Characterization of developmentally-regulated activities in axenic amastigotes of Leishmania donovani.

Leishmania donovani is an obligatory intracellular parasite which cycles between the midgut of sand flies (extracellular promastigote) and the 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 vitro differentiation of L. donovani promastigotes into amastigotes is presented, in which promastigotes are exposed to environmental changes that mimic the in vivo process. First, promastigotes are subjected to 37 degrees C + 5% CO2 for 24 h, and then are shifted to pH 5.5. Under these conditions, differentiation is completed within 120 h. In the reverse process, amastigotes are induced to differentiate back to promastigotes 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 amastigote-specific A2 gene family. They down-regulate lipophosphoglycan (LPG) synthesis and do not express it on their surface. LPG is up-regulated 2 h after inducing amastigotes to differentiate to promastigotes. Within 6 h, parasites resume the promastigote level of this molecule, although differentiation is completed only after 48 h. Axenic amastigotes also express amastigote-like metabolic activities of proline uptake, as well as thymidine and proline incorporation. In conclusion, the results indicate that the method developed for in vitro differentiation of L. donovani promastigotes to amastigotes is efficient and yields organisms resembling animal-derived amastigotes. Being able to induce in vitro differentiation of L. donovani provides us with an excellent tool to study Leishmania development and differentiation.