Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis

Abstract

The inability to synthesize heme, a well known metabolic defect of trypanosomatid protozoa, accounts for their growth requirement for heme compounds in vitro. We now extend this finding to a pathogen Leishmania mexicana amazonensis, especially the intracellular replicative stage of amastigotes in the macrophage. We measured the level of heme and its biosynthetic enzymes, aminolevulinate dehydratase and porphobilinogen deaminase in the parasites and in infected and non-infected macrophages of J774G8 line. Succinylacetone was used to inhibit heme biosynthesis. Leishmanias transform and grow only in medium containing either heme (usually supplied as hemin) or protoporphyrin IX (the latter is leishmanicidal at high concentrations). We detected 1.2, 8.5 and 25 pmol mg −1 protein of heme in amastigotes, promastigotes and macrophages, respectively. The activities of porphobilinogen deaminase and aminolevulinate dehydratase in macrophages were 70 and 2 400 pmol h −1 mg −1 protein, respectively. Leishmania-infected macrophages gave the same results and leishmanias had negligible activities of these enzymes. Succinylacetone at 10 −9-10 −3 M had no effect on leishmanias, but dose-dependently inhibited the activity of aminolevulinate dehydratase to a negligible level and lowered that of porphobilinogen deaminase in macrophages, resulting in a maximum of 66% reduction in intracellular heme. Amastigotes grew equally well in succinylacetone-treated and control untreated macrophages. The results suggest that L. m. amazonensis, incapable of heme biosynthesis, acquires heme exogenously from the culture medium, i.e., fetal bovine serum, independent of the heme synthesized by the macrophages.