Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi

(1)

Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi

¹

Myle`ne Lese´ne´chal

^a

, Laurent Duret

^b

, Maria Isabel Cano

^c

, Renato A. Mortara

^c

, Michel Jolivet

^a

, Mario E. Camargo

^d

, Jose´ Franco da Silveira

^c

,

Gla´ucia Paranhos-Baccala`

^a,

*

aUnite´ Mixte CNRS-bioMe´rieux,Ecole Normale Supe´rieure de Lyon,46,alle´e d’Italie,69364Lyon Cedex07,France

bLaboratoire de Biome´trie(CNRS,URA2055),Villeurbanne,France

cDepartment of Parasitology,Escola Paulista de Medicina,Sa˜o Paulo,Brazil

dBiolab-Me´rieux,Sa˜o Paulo,Brazil

Received 8 January 1997; received in revised form 29 April 1997; accepted 30 April 1997

Abstract

ATrypanosoma cruzigenomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas’ disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously describedT.cruzisequences. In contrast with other T.cruziantigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain. © 1997 Elsevier Science B.V.

Keywords: Cloning; Immunodominant antigen;T.cruzi

Abbre6iations:GST, Glutathione-S-transferase; ORF, open reading frame; PCR, polymerase chain reaction; PFGE, pulsed field gel electrophoresis; RACE, rapid amplification of cDNAs ends; SL, spliced leader; UTR, untranslated region.

* Corresponding author. Tel.: +33 472728590; fax: +33 472728533; e-mail: Glaucia.Baccala@ens-bma.cnrs.fr

1 Note: Nucleotide Sequence data reported in this paper are available in the GenBank™ data base under the accession number U24190 and U96914.

PIIS 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 0 6 8 - 6

(2)

1. Introduction

Chagas’ disease is one of major causes of morbid- ity and death in Latin America where it has been estimated that 16 – 18 million people are chronically infected by the parasiteTrypanosoma cruzi[1]. No definitive immunological or chemotherapeutic treatment for this illness is available. The protozoan agent has a complex life cycle which involves different evolutive forms of the parasite in the mammalian host and the insect vector. During the acute and chronic stages of the infection, the parasite presents a large number of antigenic determinants to the mammalian immune system [2 – 5].

The humoral and cellular immune response mounted by the vertebrate host is able to keep the parasitemia at low levels and prevent the reagudiza- tion after a new infection but it is unable to eradicate the parasite [6]. To better understand the biology of the T. cruzi and more importantly for its diagnosis and immunoprophylaxis, the identification and characterization of parasitic antigens is of main relevance. SeveralT.cruziantigens have been identified by the screening of genomic and cDNA expression libraries with sera from infected humans and animals and have been used for serodiagnostic purposes [7 – 14]. Most of the cloned antigens isolated with human chagasic sera are made up of tandemly arranged amino acid repeats that may be immunodominant in natural infections [4,8].

Here we describe the molecular cloning and characterization of a gene, named Tc40, that encodes a novel immunodominant T. cruzi antigen present in all developmental stages of the parasite.

We have found that gene Tc40 does not show any tandemly repeated sequence and is present in a few copies in the genome. The recombinant antigen reacted specifically with antibodies from chronic chagasic patients, in immunoblotting analysis, suggesting that it could be useful in the serodiagnosis of Chagas’ disease.

2. Materials and methods

2.1. Parasites and bacterial strains

Trypanosoma cruziepimastigotes and metacyclic

trypomastigotes were grown in liver infusion tryp- tose (LIT) liquid medium supplemented with 10%

heat-inactivated fetal calf serum at 28°C without shaking. Cell culture trypomastigotes and amastigotes were obtained from infected monolayers of Vero and HeLa cells.

E. coli DH5a(Gibco BRL, France) strain was used for cloning and also for expression of fusion protein in plasmid pGEX (Pharmacia Biotech, France) whereas Y1090 strain was used for growth and expression of phagelgt11. Phagelgt10 (Amer- sham, France) was grown in E.coliY1089 strain.

2.2. Nucleic acid isolation, DNA libraries construction and screening

DNA and RNA were isolated from T. cruzi axenic cultures by conventional protocols as reported elsewhere [15].

The recombinant clone Tc40 was isolated from a T. cruzi (G strain) genomic expression library constructed in vector lgt11 [16]. The library was screened with a pool of chronic chagasic sera essentially as described by Ozaki et al. [17]. The insert from the purified original DNA clone was subcloned into pUC19 plasmid (Gibco BRL) for sequence analysis, and into pGEX expression plasmid to produce a fusion protein with the Schisto- soma japonicum glutathione S-transferase (GST- Tc40) induced by isopropyl-b-D-thiogalactopyra- noside [18].

2.3. Sera and antibodies

Sera samples were collected from patients with chronic Chagas’ disease diagnosed by serological methods and clinical symptoms. Serological analyses were performed by indirect immunofluorescence (ImunoCruzi, Biolab-Me´rieux, Brazil) and ELISA (BioELISAcruzi, Biolab-Me´rieux, Brazil). Anti- bodies against the GST protein and the GST-Tc40 fusion protein were raised in guinea pigs by inject- ing the partially purified non recombinant and recombinant antigens extracted from polyacrylamide gels.

(3)

2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting

The GST and GST-Tc40 proteins were induced and purified onto Glutathione Sepharose 4B (Pharmacia LKB Biotechnology, France) as described above [18] in the presence of 1 mM phenylmethylsulfonyl fluoride.

Parasites were lysed in presence of a solution containing 150 mM NaCl/10 mM Tris – HCl, pH 7.5/1 mM EDTA/1% Nonidet-P40/1 mM phenylmethylsulfonyl fluoride/1 mMN-a-Tosyl-^L-lysine- chloromethyl ketone/2 U ml⁻¹ aprotinin/25 mg ml⁻¹leupeptin/25 mg ml⁻¹ antipain (Boehringer Mannheim, France). The parasite lysate was incubated at 4°C for 30 min, centrifuged for 10 min at 18 000×g at 4°C. This supernatant was used directly in the immunoblotting study, or was al- ternatively ultracentrifuged for 16 h at 4°C at 100 000×g and the pellet and supernatant were collected for electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with parasites and bacterial lysates in 10 or 12%

gels in the Laemmli [19] buffer system. Im- munoblotting was carried out by the method of Towbin et al. [20]. Bound antibodies were re- vealed either with goat anti-human IgG coupled to alkaline phosphatase (Jackson ImmunoRe- search Laboratories) or with [¹²⁵I] protein A (Amersham, France).

2.5. Immunofluorescence

For intracellular staining of parasites, Vero cells grown on glass cover slips were infected with T. cruzi. The immunofluorescence reaction was carried out at 72 – 96 h postinfection, as previously described using GST-Tc40 specific antibodies raised in guinea pig [21].

2.6. Cloning of the full-length Tc40 cDNA To clone the Tc40 cDNA, T. cruzi mRNA, G strain, was first reverse transcribed using random hexanucleotide primers, and then amplified by polymerase chain reaction (PCR). The 5% portion was generated using a sense primer-SL (5%- AACGCTATTATTAGAACAGTT-3%) deduced

from T. cruzi spliced leader (SL) sequence [22], and a reverse primer-1 (5%-TGCAGCAGCGGCA- GAAGT-3%) from Tc40 original sequence corresponding to nucleotides 1442 – 1459 of the Tc40 cDNA. The central region was obtained using a sense primer-2 (5%-CAGCCGACGGT- AGCTGCGTCCT-3%) and an anti-sense primer-3 (5%-ACATAATGGCCTCGTTCACAC-3%) from Tc40 original sequence, corresponding to nucleotides 1266 – 1287 and 2187 – 2207 of the Tc40 cDNA, respectively.

Finally, to clone the 3% Tc40 cDNA ends, T.

cruzi polyadenylated RNA, G strain, was con- verted into single-stranded cDNA according to the 3%rapid amplification of cDNAs ends (RACE) protocol [23], using a hybrid (dT)₁₇-adapter primer [(dT)₁₇-AD]. The cDNA was amplified by PCR using as gene-specific sense primer-4 (5%-CGAAGAGACCATGAACAACTT-3%) corresponding to nucleotide positions 1997 – 2017 of Tc40 cDNA, and the adapter primer-AD. The sequences of primers numbers 3 and 4 were obtained from a specific Tc40 clone isolated from a 3.7-kb EcoRI lgt10 genomic T. cruzi library which hybridized with Tc40 original insert.

The PCR experiments were performed for 35 cycles of 1 min at 94°C, 1 min at 50°C, 1 min at 72°C followed by extension of 72°C for 7 min, using 50 pmol of each primer and 100 ng T.cruzi single strand cDNA. The Taq polymerase used was obtained from Perkin Elmer Cetus, France.

The PCR products that hybridized to the Tc40 original clones were cloned into pCRII vector using TA cloning kit for PCR products (InVitro- gen, San Diego, CA) and sequenced.

2.7. In 6itro translation of Tc40cDNA

The Tc40 complete open reading frame (ORF) was obtained after assembling the 5%, central and 3%regions by PCR, as described above. The sense primer sequence included the Tc40 ‘Kozak’ consensus and the AUG start, while the anti-sense primer contained the three consecutive Tc40 stop codons. The PCR product was cloned into the pSP64 Poly(A) vector (Promega, Madison, WI), downstream the SP6 RNA polymerase promotor and was in vitro transcribed and translated by

(4)

using the TNT Coupled Transcription/Transla- tion System (Rabbit Reticulocyte Lysate, Promega). The translation ³⁵S-labeled products were separated by SDS-PAGE as described elsewhere [19].

2.8. Sequencing

The nucleotide sequence of the plasmid inserts was determined by double stranded sequencing according to the dideoxynucleotide chain termina- tion method [24], using Sequenase (Amersham, US Biochemical, France). The Tc40 sequence analysis was carried out using the MacVector 4.5 software (Kodak). The sequence was also com- pared, both at the nucleotide and at the protein level, to all major sequence databases (GenBank, EMBL, PIR, SWISS-PROT, Dbest), using BLAST similarity search softwares (BLASTN, BLASTP, TBLASTN) [25]. The databases were provided by the NCBI GENINFO(R) Experimen- tal BLAST Network Service.

2.9. Northern and Southern blot analyses

DNA restriction fragments were radiolabeled with [a-³²P]dATP using a random primer DNA labelling kit (Boehringer Mannheim). Northern and Southern blots were prepared using standard methods [15]. Hybridizations, either for DNA or RNA analysis were performed overnight at 42°C in 6×SSC (1×SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.5)/5×Denhardt’s solution (1×Denhardt’s solution: Ficoll 0.2 mg ml ⁻¹/ polyvinylpyrrolidone 0.2 mg ml ⁻¹/bovine serum albumine 0.2 mg ml⁻¹)/50% formamide/0.5%

SDS/100 mg ml⁻¹sonicated herring sperm DNA.

After hybridization with Tc40 probe, filters were washed in 2×SSC/0.1% SDS at room tempera- ture for 15 min, then in 0.1×SSC/0.5% SDS at 37°C, for 30 min and, finally at 65°C, for 30 min.

2.10.Pulsed field gel electrophoresis

The pulsed field gel electrophoresis (PFGE) samples were obtained as described [26]. Agarose blocks containing 10⁸ epimastigotes CL and G strains were prepared and stored in 0.5 M EDTA,

pH 9.0. The equivalent of 10⁷ parasites were electrophoresed at 80 V for 132 h at 13°C, with pulse times varying from 90 to 800 s. DNA chromosomal bands were transferred to nylon filter, and the blot hybridized and washed as already described.

3. Results and discussion

3.1. Isolation and characterization of the Tc40 recombinant antigen

An expression library inlgt11 vector was made directly from randomly generated fragments ofT.

cruzi nuclear DNA [16]. Approximately 50 000 recombinant phages were screened with a pool of chronic chagasic sera and forty phages expressing T. cruzi antigens were detected and purified.

Based on the signal intensity, clone Tc40 (594-bp) was chosen for further characterization. The Tc40 insert was subcloned into the expression vector pGEX in order to produce high amounts of the GST fusion protein in bacterial cultures induced with isopropyl-thiogalactoside. The reactivity of the fusion protein was analyzed by immunoblot assay using a pool of human chronic chagasic sera. As shown in Fig. 1A, clone Tc40 encodes a GST fusion protein of approximately 48 kDa (lane 1) which strongly reacted with the antibodies from chronic chagasic patients. Some proteolysis products could be observed in this antigen prepa- ration, even in the presence of a serine protease inhibitor. In the same experiment, the non recombinant GST protein failed to react with the human chagasic sera (Fig. 1A, lane 2), showing the specificity of the GST-Tc40 recombinant protein.

To identify T. cruzi native protein that share common antigenic determinants with the product of clone Tc40, immunoblots carrying T. cruzi epimastigote lysates were probed with a guinea pig monospecific antiserum against Tc40 recombinant protein (Fig. 1B). The monospecific antiserum reacted with three polypeptides of molecular masses 100, 41 and 38 kDa (Fig. 1B, lane 3). These polypeptides were also detected in comparable levels in all developmental stages of the parasite (data not shown). In contrast, anti-

(5)

Fig. 1. Characterization of Tc40 antigen. (A) Reactivity of the recombinant protein GST-Tc40 with human chagasic sera.

GST-affinity purified proteins after IPTG induction of recombinant pGEX-Tc40 (1) and nonrecombinant pGEX (2) were separated by 12% SDS-PAGE, Western blotted and probed with a pool of human chronic chagasic sera. Size markers are shown on the left.

(B) Identification ofT.cruzinative antigen related to Tc40 fusion protein. Western blot of epimastigote lysates ofT.cruzi(G strain) was incubated with the following guinea pig antisera: anti-GST serum (1), preimmune serum (2) and anti-GST-Tc40 serum (3). The arrows indicate the polypeptides specifically recognised by the anti-GST-Tc40 serum. Size markers are shown on the left. (C) Immunofluorescence localization of Tc40 epitopes in intracellularT.cruziforms. Infected Vero cells were labelled with the guinea pig anti-GST-Tc40 serum. (A, B) fluorescence and phase contrast of intracellular amastigotes; (C, D) fluorescence and phase contrast of cell-culture trypomastigotes. The arrows indicate the nucleus of Vero cells (N), the nucleus (n) and kinetoplast (k) ofT.cruzi(Bar size, 4mm).

GST control serum such as antibodies from preimmune animal failed to react with T. cruzi polypeptides (Fig. 1B, lanes 1 and 2). This result shows that the antigens recognised by the anti- GST-Tc40 serum are mostly related to Tc40.

The three polypeptides were detected by anti- GST-Tc40 antibodies even when the parasites were lysed in the presence of a mixture of protease inhibitors (serine-, cysteine- and metallo- proteinases), which is normally used to protectT.

(6)

cruzi peptides against the activity of endogenous proteases [27]. It is noteworthy that the anti-GST- Tc40 antibodies reacted with almost the same intensity with the peptides of 100 and 41 kDa (Fig. 1B, lane 3), suggesting that they are present in equivalent amounts in the cell. Available data suggest that the peptides of molecular masses 100, 41 and 38 kDa represent different molecular enti- ties that share common epitopes. However, further studies using other techniques such as Cleveland mapping would be required to confirm this hypothesis.

The cellular location of Tc40 antigen was inves- tigated by indirect immunofluoresence using formaldehyde-fixed parasites (Fig. 1C). The anti- Tc40 antibodies stained the cytoplasm of amastigotes and trypomastigotes. No reaction was obtained with the nucleus or the kinetoplast. Hu- man chagasic antibodies immunopurified on the recombinant antigen also stained the cytoplasm of T.cruzi cells giving a fluorescence pattern similar to that obtained with the guinea pig monospecific antiserum (data not shown). As expected, the anti-GST control serum did not react with T.

cruzi intact cells (amastigotes and trypomastigotes) in indirect immunofluorescence assays (data not shown). Living parasites were not labeled with anti-Tc40 antibodies suggesting that proteins recognised by these antibodies are located in intracellular structures.

3.2. Cloning and sequencing of the Tc40 gene Northern blots carrying mRNAs from epimastigote stage of G and CL strains were hybridized with the insert of Tc40 genomic clone (Fig. 2A). The probe hybridized with a transcript of about 3.9 kb in both strains. No additional bands were observed even after a longer exposure

Fig. 2. Expression of the Tc40 gene. (A) Steady-state levels of Tc40 transcripts in different T. cruzi strains. Northern blot carrying 10 mg of RNA extracted from epimastigotes of G and CL strains was probed with the radiolabelled 594-bp Tc40 fragment. Size markers are noted on the left. (B) In vitro transcription and translation of Tc40 cDNA. The in vitro translation labelled products of the Tc40 cDNA, cloned into pSP64 poly(A) vector downstream SP6 promotor were analysed by SDS-PAGE (1), Western blotted and probed with a guinea pig anti-GST serum (2) and the guinea pig anti-GST- Tc40 serum (3). Size markers, shown on the left, are in kilodaltons (kDa).

(10 days). These results indicated that the length of the gene Tc40 was at least of 3.9 kb.

In an attempt to define the entire transcribed region of Tc40 gene we have cloned three overlapping cDNA fragments corresponding to the 5%and 3%regions of Tc40 gene. The cloning strategy was based on the PCR amplification using a combination of specific lgt11-Tc40 (596-bp) and T. cruzi sequences as primers (Fig. 3B). The 5% region of gene Tc40 was amplified using a pair of primers derived from SL sequence [22] (sense) and the 594-bp genomic sequence (anti-sense) (Fig. 3A

Fig. 3. Cloning strategy, nucleotide and predicted amino acid sequences of Tc40 cDNA. (A) Schematic representation of the Tc40 lgt11 andlgt10 genomic clones. The 594-bp and 3.7-kb EcoRI Tc40 clones are represented by a striped box and a bold line, respectively. (B) Cloning strategy to obtain the Tc40 cDNA. The three overlapping fragments corresponding to the 5%, central and 3%regions of Tc40 cDNA are illustrated by dotted boxes. The arrows noted SL, 1, 2, 3, 4, AD indicate the position of PCR primers used for amplification. The resulting full-length Tc40 cDNA is represented by a white box, in which black block corresponds to the SL sequence. (C) Nucleotide and deduced amino acid sequences of Tc40 cDNA. The astericks at the end of the amino acid sequence show stop codons. The sequence of the lgt11-derived Tc40 clone is underlined. The two stretches of adenine segments and the ‘T- and GT- rich’ regions are double-underlined. The complete consensus SL is dotted-underlined.

(7)

(8)

Fig. 4. Tc40 gene structure. (A) Southern blot analysis of the Tc40 gene. 5 mg of G (1), Y (2), CL (3), Dm30 (4)T.cruziDNA and Leishmania mexicana amazonica (5) DNA were digested with the corresponding restriction enzymes and analysed by Southern blotting with the radiolabelled 594-bp Tc40 insert as a probe. Size markers are shown on the left. (B) Southern blot analysis of Tc40 gene in the G strain. Five milligrams ofT.cruziDNA were restricted with (1)HaeIII, (2)EcoRI/HaeIII, (3)EcoRI, (4)EcoRI/PstI, (5)PstI, (6)P6uII, (7)SacI, (8)P6uII/SacI and subjected to Southern blot analysis using the 594-bp Tc40 probe. (C) Southern hybridization analysis showing chromosomal locations of Tc40 gene inT.cruziG and CL strains. Chromosomal bands of G and CL epimastigotes were separated by PFGE and analysed by Southern blot hybridization with 594-bp Tc40 insert as probe.

Molecular weights of the labelled chromosomes, in megabase pairs, corresponding to G and CL strains are noted on the left and right of gel, respectively.

and 3B). The amplified cDNA fragment was 1459- bp long carrying the complete 5% region of Tc40 gene including the SL sequence. The central region of the Tc40 gene encompassing the 594-bp genomic sequence was amplified using a pair of Tc40 specific primers.

To obtain more information on the organization of gene Tc40, we have hybridized the 594- bp fragment with a genomic Southern blot carrying T. cruzi DNA digested with several restriction enzymes (see Fig. 4A and 4B). A con- served EcoRI fragment of 3.7-kb was detected in all strains tested. For this reason, a T. cruzi genomic library was constructed in lgt10 using 3.7 kb fragments obtained by complete digestion with EcoRI of T. cruzi (G strain) DNA and a recombinant phage carrying the expected 3.7-kb EcoRI genomic fragment was isolated after hy- bridization with the 594-bp clone. The region

within the lgt10 recombinant clone homologous to the 594-bp sequence was identified and sequenced using specific primers. The 3% end of the 3.7-kb EcoRI fragment was also sequenced allowing us to design primers used in the isolation of the 3% end of transcribed region (Fig. 3A and 3B). The 3% end of Tc40 gene was amplified by 3% RACE method with a hybrid d(T)17-adap- tor primer and a sense primer from an internal sequence of the 3.7-kb EcoRI genomic frag- ment. The generated product was 1405-bp long and presented a tail of 18 adenine residues which could correspond to the poly(A) tail.

Since the Northern blot suggests that the Tc40 mRNA is 3.9 kb (Fig. 2A) and the composite cDNA encompasses 3.4 kb (Fig. 3C), it is possi- ble that the 3% RACE had identified an A-rich region in the 3% untranslated region (UTR) and not identified the true poly(A) tail at the end of

(9)

the mRNA that actually could have another 0.5 kb at its 3% UTR.

The complete nucleotide sequence and the predicted amino acids of the Tc40 cDNA are dis- played in Fig. 3C. The Tc40 cDNA is 3402 bp long with an ORF of 2745 bp encoding a polypeptide of 915 amino acids with a predicted molecular mass of 100 kDa. This ORF matched with the 596 bp original insert of Tc40. It is interesting to note that no internal repeat was found in this gene, in contrast to most of cloned genes encoding T. cruzi antigens [4,8].

The first start codon, situated 266-bp downstream of the SL sequence, complied with the nucleotide sequence frequency flanking protozoan start codons [28], particularly regarding the purine at position −3. The sequence upstream of the start site contains stop codons in all three frames.

At the 3%end, three consecutive stop codons were detected 390-bp upstream of the putative poly(A) tail.

Several features could be observed in the rela- tively large untranslated regions. The 5% UTR of Tc40 presented two stretches of 11 and 13 adenine contiguous segments. Such an A-rich segment has been described in theT.cruzi PABP1 cDNAs [29]

and is thought to be, in eukaryotic organisms, involved in the autoregulation of the synthesis of these poly(A) binding protein. However, since no similarity was found between Tc40 and the PABP1 genes, we suggest that such segments could be involved in the stabilization of these and other related transcripts. The 3% Tc40 UTR contained an extensive GT-rich region at 100-bp upstream of the poly(A) tail. For instance, the dimer GT is repeated 25 times in the 3% UTR. T- and GT-rich regions are not uncommon in T. cruzi, since several authors have recently reported that similar sequences are present in the 3% UTR of many T. cruzi mRNAs [30 – 32].

As shown above the 3.7 kb-EcoRI genomic fragment cloned in lgt10 carries the 5% flanking region of the Tc40 gene (Fig. 3A). To look for putative promoter and regulatory sequences, we have sequenced a 5%region immediately upstream from the Tc40 ORF. The 1006-bp 5% flanking sequence was analyzed with the program Proscan

(version 1.7) [33] to search for potential RNA polymerase II promoter sequences. This program identified a potential TATA-box containing promoter, with a putative transcription start site at position 609. The promotor region was predicted between position 350 and 600 (position in the GeneBank accession number U96914).

The Tc40 polypeptide presents no significant similarity with other published sequences as indicated by search conducted in all major sequence databases. From the predicted amino acid sequence, in addition to 13 sites for N-glycosyla- tion, there are 17 potential sites for myristyllation.

The accessibility profile for the deduced amino acid sequence of Tc40, calculated according to the method of Parker [34], predicted two putative antigenic domains. The first one, located in the stretch of amino acids 357 – 368, is in agreement with the available experimental data, while a sec- ond one in the stretch of amino acids 594 – 622 was not analyzed in the present study. Finally, we failed to find any potential signal peptide nor sequences able to confer a transmembrane domain or potential glycophosphatidylinositol an- chor signal. These data are in agreement with an intracellular location of Tc40 protein.

3.3. Transcription and genomic organization of Tc40 gene.

Northern blot analysis showed that the insert of the Tc40 genomic clone strongly hybridized to a transcript of 3.9 kb which is large enough to encode a poypeptide of 100 000 da (Fig. 2A). To further confirm the size of the Tc40 protein, full length Tc40 mRNA was transcribed and translated in vitro. Two³⁵S-labeled polypeptides of 100 and 80 kDa were detected (Fig. 2B, lane 1), but only the 100 kDa peptide was recognised by anti- GST-Tc40 antiserum (Fig. 2B, lane 3). As expected, these polypeptides did not react with the anti-GST control serum (Fig. 2B, lane 2). Thus, the size of the protein translated in vitro corre- lated with that of the native protein found in T.

cruzi extracts and the protein encoded by the ORF shown in Fig. 3. Clearly, our results indicate that the 41 and 38 kDa peptides, also found inT.

cruzi extracts, share common epitopes with the 100 kDa protein.

(10)

The insert of the Tc40 genomic clone was hybridized with Southern blots carrying genomic DNAs from T. cruzi (G, CL, Y and DM30 strains) and Leishmania mexicana amazonensis (Fig. 4A). The probe hybridized with allT. cruzi DNA tested but not with LeishmaniaDNA, suggesting that it carries T. cruzi species-specific sequences. When T. cruzi DNA, G strain, was digested with several restriction enzymes and probed with Tc40 genomic clone (Fig. 4B), we observed a very simple pattern which is consistent with the presence of a few copies of Tc40 genes in the parasite genome.

Chromosomal mapping of Tc40 genes was carried out hybridizing the 594-bp genomic fragment with the chromosomes separated by pulsed field gel electrophoresis. Fig. 4C shows the occurrence of only one hybridizing band of 1.1 Mbp in the G strain, in agreement with the above results, while the same probe hybridized with two chromosomal bands of 0.80 and 0.70 Mbp in the CL strain.

These results suggested the existence of two allelic forms of Tc40 gene, chromosome III and IV, in the CL Brener strain [26].

3.4. Antigenic rele6ance of Tc40 recombinant antigen.

The antigenic relevance of GST-Tc40 recombinant antigen was assessed by the immunoblot assay with a large panel of human serum samples from chronic chagasic patient (n=201), non chagasic patients (leishmaniasis, toxoplasmosis, filariasis, leprosy, mononucleosis, rheumatoid arthritis, autoimmune diseases) (n=67) and healthy individuals (n=36). The Tc40 fusion protein reacted with 92% of the serum samples from chronic chagasic patients. Out of 103 non chagasic sera tested, only one serum sample gave reaction with Tc40 antigen (Table 1). These results suggest that the presence of serum antibodies to the Tc40 antigen could be specifically associated with Chagas’ disease.

Our data demonstrate that the gene structure for Tc40 protein does not carry repetitive amino acids motifs found in the majority of recombinant antigens isolated by screening of T. cruzi expression libraries with human chagasic sera. In this

context it is interesting to note that other T.cruzi proteins (ribosomal P proteins), which do not present repetitive motifs, are also antigenic in Chagas’ disease [35].

Natural humoral immune responses to manyT.

cruzi antigens appear to be largely directed to epitopes encoded by the repeat units. The strength of signals given by chronic chagasic sera in West- ern blot immunoassays on Tc40 recombinant protein indicates that antibodies against non repetitive antigens are also present in the chronic phase of Chagas’ disease. High antibody titers against repetitive amino acid motifs in the great majority of individuals living in endemic areas appear inconsistent with repetitive epitopes being the target of host-protective immune responses.

An important question to be answered is the extend to which immune response against these repeating antigenic epitopes is host protective. It

Table 1

Reactivity of chagasic and non-chagasic serum samples with Tc40 recombinant antigen (GST-Tc40) in Immunoblot assay^a

Patients’ disease No. of individ- No. of positive individuals uals assayed

Chagas’disease

9

Congenital 8

Chronic

68 74

Chronic cardiopathy

5 Digestive and cardiac/ 6

digestive forms

Indeterminate form 112 103

Other diseases

7

Toxoplasmosis 0

28

Kala-azar 1

6

Mucosal-Leishmaniasis 0

8

Filariasis 0

0 3

Leprosy

5 0

Mononucleosis

0 5

Rheumatoid Arthritis

Autoimmune disease 5 0

36 0

Normal

aStrips containing 1.25mg of purified GST-Tc40 recombinant antigen were probed with 1:100 diluted sera from patients with Chagas’ disease or with other diseases. Chagasic sera represent populations from different countries of Latin America. The Immunoblot interpretation was based on the presence or absence of the specific GST-Tc40 bands onto the strips (Fig.

1A). Positive and Negative controls were included in each experiment done.

(11)

has been suggested that it is counterproductive because it could subvert the host immune response to more critical determinants on different molecules. For this reason, it is of great interest to study the immune response against non repetitive antigens like Tc40. In our laboratory, we are mapping T- and B-cell epitopes present in Tc40 antigen using epitope libraries, overlapping amino acid sequences by gene cloning and chemical peptide synthesis.

Recently, several studies have been demon- strated thatT. cruzi recombinant antigens can be potentially used in the serological diagnosis of Chagas’ disease [7 – 9,13,36]. Tc40 recombinant peptide was used in immunoblot assays to screen standard sera classified as chagasic and non chagasic based on conventional serological tests.

The sensitivity (92%) and specificity (99%) of Tc40 antigen are comparable with that others T.

cruzirecombinant antigens carrying repetitive epitopes. The available recombinant antigens react with about 95% of the chronic chagasic sera indi- cating that additional antigenic determinants may be required to develop a truly reliable T. cruzi serodiagnostic test based on recombinant antigens. Reliability may also be increased by the combination of two or more antigens to build up a multi-antigen immunoassay. The repertoire of B-cell epitopes in a non repetitive antigen is more large than that found in the repetitive antigens. In order to improve the specificity of serodiagnostic tests, we believe that the non repetitive antigens such as Tc40 and 1F8 [14,37] should be included in the multi-antigen immunoassays.

Acknowledgements

This work was funded by bioMe´rieux. Myle`ne Lese´ne´chal is a doctoral fellow from the MRE, French Government. We thank Dr P. Dalbon and Dr P. Pion, bioMe´rieux, for oligonucleotide synthesis and for production of the polyclonal guinea pig sera. We also thank Dr P. Minoprio, Pasteur Institut, Paris (France), for permitting access toT.

cruziCL strain. We are grateful to Dr R. Baccala`, CHUV, Lausanne (Switzerland) and to Dr V.

Atrache, bioMe´rieux (France), for critical reading of the manuscript.

References

[1] WHO, Control of Chagas’ disease, Report of a WHO Expert Committee. Geneva-WHO Technical Report Se- ries. 811, 1991, 27 – 31.

[2] Franco da Silveira J, Paranhos GS, Cotrim PC, Mortara RA, Camargo ME, Rassi A, Wanderley J, Corral R, Freilij HL, Grinstein S, Degrave W. Antigens of Try- panosoma cruziwith clinical interest cloned and expressed inEscherichia coli. Mem Inst Oswaldo Cruz 1990;85:507 – 11.

[3] Reyes MB, Lorca M, Mun˜oz P, Frasch ACC. Fetal IgG specificities againstTrypanosoma cruziantigens in infected newborns. Proc Natl Acad Sci USA 1990;87:2846 – 50.

[4] Frasch ACC, Cazzulo JJ, A˚ slund L, Pettersson U. Com- parison of genes encoding Trypanosoma cruzi antigens.

Parasitol Today 1991;7:148 – 51.

[5] Krautz GM, Galva˜o LMC, Canc¸ado JR, Guevara-Es- pinoza A, Ouaissi A, Krettli AU. Use of a 24-Kilodalton Trypanosoma cruzirecombinant protein to monitor cure of human Chagas’ disease. J Clin Microbiol 1995;33:2086 – 90.

[6] Brener Z. Immune response and immunopathology in Trypanosoma cruzi infection. In: Wendel S, Brener Z, Camargo ME, Rassi A, editors. Chagas’ disease (Ameri- can Trypanosomiasis): its impact on transfusion and clinical medicine, International Society of Blood Transfusion.

1992:31 – 47.

[7] Levin MJ, Franco Da Silveira J, Frasch ACC, Camargo ME, Lafon S, Degrave WM, Rangel-Aldao R. Recombi- nant Trypanosoma cruzi antigens and Chagas’ disease diagnosis: analysis of a workshop. FEMS Microbiol Im- munol 1991;89:11 – 20.

[8] Franco da Silveira J. Trypanosoma cruzi recombinant antigens for serodiagnosis. In: Wendel S, Brener Z, Ca- margo ME, Rassi A, editors. Chagas’ disease (American Trypanosomiasis): its impact on transfusion and clinical medicine, International Society of Blood Transfusion, 1992:207 – 217.

[9] Krieger MA, Almeida E, Oelemann W, Lafaille JJ, Pereira JB, Krieger H, Carvalho MR, Goldenberg S. Use of the recombinant antigens for the accurate immunodi- agnosis of Chagas’ disease. Am J Trop Med Hyg 1992;46:427 – 34.

[10] Gruber A, Zingales B. Trypanosoma cruzi: characteriza- tion of two recombinant antigens with potential applica- tion in the diagnosis of Chagas’ disease. Exp Parasitol 1993;76:1 – 12.

[11] Carvalho MR, Krieger MA, Almeida E, Oelemann- Shikanai-Yassuda MAW, Ferreira AW, Pereira JB, Sa´ez- Alque´zar A, Dorlhiac-Llacer PE, Chamone DF, Goldenberg S. Chagas’ disease diagnosis: evaluation of

(12)

several tests in blood bank screening. Transfusion 1993;33:830 – 4.

[12] Paranhos-Bacalla GS, Santos MRM, Cotrim PC, Rassi A, Jolivet M, Camargo ME, Franco da Silveira J. Detec- tion of antibodies in sera from Chagas’ disease patients using a Trypanosoma cruzi immunodominant recombinant antigen. Parasite Immunol 1994;16:165 – 9.

[13] Pastini AC, Iglesias SR, Carricarte VC, Guerin ME, Sa´nchez DO, Frasch ACC. Immunoassay with recombinant Trypanosoma cruzi antigens potentially useful for screening donated blood and diagnosing Chagas’ disease.

Clin Chem 1994;40:1893 – 7.

[14] Godsel LM, Tibbetts RS, Olson CL, Chaudoir BM, Engman DM. Utility of recombinant flagellar calcium- binding protein for serodiagnosis of Trypanosoma cruzi infection. J Clin Microbiol 1995;33:2082 – 5.

[15] Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning.

A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

[16] Paranhos GS, Cotrim PC, Mortara RA, Rassi A, Corral R, Freilij HL, Grinstein S, Wanderley J, Camargo ME, Franco Da Silveira J. Trypanosoma cruzi: cloning and expression of an antigen recognised by acute and chronic human chagasic sera. Exp Parasitol 1990;71:284 – 93.

[17] Ozaki LS, Mattei D, Jendoubi M, Druihle P, Blisnick T, Guillote M, Puijalon O, Pereira da Silva L. Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera against Plasmodium falciparum antigens. J Immunol Methods 1986;89:213 – 9.

[18] Smith DB, Johnson KS. Single-step purification of polypeptides expressed inEscherichia colias fusions with glutathione-S-transferase. Gene 1988;67:31 – 40.

[19] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5.

[20] Towbin H, Staehelin T, Gordon J. Electrophoretic trans- fer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 1979;76:4350 – 4.

[21] Cotrim PC, Paranhos GS, Mortara RA, Wanderley J, Rassi A, Camargo ME, Franco da Silveira J. Expression in Escherichia coli of a dominant immunogen of Try- panosoma cruzirecognised by human chagasic sera. J Clin Microbiol 1990;28:519 – 24.

[22] Parsons M, Nelson RG, Watkins KP, Agabian N. Try- panosome mRNAs share a common 5% spliced leader sequence. Cell 1984;38:309 – 16.

[23] Frohman MA, Dush MK, Martin GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 1988;85:8998 – 9002.

[24] Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463 – 7.

[25] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ.

Basic local alignment search tool. J Mol Biol 1990;215:403 – 10.

[26] Cano MI, Gruber A, Vazquez M, Corte´s A, Levin MJ, Gonza´lez A, Degrave W, Rondinelli E, Zingales B, Ramirez JL, Alonso C, Requena JM, Franco da Silveira J. Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzigenome project. Mol Biochem Para- sitol 1995;71:273 – 8.

[27] Cotrim PC, Paranhos-Baccala GS, Santos MR, Mortensen C, Cano MI, Jolivet M, Camargo ME, Mor- tara RA, Franco da Silveira J. Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi. Mol Biochem Parasitol 1995;71:89 – 98.

[28] Cavener DR, Ray SC. Eukaryotic start and stop translation sites. Nucleic Acids Res 1991;19:3185 – 92.

[29] Batista JAN, Teixeira SMR, Donelson JE, Kirchhoff LV, Martins de Sa´ C. Characterization of a Trypanosoma cruzipoly(A)-binding protein and its genes. Mol Biochem Parasitol 1994;67:301 – 12.

[30] Araya JE, Cano MI, Yoshida N, Franco da Silveira J.

Cloning and characterization of a gene for the stage-specific 82-kDa surface antigen of metacyclic trypomastigotes of Trypanosoma cruzi. Mol Biochem Parasitol 1994;65:161 – 9.

[31] A˚ slund L, Carlsson L, Henriksson J, Ryda˚ker M, Toro GC, Galanti N, Pettersson U. A gene family encoding heterogeneous histone H1 proteins inTrypanosoma cruzi.

Mol Biochem Parasitol 1994;65:317 – 30.

[32] Franco FRS, Paranhos-Bacalla` GS, Yamauchi LM, Yoshida N, Franco da Silveira J. Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kDa surface antigen ofTrypanosoma cruzi metacyclic trypomastigotes. Infect Immun 1993;61:4196 – 201.

[33] Prestridge DS. Predicting Pol II promotor sequences using transcription factor binding sites. J Mol Biol 1995;249(5):923 – 32.

[34] Parker JMR, Guo D, Hodges RS. New hydrophilicity scale derived from high-performance liquid chromatogra- phy peptide retention data: correlation of predicted surface residues with antigenicity and X-ray derived accessible sites. Biochemistry 1986;25:5425 – 32.

[35] Levin MJ, Vasquez M, Kaplan D, Schijiman AG. The Trypanosoma cruziribosomal P protein family: Classifica- tion and antigenicity. Parasitol Today 1993;9:381 – 4.

[36] Moncayo A, Luquetti AO. Multicentre double blind study for evaluation of Trypanosoma cruzidefined antigens as diagnostic reagents. Mem Inst Oswaldo Cruz 1990;85:489 – 95.

[37] Engeman DM, Krause K-H, Blumin JH, Kim KS, Kirch- hoff LV, Donelson JE. A novel flagellar Ca²⁺ binding protein in trypanosomes. J Biol Chem 1989;264:18627 – 31.

.