Document Detail


Transgenic Leishmania model 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.
MedLine Citation:
PMID:  18487349     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 > or =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.
Authors:
Sujoy Dutta; Bala Krishna Kolli; Aihua Tang; Shigeru Sassa; Kwang-Poo Chang
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-05-16
Journal Detail:
Title:  Eukaryotic cell     Volume:  7     ISSN:  1535-9786     ISO Abbreviation:  Eukaryotic Cell     Publication Date:  2008 Jul 
Date Detail:
Created Date:  2008-07-09     Completed Date:  2008-08-28     Revised Date:  2013-06-05    
Medline Journal Info:
Nlm Unique ID:  101130731     Medline TA:  Eukaryot Cell     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1146-57     Citation Subset:  IM    
Affiliation:
Department of Microbiology/Immunology, Chicago Medical School/Rosalind Franklin University, North Chicago, Illinois 60064, USA.
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MeSH Terms
Descriptor/Qualifier:
Aminolevulinic Acid / metabolism,  pharmacology*
Animals
Animals, Genetically Modified / genetics,  metabolism
Azides / pharmacology
Biological Transport
Cell Membrane Permeability / radiation effects
Cell Survival / radiation effects
Cytosol / metabolism*
Flagella / drug effects,  metabolism
Humans
Leishmania / drug effects,  genetics,  metabolism*,  radiation effects
Light
Models, Animal
Phenotype
Photolysis
Porphyrias / chemically induced,  metabolism,  therapy
Proteins / metabolism*
Singlet Oxygen / metabolism*
Transport Vesicles / metabolism
Uroporphyrins / genetics,  metabolism*,  pharmacokinetics
Grant Support
ID/Acronym/Agency:
AI-20486/AI/NIAID NIH HHS; AI-68835/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Azides; 0/Proteins; 0/Uroporphyrins; 106-60-5/Aminolevulinic Acid; 17778-80-2/Singlet Oxygen
Comments/Corrections

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