Leishmania spp.: Delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

Abstract

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2 nd and 3 rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.

Delta-Aminolevulinate-Induced Host-Parasite Porphyric Disparity for Selective Photolysis of Transgenic Leishmania in the Phagolysosomes of Mononuclear Phagocytes: a Potential Novel Platform for Vaccine Delivery

“…A small aliquot from each sample was examined first under phase-contrast microscopy and then for porphyrin fluorescence by using a specific filter set (10). The remaining samples (Ͼ5 ϫ 10 6 macrophages each) were solvent extracted for analysis of porphyrins by thin-layer chromatography (TLC) using carboxymethylated porphyrins (Porphyrin Products) as standards (9,30).…”

“…During medium renewal and microscopic observations, cultures were exposed by necessity to light that was either left unchanged, as usual for such routine operations, or minimized by dimming the light sources and shortening the handling time. Cultures were experimentally illuminated with white light at ϳ6.5 mW/cm 2 for variable time periods and at porphyrin-excitable wavelengths, i.e., longwave UV light ( max ϭ 366 nm), as described previously (9,10,30), or violet light (LumaCare probe; max ϭ 400 nm). Since a desirable outcome was achieved under all experimental conditions of illumination used, they are specified for individual experiments in the figure legends.…”

“…Single transfectants (STs) with only one or the other cDNA, i.e., pX-aladϩp6.5 or pXϩp6.5-pbgd (30), were included as controls where appropriate in some experiments. All mutants were briefly grown in drug-free medium to stationary phase before being used for infection to avoid the potential cytotoxicity of the drugs being carried over to the host cells (9,10).…”

“…The substantial loss of this pathway from these organisms is thought to result from their evolution of parasitism in heme-rich environments, i.e., the gut of blood-sucking insect vectors and the phagolysosomes of mononuclear phagocytes in the mammalian reticuloendothelial system (3,4). The Leishmania defects in heme biosynthesis have been partially defined functionally by genetic complementation of the extracellular promastigote stage in vitro (9,30). Transfection of these cells with mammalian cDNAs that express the 2nd and 3rd enzymes in this pathway results in their accumulation of URO I in response to exogenously provided ALA, the product of the 1st enzyme in heme biosynthesis (30).…”

“…Among these single-cell parasites, Leishmania spp. are functionally missing at least 5 and very possibly 7 of the 8 enzymes needed to complete heme biosynthesis (9,30). The substantial loss of this pathway from these organisms is thought to result from their evolution of parasitism in heme-rich environments, i.e., the gut of blood-sucking insect vectors and the phagolysosomes of mononuclear phagocytes in the mammalian reticuloendothelial system (3,4).…”