Glyoxalase I gene deletion mutants of Leishmania donovani exhibit reduced methylglyoxal detoxification

Abstract

Background: Glyoxalase 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 findings: Analysis 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.

Conclusions: We 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.

Figure 1. Localization of GLOI in L. donovani.

A: Panel 1: confocal microscopy of wild type L. donovani transfected with pGEM7zf-αNEOα-GFP-GLOI expressing GLOI as a GFP translational fusion protein and stained with Mitotracker-Red CMX Ros dye, Panel 2: phase contrast image, Panel 3: wild type L. donovani expressing GLOI as a GFP translational fusion protein, Panel 4: merged micrograph, Panel 5: wild type L. donovani transfected with pGEM7zf-αNEOα-GFP vector alone expressing GFP, Panel 6: phase contrast image B: Panel 1: Immunoflourescence analysis by confocal micrograph of wild type L. donovani parasite transfected with pGEM7zf-αNEOα-GFP-GLOI,using anti–GLOI antibody, Panel 2: phase contrast image. Panel 3: wild type L. donovani expressing GLOI as a GFP translational fusion protein, 4: merged micrograph C: Panel 1: Immunoflourescence analysis by confocal micrograph of wild type L. donovani parasite transfected with pGEM7zf-αNEOα-GFP-GLOI using anti-GLOI antibody, Panel 2: wild type L. donovani expressing GLOI as a GFP translational fusion protein, Panel 3: stained with DAPI, Panel 4: phase contrast image, Panel 5: merged micrograph. D: Western blotting using anti-GLOI antibody. Cytosolic and mitochondrial fractions were separated from late log phase promastigotes (1×10⁸) expressing GLOI as a GFP translational fusion protein. Lane 1: recombinant GLOI, Lane 2: cytosolic fraction, Lane 3: mitochondrial fraction. The blot was also probed with mitochondrial specific anti-F1 ATP synthase α-subunit. Results are representative data from three separate experiments.

Figure 2. Schematic representation of the wild type and the hyg, neo and phleo targeted Ld GLOI locus.

SalI restriction sites were used to analyze the recombination events. The restriction fragments obtained after Sal I digestion are indicated by double headed arrows along with their respective size. Thick arrows indicate the ORF of genes as indicated. Bold arrows indicate the 5′ and 3′ flanking regions of GLOI ORF. Solid bars indicate the probes used for Southern blot analysis. Primers P 1 and P 2 were used to check the correct integration of hyg cassette and primers P 1 and P 3 were used to check the correct integration of neo cassette. Restriction sites (H- HindIII, B- BamHI, Bg - BglII, SalI) used for cloning 5′ and 3′ flank are also indicated.

Figure 3. Southern blot and PCR analysis of GLOI knockout mutants.

Southern blot analysis of wild type and mutant parasites to confirm the recombination events. Equal amount of genomic DNA (5 µg) was digested with SalI restriction enzyme and separated on a 0.8% agarose gel. The DNA was blotted on to membrane and hybridized with hyg specific probe (A), NEO specific probe (B), GLOI specific probe (C) and PHLEO specific probe (D). (+/+) represents Wild type; (+/h) represents heterozygote LdGLOI::HYG clone; (+/n) represents heterozygote LdGLOI :: NEO clone; (+/h/n) represents L. donovani double targeted transfectant, LdGLOI :: HYG :: NEO clone, (+/h-GLOI⁺) represents GLOI complementation mutant and (−/− GLOI⁺) represents GLOI null mutants rescued with an episomal copy of GLOI. M represents DNA molecular weight marker; uncut pX63-HYG and pX63-NEO and linearized pGEM7zfαNeoα-GLOI⁺ plasmids were used as positive controls for the respective blots probed with HYG, NEO and GLOI. PCR was carried out with primers P1 and P2 for checking hyg cassette integration (E, panel a) and with primers P1 and P3 for checking neo cassette integration (F, panel a). Southern blot analysis of the corresponding PCR gels probed with HYG (E, panel b) and NEO (F, panel b). G: Southern blot analysis of genomic DNA digested with SalI and probed with PHLEO probe to verify phleo cassette integration.

Figure 4. DNA content analysis.

Wild type and GLOI mutant L.donovani cell lines were analysed for DNA content using flow cytometry. A: Wild type (+/+): B: heterozygote LdGLOI :: HYG clone (+/h); C: heterozygote LdGLOI :: NEO clone (+/n);, D: GLOI complementation mutant (+/h-GLOI⁺); E: L. donovani double targeted transfectant, LdGLOI :: HYG :: NEO clone (+/h/n) were fixed and stained with propidium iodide and analysed using a Beckton Dickinson flow cytometer(FACScalibur). Representative peaks of G1 and G2 phase are shown as predicted by the CELL QUEST software. Results are representative data from three separate experiments.

Figure 5. Growth profile of promastigotes of wild type (+/+), heterozygote LdGLOI :: HYG clone (+/h); heterozygote LdGLOI :: NEO clone (+/n); L. donovani double targeted transfectant, LdGLOI :: HYG :: NEO clone (+/h/n), GLOI complementation mutant (+/h-GLOI⁺).

Growth comparison was done by inoculating stationary phase cells in modified M199 medium with 10% FBS at a density of 1×10⁶ cells/ml. Cells were counted at 24 h intervals using a Neubauer hemocytometer. Results are representative data from two separate experiments with mean±SD of triplicates in each set.