SummaryUsing as the host cell, a proline-requiring mutant of Chinese hamster ovary cell (CHO-K1), it was possible to arrest the differentiation of amastigote forms of Trypanosoma cruzi at the intermediate intracellular epimastigote-like stage. Complete differentiation to the trypomastigote stage was obtained by addition of L -proline to the medium. This effect was more pronounced using the T. cruzi CL-14 clone that differentiates fully at 33 ∞ ∞ ∞ ∞ C (permissive temperature) and poorly at 37 ∞ ∞ ∞ ∞ C (restrictive temperature). A synchronous differentiation of T. cruzi inside the host-cell is then possible by temperature switching in the presence of proline. It was found that differentiation of intracellular epimastigotes and trypomastigote bursting were proline concentration dependent. The intracellular concentration of proline was measured as well as the transport capacity of proline by each stage of the parasite. Amastigotes have the highest concentration of free proline (8.09 ± ± ± ± 1.46 mM) when compared to trypomastigotes (3.81 ± ± ± ± 1.55) or intracellular epimastigote-like forms (0.45 ± ± ± ± 0.06 mM). In spite of having the lowest content of intracellular free proline, intracellular epimastigotes maintained the highest levels of L -proline transport compared to trypomastigotes and intracellular amastigotes, providing evidence for a high turnover for the L -proline pool in that parasite stage. This is the first report to establish a relationship between proline concentration and intracellular differentiation of Trypanosoma cruzi in the mammalian host.

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The aim of the present study was to demonstrate the presence of a-Lfucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, a-L-fucosidase from the parasite. Light and electron microscopy localized the a-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the a-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that a-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-a-Lfucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-a-L-fucosidase antibodies.