Swati Pal

Characterization of Leishmania donovani Antigens Encapsulated in Liposomes That Induce Protective Immunity in BALB/c Mice

ABSTRACT Leishmania donovani promastigote membrane antigens (LAg) encapsulated in positively charged liposomes have been found to induce very significant levels of protection against experimental visceral leishmaniasis. The protectively immunized animals exhibited profound delayed-type hypersensitivity and antibody responses. The extent of protection induced by the same antigens, however, varied depending on the charge of the vesicles, with maximum induction by positively charged liposomes, followed by neutral liposomes and last negatively charged liposomes. Characterization of LAg and LAg entrapped in liposomes of different charges by Western blot analysis revealed the immunodominance of gp63 in all three vaccine preparations. The strong reactivity of antigens in a restricted antigen profile that included, in addition to gp63, 72-, 52-, 48-, 45-, 39-, and 20-kDa components in neutral and positively charged liposomes contrasted with the reactivity of a greater number of LAg components in negatively charged liposomes. Resistance to visceral leishmaniasis appears to depend on the immunity induced by gp63 and a few select antigens in association with the right liposomes. A striking similarity between the immunogenic profile of partially purified soluble antigens and that of LAg in neutral and positively charged liposomes suggests the potentiality of these antigens in future vaccine studies of L. donovani.

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca2+ level is observed due to release of Ca2+ from internal stores. Increase in cytosolic Ca2+ causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca2+ by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-l-cysteine, GSH, and l-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca2+ chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca2+-induced mitochondrial toxicity but independent of caspase-like proteases.

Ascorbate peroxidase from Leishmania major controls the virulence of infective stage of promastigotes by regulating oxidative stress.

Background Peroxidase represents a heterogeneous group of distinct enzyme family that plays extremely diverse biological functions. Ascorbate peroxidase from Leishmania major (LmAPX) has been shown to be central to the redox defense system of Leishmania. To investigate further its exact physiological role in Leishmania, we attempted to create LmAPX -knockout mutants by gene replacement in L. major strains. Methodology/Principal Findings The null mutant cell culture contains a higher percentage of metacyclic and apoptotic cells compared to both wild type and LmAPX overexpressing cells. Flowcytometric analysis reveals the presence of a higher concentration of intracellular H2O2, indicative of increased oxidative stress in parasites lacking LmAPX. IC50 value for exogenously added H2O2 shows that deletion of LmAPX in L. major renders the cell more susceptible to H2O2. Real time PCR studies demonstrate an elevated mRNA level of non-selenium glutathione peroxidase in LmAPX null mutant cell line, suggesting that these enzymes were induced to compensate the LmAPX enzyme. The null mutant cells exhibit hypervirulence after infection with macrophages as well as inoculation into BALB/c mice; in contrast, overexpressing cells show avirulence. Conclusions/Significance Collectively, these data provide strong evidence that LmAPX is an important factor for controlling parasite differentiation and survival within macrophages.

Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

ABSTRACT Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H2O2 or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca2+ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H2O2- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H2O2 or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.

Leishmania Major Ascorbate Peroxidase Overexpression Protects Cells Against Reactive Oxygen Species-Mediated Cardiolipin Oxidation

Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed, and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions revealed that LmAPX was localized within the mitochondria by its N-terminal signal sequence. Subcellular fractionation analysis of the cell homogenate by the Percoll density-gradient method and subsequent Western blot analysis with anti-LmAPX antibody further confirmed the mitochondrial localization of mature LmAPX. Submitochondrial fractionation analysis showed that the mature enzyme (~3.6 kDa shorter than the theoretical value of the whole gene) was present in the intermembrane space side of the inner membrane. Moreover, expression of the LmAPX gene was increased by treatment with exogenous H(2)O(2), indicating that LmAPX was induced by oxidative stress. To investigate the biological role of LmAPX we generated Leishmania cells overexpressing LmAPX in the mitochondria. Flow-cytometric analysis, thin-layer chromatography, and IC(50) measurements suggested that overexpression of LmAPX caused depletion of the mitochondrial ROS burden and conferred a protection against mitochondrial cardiolipin oxidation and increased tolerance to H(2)O(2). These results suggest that the single-copy LmAPX gene plays a protective role against oxidative damage.

Combination Therapy Using Sodium Antimony Gluconate in Stearylamine-Bearing Liposomes against Established and Chronic Leishmania donovani Infection in BALB/c Mice

ABSTRACT In this work we report the activity seen with combination therapy using sodium antimony gluconate in liposomes composed of egg phosphatidyl choline and stearylamine for elimination of Leishmania donovani parasites from the liver and spleen of BALB/c mice with established and chronic infections.

Role of Tryptophan-208 Residue in Cytochrome c Oxidation by Ascorbate Peroxidase From Leishmania Major-Kinetic Studies on Trp208Phe Mutant and Wild Type Enzyme

Ascorbate peroxidase from L. Major (LmAPX) is a functional hybrid between cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX). We utilized point mutagenesis to investigate if a conserved proximal tryptophan residue (Trp208) among Class I peroxidase helps in controlling catalysis. The mutant W208F enzyme had no effect on both apparent dissociation constant of the enzyme-cytochrome c complex and K(m) value for cytochrome c indicating that cytochrome c binding affinity to the enzyme did not alter after mutation. Surprisingly, the mutant was 1000 times less active than the wild type in cytochrome c oxidation without affecting the second order rate constant of compound I formation. Our diode array stopped-flow spectral studies showed that the substrate unbound wild type enzyme reacts with H(2)O(2) to form compound I (compound II type spectrum), which was quite different from that of compound I in W208F mutant as well as horseradish peroxidase (HRP). The spectrum of the compound I in wild type LmAPX showed a red shift from 409 nm to 420 nm with equal intensity, which was broadly similar to those of known Trp radical. In case of compound I for W208F mutant, the peak in the Soret region was decreased in heme intensity at 409 nm and was not shifted to 420 nm suggesting this type of spectrum was similar to that of the known porphyrin pi-cation radical. In case of an enzyme-H(2)O(2)-ascorbate system, the kinetic for formation and decay of compound I and II of a mutant enzyme was almost identical to that of a wild type enzyme. Thus, the results of cytochrome c binding, compound I formation rate and activity assay suggested that Trp208 in LmAPX was essential for electron transfer from cytochrome c to heme ferryl but was not indispensable for ascorbate or guaiacol oxidation.

Ascorbate Peroxidase Acts As a Novel Determiner of Redox Homeostasis in Leishmania

SIGNIFICANCE Reactive oxygen species (ROS) are produced as natural byproducts of metabolism and respiration. While physiological levels of ROS are required for vital cellular functions (e.g., development and proliferation), a living organism is faced with constant challenges due to accumulation or overproduction of ROS throughout its life. The life cycle of Leishmania parasite has led it to confront the highly oxidizing environment in the macrophage phagosomes, necessitating ROS homeostasis and signaling as key strategies for successful survival and pathogenicity. RECENT ADVANCES Ascorbate peroxidase from Leishmania major (LmAPX) is the only heme peroxidase identified so far in Leishmania. Structural analysis and functional characterization of LmAPX have yielded interesting and novel insight on this enzyme. The protein has been found to be a hybrid of cytochrome c peroxidase and ascorbate peroxidase. This enzyme is colocalized with cytochrome c in the inner mitochondrial membrane facing the intermembrane space and shows higher activity toward cytochrome c oxidation. CRITICAL ISSUES Overexpression of LmAPX in L. major cells confers tolerance to oxidative stress-mediated cardiolipin oxidation and consequently protects cells from extensive protein damage. LmAPX-/- mutants show higher intracellular hydrogen peroxide (H₂O₂), which might signal for cellular transformation from noninfective procyclic to infective metacyclic form and ultimately apoptosis. FUTURE DIRECTIONS Manipulation of LmAPX expression has significantly added to the present understanding of the parasites defense network against oxidative damage caused by H₂O₂. The future investigations will address more exactly the signaling pathways involved in redox homeostasis.

Role of C-terminal acidic cluster in stabilization of heme spin state of ascorbate peroxidase from Leishmania major

Architecture of hemoprotein is solely responsible for different nature of heme coordination. Here we report that substitution of the acidic surface residue Glu226 to Ala in ascorbate peroxidase from Leishmania major alters the 5 coordinate high spin (5cHS) to a 6 coordinate low spin (6cLS) form at pH 7.5. Using UV-visible spectrophotometry, we show that the sixth ligand of heme in Glu226Ala at pH 7.5 is hydroxo. When the pH is decreased to 5.5, a new species of Glu226Ala appeared that had a spectrum characteristic of a 6cHS derivative. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I was not seen in the Glu226Ala in presence of H(2)O(2). Similarly guaiacol, ascorbate and ferrocytochrome c oxidation rate was 10(3) orders less for the Glu226Ala mutants compared to the wild type. These data suggested that surface acidic residue Glu226 might play role in proper maintenance of active site conformation.

Role of proximal methionine residues in Leishmania major peroxidase.

The active site architecture of Leishmania major peroxidase (LmP) is very similar with both cytochrome c peroxidase and ascorbate peroxidase. We utilized point mutagenesis to investigate if the conserved proximal methionine residues (Met248 and Met249) in LmP help in controlling catalysis. Steady-state kinetics of methionine mutants shows that ferrocytochrome c oxidation is <2% of wild type levels without affecting the second order rate constant of first phase of Compound I formation, while the activity toward a small molecule substrate, guaiacol or iodide, increases. Our diode array stopped-flow spectral studies show that the porphyrin π-cation radical of Compound I in mutant LmP is more stable than wild type enzyme. These results suggest that the electronegative sulfur atoms of the proximal pocket are critical factors for controlling the location of a stable Compound I radical in heme peroxidases and are important in the oxidation of ferrocytochrome c.