CHARACTERIZATION OF DEVELOPMENTALLY-REGULATED ACTIVITIES IN AXENIC AMASTIGOTES OF LEISHMANIA-DONOVANI

Leishmania donovani is an obligatory intracellular parasite which cycl es between the midgut of sand flies (extracellular promastigote) and t he phagolysosomes of mammalian macrophages (intracellular amastigote). Promastigotes have been readily cultured, whereas axenic cultures of amastigotes have only recently been developed. A new method for in vit ro differentiation of L. donovani promastigotes into amastigotes is pr esented, in which promastigotes are exposed to environmental changes t hat mimic the in vivo process. First, promastigotes are subjected to 3 7 degrees C + 5% Co-2 for 24 h, and then are shifted to pH 5.5. Under these conditions, differentiation is completed within 120 h, In the re verse process, amastigotes are induced to differentiate back to promas tigotes by transferring them to promastigote growth conditions (medium 199 at pH 7.4 and 26 degrees C). Axenic amastigotes closely resemble animal-derived amastigotes. They manifest all seven proteins of the am astigote-specific A2 gene family. They down-regulate lipophosphoglycan (LPG) synthesis and do not express it on their surface. LPG is up-reg ulated 2 h after inducing amastigotes to differentiate to promastigote s. Within 6 h, parasites resume the promastigote level of this molecul e, although differentiation is completed only after 48 h. Axenic amast igotes also express amastigote-like metabolic activities of proline up take, as well as thymidine and proline incorporation. In conclusion, t he results indicate that the method developed for in vitro differentia tion of L. donovani promastigotes to amastigotes is efficient and yiel ds organisms resembling animal-derived amastigotes. Being able to indu ce in vitro differentiation of L. donovani provides us with an excelle nt tool to study Leishmania development and differentiation. (C) 1998 Elsevier Science B.V. All rights reserved.