In vitro Translation of Trypanosoma cruzi Antigens Recognized by Human Chagasic Sera

Abstract

Several reports indicate that infective (trypomastigotes) and noninfective (epimastigotes) forms of Trypanosoma cruzi display relatively simple antigenic patterns when analyzed by immunoprecipitation with hyperimmune sera from experimental animals. Thus, epimastigotes show, as their major antigen, an Mr 72,000 glycoprotein, whereas trypomastigotes (both blood forms and tissue culture-derived forms) display instead an Mr 90,000 surface glycoprotein (Nogueira et al., 1981, Journal of Experimental Medicine 153: 629-639; Snary et al., 1981, Molecular and Biochemical Parasitology 3: 343-356). These patterns become more complex when sera from chagasic patients are used in either immunoprecipitation techniques (Zingales et al., 1982, Molecular and Biochemical Parasitology 11: 111-124) or immunoblotting (Grogl and Kuhn, 1984, Journal of Parasitology 70: 822824). We have extended these studies and found that lysates of epimastigotes and tissue culturederived trypomastigotes subjected to immunoblotting with human chagasic sera, display reactive bands over a wide range of molecular weights (Mr 150,000 to Mr 12,000) (Rangel-Aldao et al., 1986, Molecular and Biochemical Parasitology 20: 25-32). Most of these antigenic bands were shared by both parasite forms and the differences in their antigenic patterns were only manifested upon antigen dilution. Trypomastigotes showed, predominantly, a group of bands in the range of Mr 150,000 to Mr 75,000, whereas epimastigotes displayed a cluster of bands in the range of Mr 72,000 to Mr 36,000 (Rangel-Aldao et al., 1986, loc. cit.). everal reports indicate that infective (trypoThis qualitative homology of epimastigotes and trypomastigotes could be exploited to produce in vitro, by the techniques of molecular biology, most of the common antigens of the infective trypomastigotes from the mRNA of epimastigotes that are not infectious and can be easily cultured in mass quantities. These antigens could then be used to develop sensitive tools for diagnosis and vaccination tests for Chagas' disease. As a prerequisite to achieve this goal, we have isolated polyadenylated (poly A+) RNA from epimastigotes, translated it in vitro in a wheat germ cell-free system, immunoblotted the translated products with a pool of sera from 10 patients with Chagas' disease, and showed that it is possible to translate in vitro antigens produced n vivo by epimastigotes. These antigens are also shared by tissue culture-derived trypomastigotes (Rangel-Aldao et al., 1986, loc. cit.). Poly A+ RNA was isolated from 1 x 1010 cultured epimastigotes (Rangel-Aldao et al., 1983, Journal of Biological Chemistry 258: 6979-6983), washed twice with 50 ml of 1% w/v NaCI, 0.6 t,g/ml cycloheximide, and 20% glycerol. The cells were resuspended in lysis buffer (50 mM TrisHC1, pH 7.4, 0.5% Triton X-100, 50% glycerol, and 1,000 units of human placental ribonuclease inhibitor) and lysed at -20 C with a Dounce homogenizer. The supernatant obtained from the lysis step was mixed with 3 volumes of a buffer solution containing 10 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.5% sodium dodecyl sulfate (SDS) and the total cytoplasmic RNA extracted with phenol-chloroform (Perry et al., 1972, Biochimica et Biophysica Acta 262: qualitative hom logy of epimastigotes and astigotes could b exploited to produce itro, by the techniques of molecular biology, of the common antigens of the infective astigotes from the mRNA of epimastis that are not infectious and can be easily red in ma s quan ties. Th se antigens could e used to develop sensitive tools for disis and va cination te ts for Chagas' di ase. rerequisite to achieve this goal, we have