Trypanosoma cruzi Morphogenesis: Preliminary Purification of an Active Fraction from Hemolymph and Intestinal Homogenate of Triatoma infestans (1986)

Differentiation of the Trypanosoma cruzi epimastigote to metacyclic form (Mtc), the parasitic stage in insect vector known to possess infective capacity, has been achieved in vitro by several authors. Insect extracts of Rhodnius prolixus (Wood and Souza, 1976, Revista do Instituto de Medicina Tropical do Sao Paulo 18: 93-96), Triatoma infestans (Isola et al., 1981, Journal of Parasitology 67: 53-58; 1983, Revista Argentina de Microbiologia 15: 181-185), or Phylosamia cynthia (Wood and Pipkin, 1969, Experimental Parasitology 24: 176-183), as well as T. infestans embryo cells (Lanar, 1979, Journal of Protozoology 26: 457-462), have been used as culture supplement. The in vitro activity of fractions obtained from hemolymph and intestinal homogenate of T. infestans, one of the most widespread vectors of this parasite, was analyzed in an attempt to determine the active components involved in the differentiation process. Epimastigotes of the Tulahuen isolate of T. cruzi maintained by weekly passages in biphasic medium were used to sow the culture media for differentiation assays. Intestinal homogenate and hemolymph of T. infestans were obtained as already reported (Isola et al., 1981, 1983, loc. cit.). Hindguts were removed and macerated in Grace medium to supplement cultures or in 0.15 M phosphate buffered saline (PBS), pH 7.2, when used for gel filtrations, in a ratio of 2.5-3.0 hindguts/ml. The suspensions were centrifuged for 30 min at 12,000 g, sterilized by filtration through 0.2 ,m pores, and kept at -70 C. Three to 5 ml of either substrate (hemolymph or intestinal homogenate) were filtered through a 100 x 2.6 cm Sephadex G-200 column at 4 C or 20-22 C, eluted with PBS 0.07 M, pH 7.2, and read at 280 nm (Dextran Blue 600K, human IgG fluorescein labelled 200K, human albumin 67K, ovoalbumin 43K, myoglobine 17.8K, and ribonuclease 13.7K were used as references). The fractions recovered were sterilized through 0.2 Aum pores, lyophilized and resuspended in Grace medium up to similar protein concentration in each fraction ranging from 0.4 to 0.7 mg/ml in different experiments. Samples of both substrates were also filtered through a 100 x 1.5 cm Sephadex G-50 column at 20-22 C, keeping elution and the fractioning process as mentioned above. The protein concentration of intestinal homogenate, hemolymph, and the fractions was determined by the method reported by Lowry et al. (1951, Journal of Biological Chemistry 193: 265-275). The protein level of intestinal homogenate was evaluated in PBS batches and a similar hindgut/ml ratio (2.5-3.0/ml) was maintained when the homogenate was prepared in Grace medium. The differentiation assays were carried out in Grace medium modified by Junker et al. (1967, Science 155: 1565-1566) (Gibco Laboratories Life Technology Inc.), Grace medium supplemented with intestinal homogenate (Isola et al., 1981, loc. cit.), Grace medium supplemented with 2% v/v hemolymph of T. infestans (Isola et al., 1983, loc. cit.), and GFx medium, prepared by supplementing Grace medium with 1 of the fractions (Fx) obtained by gel filtration (Fx-200 or Fx-50, depending on the Sephadex pore size). Each experimental variable was assayed in quintuplicate. Cultures were sown with 4 x 106 epimastigotes/ml and the percentage of differentiation to the Mtc stage was determined in at least 200 forms in wet and fixed-stained (May Griinwald-Giemsa) preparations. Cultures were examined every 48 hr during 15 days, and morphogenesis was evaluated as already reported (Isola et al., 1981, loc. cit.). The optimal protein concentration to induce Mtc differentiation was determined in Grace medium supplemented with 0.5 to 4.0 mg/ml of intestinal homogenate, as well as 0.12 to 0.50 mg/ml of the F2-50 of the same substrate in day 7. The F2-50 of intestinal homogenate was con-