TransgenicLeishmaniaModel for Delta-Aminolevulinate-Inducible Monospecific Uroporphyria: Cytolytic Phototoxicity Initiated by Singlet Oxygen-Mediated Inactivation of Proteins and Its Ablation by Endosomal Mobilization of Cytosolic Uroporphyrin

Abstract

Inherent deficiencies of Leishmania in heme biosynthesis were genetically complemented for delta-aminolevulinate-inducible biosynthesis and accumulation of light-excitable uroporphyrin. The phototoxic flagellar immobilization and cytolysis phenotypes and porphyrin mobilization noted previously were further analyzed biochemically and cytologically to delineate the mechanism of phototoxicity and detoxification in this monoporphyric model. Under optimal conditions of induction for approximately 3 days, cells remained viable but became increasingly uroporphyric, peaking at >90% of the population by approximately day 2; thereafter, a small population of less porphyric or aporphyric cells emerged. On exposure to light, the flagella of porphyric cells were immobilized in milliseconds, and singlet oxygen became detectable in their lysates. Both photosensitive phenotypes increased proportionally with the cellular uroporphyric levels and were susceptible to inhibition by azide, but not by D-mannitol. Brief irradiation of the uroporphyric cells produced no appreciable protein degradation but inactivated cytosolic neomycin phosphotransferase and significantly bleached cytosolic green fluorescent protein, which was azide reversible. These cells were irreparably photodamaged, as indicated by their subsequent loss of membrane permeability and viability. This is the first in situ demonstration that early inactivation of functional proteins by singlet oxygen initiates the cytolytic phototoxicity in uroporphyria. Detoxification appears to involve endocytic/exocytic mobilization of uroporphyrin from cytosol to "porphyrinosomes" for its eventual extracellular expulsion. This is proposed as the sole mechanism of detoxification, since it is attributable to the reversion of porphyric to aporphyric cells during uroporphyrinogenesis and repeated cycles of this event plus photolysis selected no resistant mutants, only aporphyric clones of the parental phenotypes. Further characterization of the transport system for uroporphyrin in this model is expected to benefit not only our understanding of the cellular mechanism for disposal of toxic soluble wastes but also potentially the effective management of human uroporphyria and the use of uroporphyric Leishmania for vaccine/drug delivery.

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

“…Infected and noninfected cultures with and without ALA treatment were withdrawn daily. 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 immediate product expected is porphobilinogen (PBG), which in the absence of the downstream enzyme, i.e., uroporphyrinogen cosynthase, condenses and cyclizes spontaneously to form URO I instead of URO III (31). URO I is a nonmetabolizable by-product that is water soluble and is released without reuptake by cells, including Leishmania (10). When exposed to light, uroporphyric promastigotes are lysed, as the cytosolic URO is excited to generate singlet oxygen and other leishmanolytic ROS (10).…”

“…URO I is a nonmetabolizable by-product that is water soluble and is released without reuptake by cells, including Leishmania (10). When exposed to light, uroporphyric promastigotes are lysed, as the cytosolic URO is excited to generate singlet oxygen and other leishmanolytic ROS (10).…”