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 amazonensis, a causative agent of cutaneous leishmaniasis, is susceptible in vitro to lightmediated cytolysis in the presence of or after pretreatment with the photosensitizer aluminum phthalocyanine chloride. Cytolysis of both promastigotes and axenic amastigotes required less photosensitizer (e.g., one g · ml ؊1 ) and a lower light dose (e.g., 1.5 J · cm ؊2 ) than did the mammalian cells examined for comparison. Exposure of Leishmania cells to the photosensitizer alone had little effect on their viability, as judged from their motility, growth, and/or retention of green fluorescent proteins genetically engineered for episomal expression. Fluorimetric assays for cell-associated and released green fluorescence proteins proved to be even more sensitive for the evaluation of cell viability than microscopy for the evaluation of motility and/or integrity. Axenic amastigotes pretreated with the photosensitizer infected macrophages of the J774 line but were lysed intracellularly when the infected cells were exposed to light. Addition of the photosensitizer to the already infected cells produced no effect on their intracellular parasites. However, light irradiation lysed these macrophages and also those infected with parasites preincubated with the photosensitizer at a concentration of 5 g · ml ؊1 or higher. Photosensitized Leishmania cells are highly susceptible to cytolysis, apparently due to the generation of reactive oxidative species on light illumination, suggestive of inefficiency of their antioxidant mechanisms. Efficient delivery of photosensitizers to intracellular Leishmania is expected to increase their therapeutic potentials against leishmaniasis.

Leishmania amazonensis was found to release nucleoside diphosphate kinase (NdK) -a stable enzyme capable of decreasing extracellular ATP. The release of this enzyme from Leishmania results in its progressive accumulation extracellularly as they replicate, peaking at the stationary phase in vitro. The released NdK is immunoprecipitable and constitutes ~40% of its total activities and proteins. The retention of a known cytosolic protein by wild type cells and a fluorescent protein by DsRed transfectants at stationary phase, which release NdK, indicates that this is a spontaneous event, independent of inadvertent cytolysis. Recombinant products of Leishmania NdK prepared were enzymatically and immunologically active. Both recombinant and native Leishmania NdK utilized ATP to produce expected nucleoside triphosphates in the presence of nucleoside diphosphates in excess. Both native and recombinant Leishmania NdK were also found to prevent ATP-induced cytolysis of J774 macrophages in vitro, as determined by assays for lactate dehydrogenase release from these cells and for their mitochondrial membrane potential changes. The results obtained thus suggest that Leishmania NdK not only serves its normal house-keeping and other important functions true to all cells, but also prevents ATP-mediated lysis of macrophages, thereby preserving the integrity of the host cells to the benefit of the parasite.

Photodynamic therapy, unlikely to elicit drug-resistance, deserves attention as a strategy to counter this outstanding problem common to the chemotherapy of all diseases. Previously, we have broadened the applicability of this modality to photodynamic vaccination by exploiting the unusual properties of the trypanosomatid protozoa, Leishmania, i.e., their innate ability of homing to the phagolysosomes of the antigen-presenting cells and their selective photolysis therein, using transgenic mutants endogenously inducible for porphyrin accumulation. Here, we extended the utility of this host-parasite model for in vitro photodynamic therapy and vaccination by exploring exogenously supplied photosensitizers. Seventeen novel phthalocyanines (Pcs) were screened in vitro for their photolytic activity against cultured Leishmania. Pcs rendered cationic and soluble (csPcs) for cellular uptake were phototoxic to both parasite and host cells, i.e., macrophages and dendritic cells. The csPcs that targeted to mitochondria were more photolytic than those restricted to the endocytic compartments. Treatment of infected cells with endocytic csPcs resulted in their accumulation in Leishmania-containing phagolysosomes, indicative of reaching their target for photodynamic therapy, although their parasite versus host specificity is limited to a narrow range of csPc concentrations. In contrast, Leishmania pre-loaded with csPc were selectively photolyzed intracellularly, leaving host cells viable. Pre-illumination of such csPc-loaded Leishmania did not hinder their infectivity, but ensured their intracellular lysis. Ovalbumin (OVA) so delivered by photo-inactivated OVA transfectants to mouse macrophages and dendritic cells were co-presented with MHC Class I molecules by these antigen presenting cells to activate OVA epitope-specific CD8+T cells. The in vitro evidence presented here demonstrates for the first time not only the potential of endocytic csPcs for effective photodynamic therapy against Leishmania but also their utility in photo-inactivation of Leishmania to produce a safe carrier to express and deliver a defined antigen with enhanced cell-mediated immunity.

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.