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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.

ABSTRACT: Leishmania double transfectants (DTs) expressing the 2nd and 3rd enzymes in the heme biosynthetic pathway were previously reported to show neogenesis of uroporphyrin I (URO) when induced with delta-aminolevulinate (ALA), the product of the 1st enzyme in the pathway. The ensuing accumulation of URO in DT promastigotes rendered them light excitable to produce reactive oxygen species (ROS), resulting in their cytolysis. Evidence is presented showing that the DTs retained wild-type infectivity to their host cells and that the intraphagolysosomal/parasitophorous vacuolar (PV) DTs remained ALA inducible for uroporphyrinogenesis/photolysis. Exposure of DT-infected cells to ALA was noted by fluorescence microscopy to result in host-parasite differential porphyrinogenesis: porphyrin fluorescence emerged first in the host cells and then in the intra-PV amastigotes. DT-infected and control cells differed qualitatively and quantitatively in their porphyrin species, consistent with the expected multi- and monoporphyrinogenic specificities of the host cells and the DTs, respectively. After ALA removal, the neogenic porphyrins were rapidly lost from the host cells but persisted as URO in the intra-PV DTs. These DTs were thus extremely light sensitive and were lysed selectively by illumination under nonstringent conditions in the relatively ROS-resistant phagolysosomes. Photolysis of the intra-PV DTs returned the distribution of major histocompatibility complex (MHC) class II molecules and the global gene expression profiles of host cells to their preinfection patterns and, when transfected with ovalbumin, released this antigen for copresentation with MHC class I molecules. These Leishmania mutants thus have considerable potential as a novel model of a universal vaccine carrier for photodynamic immunotherapy/immunoprophylaxis.

SUBMITTER: Dutta S

PROVIDER: S-EPMC3318301 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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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.

Dutta Sujoy S Chang Celia C Kolli Bala Krishna BK Sassa Shigeru S Yousef Malik M Showe Michael M Showe Louise L Chang Kwang-Poo KP

Eukaryotic cell 20120203 4

Leishmania double transfectants (DTs) expressing the 2nd and 3rd enzymes in the heme biosynthetic pathway were previously reported to show neogenesis of uroporphyrin I (URO) when induced with delta-aminolevulinate (ALA), the product of the 1st enzyme in the pathway. The ensuing accumulation of URO in DT promastigotes rendered them light excitable to produce reactive oxygen species (ROS), resulting in their cytolysis. Evidence is presented showing that the DTs retained wild-type infectivity to th ...[more]

PMID: 22307976

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Project description: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.

Dataset Information

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.

Publications

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.

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