Genetic Rescue of Leishmania Deficiency in Porphyrin Biosynthesis Creates Mutants Suitable for Analysis of Cellular Events in Uroporphyria and for Photodynamic Therapy

Abstract

Leishmania was found deficient in at least five and most likely seven of the eight enzymes in the heme biosynthesis pathway, accounting for their growth requirement for heme compounds. The xenotransfection of this trypanosomatid protozoan led to their expression of the mammalian genes encoding ␦-aminolevulinate (ALA) dehydratase and porphobilinogen deaminase, the second and the third enzymes of the pathway, respectively. These transfectants still require hemin or protoporphyrin IX for growth but produce porphyrin when ALA was supplied exogenously. Leishmania is thus deficient in all first three enzymes of the pathway. Uroporphyrin I was produced as the sole intermediate by these transfectants, further indicating that they are also deficient in at least two porphyrinogen-metabolizing enzymes downstream of porphobilinogen deaminase, i.e. uroporphyrinogen III co-synthase and uroporphyrinogen decarboxylase. Pulsing the transfectants with ALA induced their transition from aporphyria to uroporphyria. Uroporphyrin I emerged in these cells initially as diffused throughout the cytosol, rendering them sensitive to UV irradiation. The porphyrin was subsequently sequestered in cytoplasmic vacuoles followed by its release and accumulation in the extracellular milieu, concomitant with a reduced photosensitivity of the cells. These events may represent cellular mechanisms for disposing soluble toxic waste from the cytosol. Monocytic tumor cells were rendered photosensitive by infection with uroporphyric Leishmania, suggestive of their potential application for photodynamic therapy.

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

“…This decline in cellular URO I is coupled with an increase in the level of this porphyrin found in the extracellular milieu at >0.5 nmoles/ml (Fig. 4C Released), suggestive of extracellular efflux, as seen previously when the L. amazonensis double-transfectants were examined under growing conditions in culture medium (Sah et al, 2002). Under both growing and non-growing conditions, URO I was released by viable cells into the extracellular milieu, independent of inadvertent cytolysis.…”

“…The kinetics of uroporhyrin neogenesis follows the same pattern under both conditions for all species, although the maximal porphyrin levels reached may vary. We have previously proposed that mobilization of cytosolic URO into vacuoles followed by its extracellular efflux may be related to soluble toxic waste disposal (Sah et al 2002). Of interest for further investigation is the leishmanial mechanism of URO efflux, for which ABC transporters have been suggested to play a role in mammalian cells (Latunde-Dada et al, 2006).…”

“…Single-and double-transfection of these cells by electroporation with pX-alad and/ or p6.5-pbgd, selection for transfectants and their maintenance followed published procedures (Sah et al, 2002). Stable lines of single-and double-transfectants were all maintained in the presence of appropriate selective pressures, e. g. 100 ug/ml of G418 (for pX-alad) and/or 20 ug/ml of tunicamycin (for p6.5-pdgd) (except those of L. infantum, for which the selective pressure was lowered to 5-10 ug/ml of tunicamycin and 50 ug/ml of G418).…”

“…1). Development of uroporphyria in these transfectants in the presence of exogenous delta-aminolevulinate (ALA) led us to suggest that this New World Leishmania is functionally defective in at least the first five and possibly the first seven of the eight enzymes known in heme biosynthetic pathway (Sah et al, 2002) (Fig. 1).…”

“…Earlier, this deficiency has been delineated genetically by transfecting Leishmania amazonensis with mammalian cDNAs, encoding the 2 nd and 3 rd enzymes of the biosynthetic pathway for tetrapyrroles (Sah et al, 2002) (Cf. Fig.…”