Tc45, A DIMORPHIC TRYPANOSOMA CRUZI ... - CiteSeerX

4 downloads 76853 Views 413KB Size Report
asite DNA. Mol Biochem Parasitol 25: 175–184. 30. Young RA, Mehra V, Sweetser D, Buchanan T, Clark-Curtiss J,. Davis RW, Bloom BR, 1985. Genes for the ...
Am. J. Trop. Med. Hyg., 63(5, 6), 2000, pp. 306–312 Copyright 䉷 2000 by The American Society of Tropical Medicine and Hygiene

Tc45, A DIMORPHIC TRYPANOSOMA CRUZI IMMUNOGEN WITH VARIABLE CHROMOSOMAL LOCALIZATION, IS CALRETICULIN ´ N, LORENA FERREIRA, CLAUDIO PE ´ REZ, ALICIA COLOMBO, MARI´A C. MOLINA, JUAN CARLOS AGUILLO ¨ RN, ANNE WALLACE, ALDO SOLARI, PILAR CARVALLO, MARIO GALINDO, NORBEL GALANTI, ANDERS O ROSARIO BILLETTA, AND ARTURO FERREIRA Programa de Inmunologı´a, Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; Programa de Biologı´a Celular y Molecular, Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; Programa de Gene´tica Humana, Instituto de Ciencias Biome´dicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden

Abstract. We demonstrate that Tc45, a polypeptide described as an immunogenetically restricted Trypanosoma cruzi antigen in mice, is calreticulin, a dimorphic molecule encoded by genes with variable chromosomal distribution. Previously we showed that IgG from A.SW (H2s) mice immunized with T. cruzi trypomastigotes or epimastigotes and sera from infected humans recognize Tc45, a 45 kD parasite polypeptide. Herein we describe the cloning, sequencing, and expression of the Tc45 gene. A 98% homology in the deduced amino acid sequence was found with a T. cruzi calreticulin-like molecule and 41% with Leishmania donovani and human calreticulin. In the T. cruzi CL Brener clone and in the Tulahue´n strain, the gene is located in two and four chromosomes, respectively. Calreticulin was detected in several T. cruzi clones, in the Tulahue´n strain, and in T. rangeli, displaying alternative 43 and 46 kD forms. nized the Tc45 polypeptide.17,18 It remains to be determined whether in the murine model the relationship between resistance to acute infection and recognition of Tc45 is causal. In the context of the human infection, Tc45 is immunogenic in humans infected with T. cruzi. Thus, in immunometric assays 100% variable reactivity with Tc45 was detected in 69 human sera positive for the infection as determined by immunoradiometric and immunowesternblotting (IWB) assays.7 To contribute to the understanding of the immunogenic and diagnostic values of the T. cruzi antigen Tc45, we present data on the genetic cloning, sequencing, and expression of the encoding gene. We also show data on biochemical characterization of the native Tc45 protein and the chromosome localization of the coding gene. Based on the high homology between the cloned Tc45 DNA sequence reported here and that recently reported for a calreticulin-like molecule in T. cruzi22 and other calreticulin genes for other species, we propose that Tc45 is indeed T. cruzi calreticulin.

INTRODUCTION

Trypanosoma cruzi, an obligate intracellular parasite, is the causal agent of Chagas’ disease. Close to 20 million people are infected with the parasite in Latin America. To date, about 90 T. cruzi antigens have been biochemically characterized and about 30% of their coding genes have been cloned, sequenced, and expressed.1–6 Although important contributions have been made to better define their potential role as immunogens or diagnostic tools, no functional characterization has been defined for the majority of the described parasite molecules.1–13 The immunological functions of calreticulin in mammals have been delineated to some extent, in particular as a chaperone molecule in the process of antigen presentation to CD8⫹ T cells. Thus, when ␤2-microglobulin binds to the major histocompatibility complex (MHC) class I ␣ chain, the partially folded ␣␤2-microglobulin heterodimer dissociates from calnexin and binds calreticulin.14–16 The findings previously reported by this laboratory,7,17,18 have characterized the immunogenicity and diagnostic value of T. cruzi calreticulin, then known as Tc45. These results plus those reported here open a wide range of possibilities to study the role of this immunologically relevant molecule in parasite infectivity and its potential immunoprophylactic and immunodiagnostic value. Although the immune response to a variety of simple antigens is controlled by genes located in the H2 region,19,20 the response against complex organisms such as the intracellular parasite T. cruzi is probably controlled by multiple genes in the host genome, including the MHC. In the murine model, the A.SW (H2s) and A.CA (H2f) congenic strains differ in their sensitivity to an acute infection with blood trypomastigotes of the T. cruzi Tulahue´n strain.21 A.SW mice survive indefinitely, while A.CA mice die about 15 days post-infection during an acute parasitemia. Passive transfer of purified IgG from immune A.SW animals protects A.CA mice challenged with a lethal dose of parasites.21 Interestingly, IgG from immune A.SW but not A.CA mice recog-

MATERIALS AND METHODS

Parasites. Epimastigotes of cryopreserved T. cruzi clones NR cl3, Nicaragua, SC43 cl2, V86, Esmeraldo cl3, MN cl2, SO3 cl5, P11 cl3, and CL Brener, and also the LDG strain from T. rangeli, were grown in liquid medium.23 Cultured epimastigotes from the T. cruzi Tulahue´n strain were grown in liver infusion tryptose culture medium24 and harvested at the exponential growth phase. Parasite antigenic extracts. Parasites were subjected to sonication, 1 mM of N␣-p-tosyl-L-lysine chloromethyl ketone as a protease inhibitor was added, and the material centrifuged and stored at ⫺70⬚C.5 Monoclonal antibody E2G7 anti-Tc45. This monoclonal antibody (mAb) was generated according to standard procedures25 with minor modifications.7 A BALB/c adult female mouse was immunized with Tc45 purified by two independent FPLC chromatographic criteria, ionic exchange, and hydroxylapatite adsorption. This material was recognized by

306

TRYPANOSOMA CRUZI TC45 IS CALRETICULIN

307

FIGURE 1. Electrophoretic dimorphism of Tc45 in Trypanosoma cruzi and Trypanosoma rangeli. Half an hour (A) and one hour (B–C) runs, after the exit of the sodium dodecyl sulfate polyacrylamide gel electrophoretic front are shown. The proteins were transferred onto nitrocellulose filters and detection of Tc45 was accomplished with a mAb followed by a goat anti-mouse IgG, radiolabelled with I.125 (A–B) Tracks 1–6, 8–9: NR cl3, Nicaragua, SC43 cl2, V86, Esmeraldo cl3, MN cl2, SO3 cl5 and P11 cl3, all T. cruzi clones, respectively. Track 7: LDG T. rangeli strain. (C) Tracks 1–3: Esmeraldo cl3, Tulahue´ n T. cruzi strain and SC43 cl2, respectively.

the A.SW but not A.CA congenic strains.18 The BALB/c mouse received three Tc45 injections, 10 ␮g each, one week apart. The first one (40 ␮L, prepared with equal volumes of saline and Complete Freund Adjuvant) was done in the rear foot pads. The second and third immunizations were administered subcutaneously in the dorsal region and intravenously, respectively. After the second injection, the animal was bled and the presence of anti-Tc45 IgG was verified by IWB. Four days after the last immunization, splenocytes were fused with the BALB/c mouse myeloma subline P3/ x63.Ag8.653.26 Positive supernatants were monitored by immunoradiometric assay against purified Tc45 and developed with gamma chain-specific polyclonal antibodies. Recognition of Tc45 by positive supernatants was confirmed by IWB against rabbit and murine anti-Tc45 polyclonal antibodies. The mAb was purified from murine ascites by SepharoseProtein G (Sigma, St. Louis, MO).27 The maintenance and care of experimental animals complied with the National Institutes of Health guidelines for the humane use of laboratory animals. Electrophoretic dimorphism of Tc45. Protein extracts from T. cruzi and T. rangeli were separated in 10% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Proteins were transferred onto nitrocellulose filters and Tc45 was detected with the E2G7 mAb followed by an affinity purified and 125I-labelled heavychain specific anti-mouse goat IgG. Peptide microsequencing. Briefly, Tc45 was purified by two HPLC criteria, ionic exchange and hydroxylapatite adsorption. The positive fractions monitored by IWB were pooled for further analysis. Sequencing was performed as described.17,28 Electroblotted purified Tc45 was stained with Ponceau S, cut out from the nitrocellulose sheet, and destained. The nitrocellulose-bound polypeptide was enzymatically digested in situ with trypsin. Tc45 fragments were sep-

arated by reverse phase HPLC. Amino acid sequence analysis was performed on an automated Applied Biosystems model 477A sequencer equipped with on-line phenylthiohydantoin amino acid analyzer, model 120A. Genetic cloning and expression of Tc45. Isolation of the Tc45 gene was carried out by conventional immunoscreening of a genomic library from Miranda/76 T. cruzi29 constructed in the bacteriophage ␭gt-1130 using the E2G7 mAb. Briefly, fusion-protein expression by ␭gt-11-infected Escherichia coli Y-1090 was induced by incubation with nitrocellulose filters soaked in isopropyl thio-␤-D-galactoside (IPTG). Wild-␭gt-11 phage was used as a negative control. The filters were then blocked, incubated with the E2G7 mAb, washed and developed with ␥-chain specific affinitypurified and radiolabelled rabbit anti-mouse IgG. The filters were then subjected to radioautography. Positive phage plaques were subjected to six further immunoscreenings until 100% plaque positivity was obtained. Protein over-expression was achieved according to standard procedures.31 Sequencing of the Tc45 coding DNA. Sequencing was performed following PCR amplification of the Tc45 1.6 kb EcoRI insert from the ␭gt-11 isolated clone using forward (5⬘d-GGTGGCGACGACTCCTGGAGCCCG-3⬘) and reverse (5⬘d-TTGACACCAGACCAA-CTGGTAATG-3⬘) primers (Promega, Madison, WI) flanking the ␭gt-11 insertion site. Amplification products were resolved in low melting point agarose gels, purified (Wizard娂 PCR Preps DNA Purification System, Promega) and sequenced (ds DNA Cycle Sequencing System, Gibco, New York) according to manufacturer’s instructions.32 Internal primers were prepared based on the preliminary sequence until the full sequence of the 1.6 kb EcoRI fragment was obtained. Chromosome localization of the Tc45 gene. The Tc45 coding DNA sequence was amplified by PCR (using the primers described above) and a radiolabeled probe was ob-

308

´ N AND OTHERS AGUILLO

tained according to standard procedures.29 Tulahue´ n and CL Brener epimastigote chromosomes33 were separated by Pulse Field Gel Electrophoresis (PFGE) (CHEF DR II Bio-Rad) using standard procedures.34–36 The DNA was transferred onto nylon membranes. The Tc45 radioactive DNA probe was hybridized under high stringency conditions and the membrane was exposed to radioautography.34,36 Tc45 sequence analysis. Homologies with other described genes were determined by using the BLAST search algorithm at the NCBI Data Base.37 Signal peptide sequence, cleavage site, molecular weight, isoelectric point, and glycosylation sites were predicted using an artificial neuronal network algorithm available on-line at the Center for Biological Sequences Analysis.38 T cell epitopes were predicted using the BioInformatics and Molecular Analysis Section (BIMAS) Data Base.39 RESULTS

Electrophoretic dimorphism of Tc45. To determine the presence of Tc45 in several T. cruzi clones, the Tulahue´ n strain and T. rangeli, we used the E2G7 mAb as a probe in IWB. Results of three representative experiments are shown in Figures 1A–C. As shown in Figure 1A–B, a 46 kD form is present in the Nicaragua, Esmeraldo cl3, and SO3 cl5 clones from T. cruzi (Tracks 2, 5, and 8, respectively) and in the LDG strain of T. rangeli (Track 7). An alternative 43 kD electrophoretic form is present in T. cruzi clones NR cl3, SC43 cl2, V86, MN cl2, and P11 cl3 (Tracks 1, 3, 4, 6, and 9, respectively). This dimorphism is more evident in a second experiment where the electrophoresis was extended one hour after the front exit (Figure 1B). Tc45 from the Tulahue´ n strain of T. cruzi displays a 43 kD mobility as shown in a third experiment (Figure 1C; Track 2), where the Esmeraldo cl3 (46 kD form, Track 1) and SC43 cl2 (43 kD form, track 3) clones, were used as internal controls. Cloning, sequencing, and expression of Tc45. To search for structural and functional homologies of Tc45, we cloned its gene, starting from Miranda/76 epimastigote DNA. As shown in Figure 2, the bacterial lysate containing recombinant Tc45 was subjected to IWB and probed with the antiTc45 E2G7 mAb (in Tracks 3–4, three times more lysate was loaded). Recombinant Tc45 was detected mostly in its free form with a relative molecular mass around 45 kD (most likely because of spontaneous rupture of the peptide bond joining Tc45 with the fusion protein ␤-galactosidase) (Tracks 1 and 3). However, as expected, Tc45 was also detected bound to the fusion ␤-galactosidase protein (about 186 kD for the complex). The 35 kD band, better observed in Track 3, most likely corresponds to a fragment of Tc45, a finding consistent with the high susceptibility to proteolytic digestion (results not shown). As expected, the absence of IPTG prevented the expression of Tc45 (Tracks 2 and 4). The expression in E. coli confirms the molecular mass observed in the native protein. The deduced amino acidic sequence of Tc45 is shown in Figure 3. The ␭gt-11 isolated clone contained an insert of 1,681 bp with an open reading frame of 1,209 bp between nucleotides 2 and 1,210, encoding a 401 amino acid polypeptide. The predicted molecular weight and isoelectric point of the mature polypeptide are 44,506 and 4.80, re-

FIGURE 2. Expression of recombinant Tc45. The Tc45 DNA coding segment was expressed in Escherichia coli Y-1089, infected with the recombinant ␭gt-11 phage. The proteins were transferred onto nitrocellulose membranes and detection of Tc45 was accomplished with a mAb followed by a goat anti-mouse IgG, radiolabelled with I.125 Tracks 1 and 3: Tc45 fused to ␤-galactosidase (186 kD), free (45 kD) and partially degraded (35 kD). Tracks 2 and 4: results obtained in the absence of IPTG. In Tracks 3–4 three times more bacterial lysate was loaded than in Tracks 1–2.

spectively.38 A putative signal peptide sequence corresponding to residues 1–20 has been calculated.38 The sequence displays only one potential O-glycosylation site (amino acid 114).38 Interestingly, at the amino acid level, 98, 41, and 41% homologies exist with calreticulin from T. cruzi (Tulahue´ n strain),7 Leishmania donovani,40 and humans,41 respectively. Further analysis of the Tc45 sequence shows a 67% homology with the central region of human calreticulin. Moreover, in the sequence obtained in our laboratory, a full homology was observed with three biochemically microsenquenced peptides (KVYFHEEF, MEHWTTSK, CGGGYIK) obtained from native Tulahue´ n Tc45. These peptides correspond to positions 22–29, 31–39, and 104–110, respectively. Chromosome localization of the Tc45 gene. Since differential localization of genes and their subsequent expression may be related with different parasite stages or with infectivity, we attempted to determine the epimastigote chromosomal localization of the gene coding for Tc45/calreticulin using PFGE. As shown in Figure 4 (Tracks 7–8) for the CL Brener clone, the Tc45 gene is located on two different chromosomes (about 1,125 and 2,000 kbp), while in

TRYPANOSOMA CRUZI TC45 IS CALRETICULIN

309

FIGURE 3. Comparison of amino acid sequences of Tc45 with calreticulin from different origins. A) Tc45 from Trypanosoma cruzi, Miranda/ 76 clone; B) Calreticulin-like molecule from T. cruzi, Tulahue´ n strain; C) Calreticulin from Leishmania donovani and, D) Human calreticulin. Shaded squares correspond to full homologies with three microsequenced Tc45 peptides, encompassing 23 aa. The putative signal peptide spans amino acids 1–20. (The clone that generated this sequence did not contain the first five base pairs, a fact irrelevant for the purposes of this report). Dashes represent spaces introduced for sequence alignment. This sequence information is available from NCBI’s Gen Bank under accession number AF162779.

the Tulahue´ n strain (Tracks 9–10) four different chromosomes bear the relevant sequences (between 1,125–2,000 kbp, approximately). In both, it seems that a third and fifth chromosomes (about 1,700 and 2,400 kbp, respectively) carry a significantly lower number of Tc45 gene copies. All radioactive bands have a corresponding signal in the ethidium bromide-stained gel (Tracks 2–3 and 4–5, respectively). DISCUSSION

In contrast to the approach used to identify most of the Ags described to date in T. cruzi, Tc45 was first detected by utilizing its immunogenetic restriction in congenic mouse strains.17,18 A high affinity mAb (E2G7) against Tc45 allowed us to start the biochemical characterization of this polypeptide. Assays with potential diagnostic value for the human infection have also been developed.7 Although we have not characterized the biochemical or functional nature of the Tc45 dimorphism shown in Figure 1, we have determined that it is not altered by treatment with sodium periodate (results not shown). Additionally, since a single potential glycosylation site is present at the amino acid level, post-translational glycosylation seems an unlikely cause for the observed differences in apparent molecular weights among T. cruzi clones, strains, and species (Figure

1). Given the lack of intronic/exonic genomic organization in T. cruzi, alternative mRNA splicing may be ruled out as cause for the observed dimorphism. It remains to be determined if this dimorphism is also present in different stages of the parasite cycle. When the deduced amino acid sequence of Tc45 is compared with those of calreticulin from T. cruzi,22 L. donovani,40 and humans,41 important homologies are detected. Additionally, if we compare the sequence recently reported for a Tulahue´ n calreticulin-like molecule22 with that of the Miranda/76 clone we find discrepancies in only four amino acids (Arg61→Ser, Met129→Val, Asn382→Lys, and Asn388→Val). This is most likely due to strain/clone variations in the sequence of the calreticulin gene. Moreover, in order to confirm that the cloned gene corresponds to the native Tc45,17,18 three peptides spanning a total of 23 amino acids obtained from native Tc45 were biochemically microsequenced. Full homologies with the sequences deduced from the Tc45 gene were observed. Finally, Tc45 is about 50% homologous with RAL, a 41 kD calreticulin associated Ag from Litomosoides sigmodontes.42 Taken together these data support the conclusion that Tc45 is T. cruzi calreticulin. Although CD8⫹ T cells are crucial in parasite control and survival following infection, few studies have been performed to identify antigens recognized by cytotoxic T cells (CTLs) in T. cruzi.43–46 Contingent to its expression in amas-

310

´ N AND OTHERS AGUILLO

FIGURE 4. Molecular karyotype of Trypanosoma cruzi CL Brener and Tulahue´ n after PFGE. Ethidium bromide stained chromosomal patterns (Tracks 1–5) and Southern blot using a Tc45/calreticulin DNA probe (tracks 6–10). Tracks 2–3 and 4–5 correspond to the CL Brener and Tulahue´ n kariotypes, respectively. Tracks 7–8 and 9–10, correspond to the respective hybridization patterns. In Tracks 2–3 and 4–5; 20 ⫻ 106 and 30 ⫻ 106 parasites were loaded, respectively. Tracks 1 and 6 correspond to the Saccharomyces cerevisiae chromosomal marker. In Tracks 1–10, the top band corresponds to DNA that did not enter the gel.

tigotes, calreticulin could be a target for CTLs. In agreement with this possibility, HLA and H2 binding peptide motif searches identify in Tc45 several putative high-affinity T-cell epitopes (Figure 3). As an example, for the human HLA-A2 class I molecule, two such epitopes (positions 10–19 and 65–74), and five for the mouse H2Kd (positions: 5–13, 24– 32, 107–115, 232–240, and 317–325) were identified.39 Calreticulin has been defined as a high-capacity, calciumand peptide-binding heat-shock protein,47 interacting with peptides transported into the endoplasmic reticulum by molecules associated with antigen processing.48 However, its role in T. cruzi seems to be independent from calnexin (whose gene may be absent in T. cruzi genome) and may be associated with glycoprotein folding.22 Trypanosoma cruzi infection of macrophages seems to inhibit MHC class II49 but not class I presentation,45 however, the possibility that T. cruzi calreticulin is able to interact with mammalian class I MHC molecules, modulating the presentation of parasite peptides to CTLs, cannot be ruled out. In this regard, the presence of a signal peptide in the calreticulin sequence38 favors the possibility that this protein could access the endoplasmic reticulum of the mammalian host cells. In epimastigotes, most likely a diploid stage in the parasite cycle,2,35,45,50–52 the results shown in Figure 4 suggest the participation of one and two pairs of homologous chromosomes in the CL Brener clone and Tulahue´ n strain, respectively. An alternative explanation, valid for the non-cloned Tulahue´ n strain, is that the four relevant chromosomes correspond to the presence of at least two clones, each one providing one

pair of homologous chromosomes. The localization of the calreticulin gene in different chromosomes could be explained by gene transposition or by duplication and translocation. In both CL Brener and Tulahue´ n parasites, it seems that a third and fifth chromosome carry a significantly lower number of calreticulin gene copies or they may represent cross-hybridizing sequences. Perhaps T. cruzi calreticulin is not an exception in that the majority of genes described thus far have a variable number of tandemly repeated copies per chromosome.1,3,4,53 Additionally, it is worth noting that the differential expression of these genes could be related with different parasite stages and/or with differences in their infectivity. Acknowledgments: We thank Dr. Carlos Frasch for providing us with the Miranda/76 T. cruzi ␭gt-11 library, Dr. Ulf Hellman for the facilities and training to perform the microsequencing experiments, Dr. Antonio Morello for the cultured epimastigotes, Dr. Flavio Salazar for important advice in the preparation of this manuscript, and Dr. Viviana Ferreira for editorial suggestions. Financial support: This work was supported by Research Grants 1970878 from National Fund for Scientific and Technological Research (Chile), the Swedish Agency for Research Cooperation with Developing Countries (Sweden) and United Nations Educational Scientific and Cultural Organization/Latinoamerican Network of Immunological Laboratories. Authors’ addresses: Juan Carlos Aguillo´ n, Lorena Ferreira, Claudio Pe´ rez, Alicia Colombo, Marı´a Carmen Molina, Anne Wallace, Rosario Billetta, and Arturo Ferreira, Programa de Inmunologı´a. Aldo Solari, Mario Galindo, and Norbel Galanti, Programa de Biologı´a Celular y Molecular. Pilar Carvallo, Programa de Gene´ tica Humana.

TRYPANOSOMA CRUZI TC45 IS CALRETICULIN

Instituto de Ciencias Biome´ dicas (ICBM), Facultad de Medicina, Universidad de Chile. Casilla 13898, Correo 21, Santiago, Chile. FAX: 56-2-7353346. Email: [email protected]. Anders ¨ rn, Microbiology and Tumor Biology Center, Karolinska Institute, O Stockholm, Sweden.

17.

18. REFERENCES

1. Swindle J, Ajioka J, Eisen H, Sanwal B, Jacixuemot C, Browder C, Buck G, 1988. The genomic organization and transcription of the ubiquitin genes of Trypanosoma cruzi. EMBO J 7: 1121–1127. ˚ slund L, Frasch ACC, Pettersson 2. Campetella O, Henriksson J, A U, Cazzulo JJ, 1992. The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes. Mol Biochem Parasitol 50: 225–234. ˚ slund L, Porcel B, Segura EL, Henriks3. Bontempi EJ, Bua J, A ¨ rn A, Pettersson U, Ruiz AM, 1993. Isolation and son J, O characterization of a gene from Trypanosoma cruzi encoding a 46-kilodalton protein with homology to human and rat tyrosine aminotransferase. Mol. Biochem Parasitol 59: 253– 262. ˚ slund L, Carlsson L, Henriksson J, Rydaker M, Toro GC, Gal4. A anti N, Pettersson U, 1994. A gene family encoding heterogeneus histone H1 proteins in Trypanosoma cruzi. Mol Biochem Parasitol 65: 317–330. 5. Krautz GM, Peterson JD, Godsel LM, Krettli AU, Engman DM, 1998. Human antibody responses to Trypanosoma cruzi 70kD heat-shock proteins. Am J Trop Med Hyg 58: 137–143. 6. Kaplan D, Baldi C, Chiaramonte MG, Ferna´ ndez MM, Levin MJ, Malchiodi E, Baldi A, 1998. Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi. Biochem Biophys Res Commun 253: 53–58. 7. Aguillo´ n JC, Harris R, Molina MC, Colombo A, Cortes C, Her¨ rn A, Ferreira A, 1997. Recognition mosilla T, Carren˜ o P, O of an immunogenetically selected Trypanosoma cruzi antigen by seropositive chagasic human sera. Acta Tropica 63: 159– 166. 8. Gazzinelli RT, Galvao LMC, Krautz G, Lima APCA, Canc¸ ado JR, Scharfstein J, Kretti AU, 1993. Use of Trypanosoma cruzi purified glycoprotein GP57/51 or trypomastigote-shed antigens to assess cure for human Chagas’ disease. Am J Trop Med Hyg 49: 625–635. 9. Godsel LM, Tibbetts RS, Olson CL, Chaudoir BM, Engman DM, 1995. Utility of recombinant flagellar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection. J Clin Microbiol 33: 2082–2085. 10. Reyes MB, Lorca M, Mun˜ oz P, Frasch ACC, 1990. Fetal IgG specificities against Trypanosoma cruzi antigens in infected newborns. Proc Natl Acad Sci USA 87: 2846–2850. 11. Aznar C, Lopez-Bergami P, Brandariz S, Mariette C, Liegeard P, Alves MD, Barreiro EL, Carrasco R, Lafon S, Kaplan D, 1995. Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection. FEMS Immunol Med Microbiol 12: 231–238. 12. Umezawa ES, Shikanai-Yasuda MA, Gruber A, Pereira-Chioccola VL, Zingales B, 1996. Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas’ disease in Brazil (Catole do Rocha, Paraiba). Mem Inst Oswaldo Cruz 91: 87–93. 13. Alberti AE, Fachado Carvajales A, Montalvo AM, Izquierdo Perez LA, Fonte Galindo L, 1998. Cysteine-dependent protease in Trypanosoma cruzi useful for the diagnosis of Chagas’disease. Rev Cub Med Trop 50: 75–81. 14. Pamer E, Cresswell P, 1998. Mechanisms of MHC class I-restricted antigen processing. Ann Rev Immunol 16: 323–358. 15. Sandasivan B, Lehner PJ, Ortmann B, Spies T, Cresswell P, 1996. Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. Immunity 5: 103–114. 16. Yewdell JW, Bennink JR, 1999. Immunodominance in major

19.

20. 21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

311

histocompatibility complex class I-restricted T lymphocyte responses. Ann Rev Immunol 17: 51–88. Ramos R, Juri MA, Ramos A, Hoecker G, Lavandero S, Pen˜ a P, Morello A, Repetto Y, Aguillo´ n JC, Ferreira A, 1991. An immunogenetically defined and immunodominant Trypanosoma cruzi antigen. Am J Trop Med Hyg 44: 314–322. Aguillo´ n JC, Bustos C, Vallejos P, Hermosilla T, Morello A, ¨ rn A, Ferreira A, 1995. Purification Repetto Y, Hellman U, O and preliminary sequencing of Tc45, an immunodominant Trypanosoma cruzi antigen: Absence of homology with cruzipain, cruzain, and a 46-kilodalton protein. Am J Trop Med Hyg 53: 211–215. Lawson CM, O’Donoghue H, Bartholomaeus WN, Reed WD, 1991. Genetic control of mouse cytomegalovirus-induced myocarditis. Immunology 69: 20–26. Janeway C, 1991. Mls: makes a little sense. Nature 349: 459– 461. Juri MA, Ferreira A, Ramos A, Hoecker G, 1990. Non-lytic antibodies in H-2-controlled resistance to Trypanosoma cruzi. Bras J Med Biol Res 23: 685–695. Labriola C, Cazzulo JJ, Parodi AJ, 1999. Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins. Mol Biol Cell 10: 1381–1394. Tibayrenc M, Neubauer K, Barnabe C, Guerrini F, Skarecky D, Ayala F, 1993. Genetic characterization of six parasitic protozoa: parity between random-primer DNA typing and multilocus enzyme electrophoresis. Proc Natl Acad Sci USA 90: 1335–1339. Morato MJ, Brener Z, Canc¸ ado JR, Nun˜ ez RM, Chiari E, Gazzinelli G, 1986. Cellular immune responses of chagasic patients to antigens derived from different Trypanosoma cruzi strains and clones. Am J Trop Med Hyg 35: 505–511. Ko¨ hler G, Milstein C, 1975. Continuous cultures of fused cells secreting antibodies of predefined specificity. Nature 256: 495–497. Gavilondo J, 1995. Hibridomas por fusio´ n celular. In: Anticuerpos Monoclonales. Teorı´a y Pra´ ctica. Jorge Gavilondo Cowley, ed. Biotecnologı´a Aplicada, La Habana. 13–18. Bjo¨ rck L, Kastern W, Lindahl G, Widebo¨ ck K, 1987. Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG. Mol Immunol 24: 1113–1122. Aebersold RH, Leavitt J, Saavedra RA, Hood LE, Kent SB, 1987. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci USA 84: 6970–6974. Iba´ n˜ ez CF, Affranchino JL, Frasch ACC, 1987. Antigenic determinants of Trypanosoma cruzi defined by cloning of parasite DNA. Mol Biochem Parasitol 25: 175–184. Young RA, Mehra V, Sweetser D, Buchanan T, Clark-Curtiss J, Davis RW, Bloom BR, 1985. Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae. Nature 316: 450–452. Sambroock J, Fritsch EF, Maniatis T, 1989. Molecular Cloning. A Laboratory Manual. Second edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Sanger F, Nicklen S, Coulson AR, 1977. DNA sequencing with chain-terminating inhibitor. Proc Natl Acad Sci USA 74: 5463–5467. Cazzulo JJ, Franke de Cazzulo BM, Engel JC, Cannata JJB, 1985. End products and enzyme levels of aerobic glucose fermentation in trypanosomatids. Mol Biochem Parasitol 16: 329–343. Santos MR, Cano MI, Schijman A, Lorenzi H, Va´ zquez M, Levin ML, Ramirez JL, Brandao A, Degrave WM, da Silva JF, 1997. The Trypanosoma cruzi genome project: nuclear karyotype and gene mapping of clone CL Brener. Mem Inst Oswaldo Cruz 92: 821–828. Henriksson J, Porcel B, Rydaker M, Sabaj V, Galanti N, Cazzulo JJ, Frasch AC, Pettersson U, 1995. Chromosome specific markers reveal conserved linkage groups in spite of extensive

312

36.

37.

38.

39.

40.

41.

42. 43.

44.

´ N AND OTHERS AGUILLO

chromosomal size variation in Trypanosoma cruzi. Mol Biochem Parasitol 73: 63–74. Requena JM, Lo´ pez MC, Jimenez-Ruiz A, de la Torre JC, Alonso C, 1988. A head-to-tail tandem organization of hsp70 genes in Trypanosoma cruzi. Nucleic Acids Res 16: 1393– 1406. Altschul SF, Madden TL, Scha¨ ffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. Nielsen H, Engelbrecht J, Brunak S, von Heijne G, 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10: 1–6. Parker KC, Bednarek MA, Coligan JE, 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 152: 163–175. Pogue GP, Joshi M, Lee NS, Dwyer DM, Kenney RT, Gam AA, Nakhasi HL, 1996. Conservation of low-copy gene loci in Old World leishmanias identifies mechanisms of parasite evolution and diagnostic markers. Mol Biochem Parasitol 81: 27– 40. Rokeach LA, Haselby JA, Meilof JF, Smeenk RJ, Unnasch TR, Greene BM, Hoch SO, 1991. Characterization of the autoantigen calreticulin. J Immunol 147: 3031–3039. Smith MJ, 1992. A C. elegans gene encodes a protein homologous to mammalian calreticulin. DNA Seq 2: 235–240. Wizel B, Nunes M, Tarleton RL, 1997. Identification of Trypanosoma cruzi trans-sialidase family members as targets of protective CD8⫹ CTL responses. J Immunol 159: 6120– 6130. Low HP, Santos MA, Wizel B, Tarleton RL, 1998. Amastigote

45.

46.

47. 48. 49.

50.

51. 52. 53.

surface proteins of Trypanosoma cruzi are targets for CD8⫹ CTL. J Immunol 160: 1817–1823. Buckner FS, Wipke BT, van Voorhis WC, 1997. Trypanososma cruzi infection does not impair major histocompatibility complex class I presentation of antigen to cytotoxic T lymphocytes. Eur J Immunol 27: 2541–2548. Tarleton RL, Sun J, Zhang L, Postan M, 1994. Depletion of Tcell subpopulations results in exacerbation of myocarditis and parasitism in experimental Chagas’ disease. Infect Immun 62: 1820–1829. Nauseef WM, McCormick SJ, Clark RA, 1995. Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase. J Biol Chem 270: 4741–4747. Basu S, Srivastava PK, 1999. Calreticulin, a peptide-binding chaperone of the endoplasmic reticulum, elicits tumor- and peptide-specific immunity. J Exp Med 189: 797–802. La Flamme AC, Kahn SJ, Rudensky AY, van Voorhis WC, 1997. Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation. Eur J Immunol 27: 3085–3094. ˚ slund L, Macina RA, Franke de Cazzulo BM, Henriksson J, A Cazzulo JJ, Frasch ACC, Pettersson U, 1990. Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demostration of karyotype variability in Trypanosoma cruzi. Mol Biochem Parasitol 42: 213–224. Gibson WC, Miles MA, 1986. The karyotype and ploidy of Trypanosoma cruzi. EMBO J 5: 1299–1305. ˚ slund L, Pettersson U, 1996. Karyotype variHenriksson J, A ability in Trypanosoma cruzi. Parasitol Today 12: 108–114. Dragon EA, Sias SR, Kato EA, Gabe JD, 1987. The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein. Mol Cell Biol 7: 1271–1275.