Abstract
BackgroundGlyoxalase I is a metalloenzyme of the glyoxalase pathway that plays a central role in eliminating the toxic metabolite methyglyoxal. The protozoan parasite Leishmania donovani possesses a unique trypanothione dependent glyoxalase system.Principal FindingsAnalysis of the L. donovani GLOI sequence predicted a mitochondrial targeting sequence, suggesting that the enzyme is likely to be targeted to the mitochondria. In order to determine definitively the intracellular localization of GLOI in L. donovani, a full-length GLOI gene was fused to green fluorescent protein (GFP) gene to generate a chimeric construct. Confocal microscopy of L. donovani promastigotes carrying this chimeric construct and immunofluorescence microscopy using anti-GLOI antibodies demonstrated that GLOI is localized in the kinetoplast of the parasite apart from the cytosol. To study the physiological role of GLOI in Leishmania, we first created promastigote mutants heterozygous for GLOI by targeted gene replacement using either hygromycin or neomycin phosphotransferases as selectable markers. Heterozygous mutants of L. donovani display a slower growth rate, have lower glyoxalase I activity and have reduced ability to detoxify methylglyoxal in comparison to the wild-type parasites. Complementation of the heterozygous mutant with an episomal GLOI construct showed the restoration of heterozygous mutant phenotype nearly fully to that of the wild-type. Null mutants were obtained only after GLOI was expressed from an episome in heterozygous mutants.ConclusionsWe for the first time report localization of GLOI in L. donovani in the kinetoplast. To study the physiological role of GLOI in Leishmania, we have generated GLOI attenuated strains by targeted gene replacement and report that GLOI is likely to be an important gene since GLOI mutants in L. donovani showed altered phenotype. The present data supports that the GLOI plays an essential role in the survival of this pathogenic organism and that inhibition of the enzyme potentiates the toxicity of methylglyoxal.
Highlights
Leishmaniasis constitutes a wide spectrum of diseases ranging from the simple self-limiting cutaneous form to the debilitating visceral form, which is often fatal if left untreated
To study the physiological role of glyoxalase I (GLOI) in Leishmania, we have generated GLOI attenuated strains by targeted gene replacement and report that GLOI is likely to be an important gene since GLOI mutants in L. donovani showed altered phenotype
In the present study we report that L. donovani promastigotes carrying a GLOI-green fluorescent protein (GLOI-GFP) chimeric construct display localization of GLOI in the kinetoplast of the parasite apart from the cytosol
Summary
Leishmaniasis constitutes a wide spectrum of diseases ranging from the simple self-limiting cutaneous form to the debilitating visceral form, which is often fatal if left untreated. The protozoan parasite Leishmania donovani is the major causative agent of visceral leishmaniasis. The glyoxalase system is a ubiquitous thiol-dependent detoxification pathway [1]. This system comprises of two enzymes, glyoxalase I (GLOI) (lactoylglutathione lyase, EC 4.4.1.5) and glyoxalase II (GLOII) (hydroxyacylglutathione hydrolase, EC 3.1.2.6). Glyoxalase I is a single copy gene and is placed at a single chromosomal band of ,2.2 Mb. Multiple sequence alignment and homology modeling clearly showed that there is a crucial difference in the active site of human and Leishmania glyoxalase I enzymes suggesting that the glyoxalase I may be a potential target for drug design [5]. The protozoan parasite Leishmania donovani possesses a unique trypanothione dependent glyoxalase system
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