Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy.
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 delta-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.
Sah
Jerome Franklin
JF
Department of Microbiology, University of Health Sciences, Chicago Medical School, North Chicago, Illinois 60064, USA.
Ito
Hiroya
H
Kolli
Bala Krishna
BK
Peterson
Daniel A
DA
Sassa
Shigeru
S
Chang
Kwang-Poo
KP
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers; D011163:Hydroxymethylbilane Synthase / Q000235:genetics* / Q000378:metabolism; D007891:Leishmania / Q000201:enzymology* / Q000235:genetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D010778:Photochemotherapy; D000623:Porphobilinogen Synthase / Q000235:genetics* / Q000378:metabolism; D011164:Porphyrias / Q000473:pathology*; D011166:Porphyrins / Q000096:biosynthesis*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D017931:DNA Primers; D011166:Porphyrins; D011163:Hydroxymethylbilane Synthase; D000623:Porphobilinogen Synthase