBB cl3 (DTU IIb) ...................................T....................E..............A........Y cl7 (DTU IIb) ...................................T....................E..............A........M6241 cl6 (DTU IIc) ...T...............................T........L..........................A........X110/8 (DTU IIc) ...T...............................T........L..........................A.......NBug2148 cl1 (DTU IId) ...T...............................T........L..........................A........MN cl2 (DTU IId) ...................................T...................................A........Cl Brener (DTU IIe) ...................................T...................................A........Tula cl2 (DTU IIe) ...................................T........X...........X..............A........B3 (T. marinkellei) ...K...............................T.......IM........Y...M......................B7 cl2 (T. marinkellei) ...K...............................T.......IM........Y..NM.............A.......K

***+******************************..*******.:********.**+.*************.*******:

B. Oury et al. / Experimental Parasitology 111 (2005) 198–206 203

remained unable to infect man. Therefore we can expectthat Tc52, one of the factors of virulence for humans inT. cruzi, could be widely involved in the process of adap-tation and one or several speciWc mutations might mod-ulate the functions of the enzyme and partiallydetermine the pathogenicity character unless the expres-sion of Tc52 is regulated at the transcriptional or post-transcriptional level.

To evidence the intraspeciWc diversity of T. cruziclones, a neighbor-joining tree was built using thesequence of the protein TDR1, homologue of Tc52 inL. major, as outgroup (Fig. 3). The dendrogram evi-denced two clusters corresponding to both species,T. cruzi and T. marinkellei. Within T. cruzi, the twomajor lineages, DTU I and II, were individualized. Like-wise, within DTU II, DTU IIa was clearly separatedfrom other sub-DTU IIs b–e. If clones belonging toDTU IIb and IIc were, respectively, associated, clonesattributed to DTU IId and IIe were distributed in diVer-ent clusters. So the clone MN cl2 from DTU IId and thetwo clones, Cl Brener and Tula cl2, belonging to DTUIIe were close to DTU IIb. And the clone Bug2148 cl1from DTU IId was found associated with DTU IIc. Thisclustering was consistent with the fact that DTU IId andIIe gather hybrid genotypes which putative parentsbelong to DTU IIb and IIc (Barnabé et al., 2000). So it isnoteworthy that the intraspeciWc hierarchy obtainedfrom the polymorphism of Tc52 protein sequences,matches with results reported by Machado and Ayala(2001) and Gaunt et al. (2003).

A number of residues of human (Reinemer et al.,1992) and rat �-GST (Reinemer et al., 1991) as well asrat �-GST (Ji et al., 1992), have been shown to be ofmajor importance for GSH binding while mutations ofother residues appear to aVect GSH binding activity orenzymatic function (Manoharan et al., 1992). Compari-son of Tc52 sequence from the T. cruzi P209 cl1 clone tothe GST sequences allowed identifying functional resi-dues of Tc52 (Table 2). Most of the residues importantfor GSH binding or enzymatic function in �-GST, havetheir corresponding counterparts in the two Tc52sequence halves (Tc52N and Tc52C), either identical orsimilar except for Tyr7 which is replaced by Met11in Tc52N. Tyr6 (rat �-GST) or Tyr7 (human and rat

Fig. 2. Multiple alignments of the Tc52 amino-acid sequences from diVerenlei in comparison to the reference sequence from P209 cl1 clone. Only mureference T. cruzi P209 cl1 clone are replaced by dots. (X) refers to irresolutions. In the consensus sequence below the alignment, (¤) displays an isequence of reference T. cruziP209 cl1 clone are mentioned by (.), (:) or (+three aa, respectively, in all clones surveyed. Dark grey bars correspond totion in the human �-GST (Manoharan et al., 1992). Light grey bars indic1992) and rat (Reinemer et al., 1991), and the rat �-GST (Ji et al., 1992). AY195575 (OPS21 cl11); AY238345 (P209 cl1); AY238346 (CanIII cAY238350 (M6241 cl6); AY238351 (X110/8); AY238352 (Bug2148 cl1AY238356 (B3), and AY238357 (B7 cl2).

�-GST) has a key role in GSTs for GSH deprotonationand activation (KarshikoV et al., 1993; Liu et al., 1992).

Comparison of Tc52 sequences from T. cruzi clonesbelonging to diVerent clones showed aa substitutions atpositions corresponding to the putative residues playinga role in GSH binding and/or enzymatic function(Fig. 2). For example, the following aa Trp266, Thr275,and Gln283, in the reference sequence P209 cl1 aresubstituted by Gly, Leu, and Arg, respectively, in thesequences of CanIII cl1, OPS21 cl11, and M6241 cl6,X110/8 or Bug2148 cl1. Two aa substitutions aVectalso positions involved in GSH binding in bothT. marinkellei sequences (positions 52 and 327). More-over, among the positions aVected by mutations, 10 are

Fig. 3. Neighbor-joining tree derived from the distance matrix andbased on Tc52 and TDR1 protein sequences, depicting the phyloge-netic relationships between T. cruzi, T. marinkellei, and L. majorclones. L. major represented by TDR1 sequence was used as outgroupto root the tree.

0.1

CBB cl3 (DTU2b)

Y cl7 (DTU 2b)

Cl Brener (DTU 2e)

Mn cl2 (DTU2d)

Tula cl2 (DTU2e)

M6241 cl6 (DTU2c)

X110/8 (DTU2c)

Bug2148 cl1 (DTU2d)

CanIII cl1 (DTU2a)

DogTheis (DTU2a)

P209 cl1 (DTU1)

OPS21 cl11 (DTU1)

B3 (T. marinkellei)

B7 cl2 (T. marinkellei)

TDR1(L. major)

T. cruzi genetic variants and another related parasite species, T. marinkel-tions are displayed. Residues identical to their corresponding ones in theion between two aa. Numbers above alignments indicate the residue posi-ntity between all clones. Mutations evidenced by comparison with the which indicate the substitution of the reference residue by one, two, andhe putative residues playing a role in GSH binding or in enzymatic func-e aa involved in GSH binding in the �-GSTs from man (Reinemer et al.,quences are available on GenBank under the following Accession Nos.:

); AY238347 (Dog Theis); AY238348 (CBB cl3); AY238349 (Y cl7);; AY238353 (Mn cl2); AY238354 (Cl Brener); AY238355 (Tula cl2);

t tat

de) t

atSel1)

204 B. Oury et al. / Experimental Parasitology 111 (2005) 198–206

located in the close environment (§ 2) of putative resi-dues involved in GSH binding and/or enzymatic func-tion for T. cruzi (positions 55, 71, 100, 106, 195, 265,270, 278, 285, and 286) and 11 for T. marinkellei (posi-tions 58, 64, 102, 106, 195, 236, 265, 278, 286, 321, and329).

The � class of GSTs show a speciWc Ser11 residue inplace of the N-terminal tyrosine found in classes �, �,

Table 2Residues of human �-GST, rat �-GST, and �-GST important for GSHbinding or enzymatic function (Ji et al., 1992; Manoharan et al., 1992;Reinemer et al., 1991, 1992) and their counterparts in the two Tc52halves, Tc52N and Tc52C, from the reference T. cruzi P209 cl1

Substitutions are mentioned in italics. Hyphens indicate that equivalentaa in Tc52 sequence from the reference T. cruzi Y strain were not foundin rat �-GST and rat �-GST sequences compared with the human �-GST one.

Human �-GST

Rat �-GST Rat �-GST Tc52

N-terminal C-terminal

Tyr7 Tyr7 Tyr6 Met11 Tyr235Arg13 Arg13 Leu12 Arg17 Arg241Trp38 Trp38 Trp45 Trp43 Trp266Lys42 Lys42 Lys49 Lys45 Arg268Gln49 Gln49 Asn58 Thr52 Thr275Leu50 ¡ Leu59 Val53 Val276Pro51 Pro51 Pro60 Pro54 Pro277Asp57 ¡ ¡ Asp59 Gln283Gly58 ¡ ¡ Gly60 Gly284Gln62 Gln62 Gln71 Glu66 Glu289Ser63 Ser63 Ser72 Ser67 Ser290Ile68 ¡ ¡ Ile70 Ile293His71 ¡ ¡ Arg72 His295Glu95 Glu95 ¡ Glu101 Glu323Asp96 ¡ Asp105 Asp104 Asp327Arg182 ¡ ¡ Arg194 Lys425

and � (Landi, 2000). This diVerence confers propertiesspeciWc to � class of GSTs compared to the others (�, �,and �). Regarding the process of GSH detoxiWcation, the� class GSTs have higher enzymatic activity and aYnityfor GSH than the �, �, and � classes, whereas the aYni-ties towards conjugate products are much lower in thecase of � class GSTs than in the others. The fact thatSer11 or Tyr6-7 is replaced in the Tc52N by Met11 forP209 cl1 (DTU I) clone, Lys11 or Ile11 for the OPS21cl11 (DTU I) and X110/8 (DTU IIc) clones, respectively,might suggest diVerent properties for Tc52N comparedto Tc52C where the tyrosine is conserved in the position235 in all DTUs. Furthermore, the residue Gln49 in the�-GST is replaced by Thr52 (Tc52N) and Thr275(Tc52C), but this seems also to be the case in � GSTs-1-1of Drosophila melanogaster (Toung et al., 1990). More-over, the presence of Glu66 (Tc52N) and Glu289(Tc52C) in place of Gln62/Gln71 in �-GST and �-GST,respectively, also occurs in diVerent other GSTsmolecules.

Complementary experiments using Western blottingand anti-Tc52 speciWc immune serum were performed toexamine the presence of the Tc52 reactive epitopes intotal extracts from clones of diVerent Trypanosomati-dae: T. cruzi, T. marinkellei, T. rangeli, T. brucei gamb-iense, and L. infantum. As shown in Fig. 4, a major bandof molecular mass 52 kDa could be visualized as previ-ously reported (Ouaissi et al., 1995), in most of T. cruziextracts (DTU I, IIa, IIc, IId, and IIe). A lower band ofmolecular mass 45 kDa could be seen in the case of DTUIIb, suggesting therefore that the amino-acid changeshave no eVect on the immune recognition by the anti-bodies although they might have an impact on the enzy-

Fig. 4. Western blot of diVerent parasite extracts from T. cruzi genetic variants and various related Trypanosomatidae: T. marinkellei, T. rangeli,T. brucei gambiense, and L. infantum. Proteins were separated by SDS–PAGE (12%) and immunoblotted with an anti-Tc52 immune serum asdescribed by Ouaissi et al. (1995). Blot (A) gathers proWles of T. cruzi clones belonging to DTU I, DTU IIa, and DTU IIc-e. Blot (B) displays proWlesobtained with two clones representing T. cruzi/DTU IIb and T. marinkellei, respectively. Blot (C) shows proWles obtained for T. rangeli, T. bruceigambiense, and L. infantum. T. cruzi genetic variants were represented by: P209 cl1 for the DTU I, CanIII cl1 for the DTU IIa, CBB cl3 for the DTUIIb, X110/8 for the DTU IIc, Mn cl2 for the DTU IId, Tula cl2 for the DTU IIe, and T. marinkellei by B3.

IIb T. ran

geli

T. mar

inke

llei

L. infa

ntum

T. bru

cei g

ambien

se

MW MWIIcMW I IIdIIa IIekDa

64

50

36

30

A

B

C

B. Oury et al. / Experimental Parasitology 111 (2005) 198–206 205

matic function. There was a high variation in term ofTc52 protein synthesis between clones from diVerentgenetic subdivisions, the signiWcance of which awaits fur-ther investigations. In the case of T. marinkellei, the reac-tive bands range from 39 to 56 kDa whereas in the caseof T. rangeli and T. brucei gambiense extracts, majorbands of 45, 56, and 47 kDa, respectively, could be evi-denced. Moreover, the immune serum also reacted witha polypeptide of molecular mass of around 54 kDa in thecase of L. infantum extracts. This observation is in agree-ment with the expected molecular mass of theL. infantum putative homologue identiWed in the proteindatabases (www.geneb.org). Furthermore, immune reac-tivity of our anti-Tc52 mouse immune serum was alsoobserved against the L. major recombinant his-tag fusedTDR1 protein (data not shown). Some variation in themolecular mass of Tc52 could be observed in certainpreparations. This might be due to the non-completereduction of the samples. In fact Tc52 has a nativemolecular mass of t100 kDa (Moutiez et al., 1995).Moreover, gel Wltration analysis of L. major TDR1showed that the protein has a native molecular mass of155 kDa consistent with the notion of the native proteinbeing a trimer. Furthermore, during our experiments, wehave recently observed that Tc52, when incubated at37 °C under various acidic pH buVers, was split generat-ing a number of peptides varying in size, a major degra-dation product being a 28 kDa peptide (Borges et al.,2003). This might explain the appearance of multiplebands and even lower molecular weight peptides inWestern blots.

In summary, the present study provided the Wrst evi-dence for the Tc52 gene sequence polymorphism amongthe T. cruzi clones. Moreover, if our data do not excludethe possible existence of Tc52 homologues in the otherparasites of Trypanosomatidae family, further enzy-matic assays with diVerent substrates, pHs, and complexreaction kinetics are needed to demonstrate the absenceor presence of “Tc52-like” protein with a thio:disulWdeoxydoreductase activity.

The sequence divergence in T. cruzi Tc52 from natu-ral clones aVects some putative residues important forGSH binding and or enzymatic function. However, theimmune recognition of the Tc52 target molecule washighly conserved among parasites clones. Although it istempting to speculate that the immune protectionobserved with the Tc52 native protein from the Y strainas well as the thioltransferase activity could be extrapo-lated to the other T. cruzi isolates, further investigationswill be needed to ascertain this point.

Acknowledgments

This work received Wnancial support from InstitutNational de la Santé et de la Recherche Médicale and

Institut de Recherche pour le Développement. F.T. is arecipient of a MRT fellowship. The authors are gratefulto Mr. Christian Barnabé for providing the T. cruziclones and helping in culture. We are grateful to Profes-sor Graham H. Coombs, Division of Infection andImmunity, University of Glasgow, UK, for providing usthe L. major TDR1 recombinant protein.

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