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

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.In multicellular organisms, mitochondria are the major physiological source of reactive oxygen species (ROS) within cells and also are important checkpoints for the control of programmed cell death (27). There are increasing numbers of reports that describe apoptosis- or programmed cell death-like processes in unicellular organisms also, such as trypanosomatids (4, 60), bacteria (20, 25), yeasts (34), and Plasmodium (3). Among the kinetoplastid parasites, Trypanosoma and Leishmania are the most carefully studied genera where apoptotic features are well established (49). Several reports have shown that mitochondrial dysfunction or an imbalance of antioxidant homeostasis causes an increase in mitochondrion-generated ROS, which include H₂O₂, superoxide radical anions, singlet oxygen, and hydroxyl radicals. These species have all been implicated in apoptosis (16, 26, 28, 41). Increasing evidence has been presented to support that ROS homeostasis regulates two major types of important physiological processes and exerts diverse functions within cells. One type of function includes damage or oxidation of cellular macromolecules (DNA, proteins, and lipids), which can lead to necrotic cell death or protein modification (7). The second type of function includes the activation of cellular signaling cascades that regulate proliferation, detoxification, DNA repair, or apoptosis (11). The detoxification of toxic mitochondrial ROS in cells occurs through a variety of cellular antioxidant enzymes, such as superoxide dismutase, which detoxifies cells from superoxide released into the mitochondrial matrix, and several other antioxidant proteins, such as catalase, glutathione (GSH) peroxidase, and peroxiredoxins, which are known to catalyze further degradation of H₂O₂ (44). During its life cycle, the Leishmania sp. encounters a pool of ROS that is generated either by its own physiological processes or as a result of host immune reaction and drug metabolism. However, unlike most eukaryotes, Leishmania lacks catalase- and selenium-containing GSH peroxidases, enzymes that play a front-line role in detoxifying ROS. Hence, the mechanism by which it resists the toxic effects of H₂O₂ remains poorly understood.Recently, we cloned, expressed and characterized the unusual heme-containing ascorbate peroxidase from Leishmania major (LmAPX) and observed that the expression of LmAPX is increased when Leishmania cells are treated with exogenous H₂O₂ (1, 18). This enzyme is a functional hybrid between cytochrome c peroxidase and APX, owing to its ability to use both ascorbate and cytochrome c as reducing electron donors (58). Colocalization studies by confocal microscopy, submitochondrial fractionation analysis of the isolated mitochondria, and subsequent Western blot analysis with anti-LmAPX antibody have confirmed that the mature enzyme is present in intermembrane space side of the inner membrane. It has also been shown that overexpression of LmAPX causes a decrease in the mitochondrial ROS burden, an increase in tolerance to H₂O₂, and protection against cardiolipin oxidation under oxidative stress (18). Although previous studies have shown that Leishmania species use superoxide dismutase (23), peroxiredoxins (8), intracellular thiols (14), lipophosphoglycan (13), trypanothione (5), HSP 70 (a heat shock protein) (36), tryparedoxin peroxidase (29), and APX (18) for detoxification of ROS, it is still unclear how the antioxidants protect against oxidative stress-induced apoptotic events in the unicellular organism Leishmania.Since the LmAPX protein is localized in the mitochondria, we hypothesized that it would be a key protein for the maintenance of mitochondrial functions due to its antioxidant properties via its ROS-scavenging function (18). To test this hypothesis, we overexpressed LmAPX in Leishmania major cells and investigated whether overexpression of LmAPX can confer resistance to oxidant-mediated mitochondrial damage as well as oxidative stress-induced cell death. In this study, we provide evidence that the overexpression of LmAPX in Leishmania cells can indeed protect against camptothecin (CPT) or H₂O₂-mediated mitochondrial damage as measured by various parameters, including disruption of mitochondrial membrane potential (Δψ_m), decrease of ATP production, and cytochrome c and endonuclease G release from mitochondria. Cells overexpressing LmAPX were also protected against oxidative stress-induced protein carbonylation, DNA fragmentation, and apoptosis. To the best of our knowledge, this is the first report of a mitochondrial hemeperoxidase that controls the ROS-induced mitochondrial death pathway.     

More membranes, more proteins: complex protein import mechanisms into secondary plastids

Plastids are found across the tree of life in a tremendous diversity of life forms. Surprisingly they are not limited to photosynthetic organisms but also found in numerous predators and parasites. An important reason for the pervasiveness of plastids has been their ability to move laterally and to jump from one branch of the tree of life to the next through secondary endosymbiosis. Eukaryotic algae have entered endosymbiotic relationships with other eukaryotes on multiple independent occasions. The descendants of these endosymbiotic events now carry complex plastids, organelles that are bound by three or even four membranes. As in all endosymbiotic organelles most of the symbiont's genes have been transferred to the host and their protein products have to be imported into the organelle. As four membranes might suggest, this is a complex process. The emerging mechanisms display a series of translocons that mirror the divergent ancestry of the membranes they cross. This review is written from the viewpoint of a parasite biologist and seeks to provide a brief overview of plastid evolution in particular for readers not already familiar with plant and algal biology and then focuses on recent molecular discoveries using genetically tractable Apicomplexa and diatoms.     

Treatment of spentwash using chemically modified bagasse and colour removal studies

Studies were carried out on derivatisation of bagasse into an ion exchange material and application of this chemically modified bagasse in the treatment of distillery wastewater. It was found that CHPTAC bagasse with HCl treatment and DEAE-bagasse in its free base form were most effective in colour removal and the mechanism of colour removal indicated significant contribution of both, the conventional ion exchange and the chemical sorption.     

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 Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) 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 Ca(2+) 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 Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.     

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C₃₀ carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.     

Phylogeography of the spring and fall waves of the H1N1/09 pandemic influenza virus in the United States

Spatial variation in the epidemiological patterns of successive waves of pandemic influenza virus in humans has been documented throughout the 20th century but never understood at a molecular level. However, the unprecedented intensity of sampling and whole-genome sequencing of the H1N1/09 pandemic virus now makes such an approach possible. To determine whether the spring and fall waves of the H1N1/09 influenza pandemic were associated with different epidemiological patterns, we undertook a large-scale phylogeographic analysis of viruses sampled from three localities in the United States. Analysis of genomic and epidemiological data reveals distinct spatial heterogeneities associated with the first pandemic wave, March to July 2009, in Houston, TX, Milwaukee, WI, and New York State. In Houston, no specific H1N1/09 viral lineage dominated during the spring of 2009, a period when little epidemiological activity was observed in Texas. In contrast, major pandemic outbreaks occurred at this time in Milwaukee and New York State, each dominated by a different viral lineage and resulting from strong founder effects. During the second pandemic wave, beginning in August 2009, all three U.S. localities were dominated by a single viral lineage, that which had been dominant in New York during wave 1. Hence, during this second phase of the pandemic, extensive viral migration and mixing diffused the spatially defined population structure that had characterized wave 1, amplifying the one viral lineage that had dominated early on in one of the world's largest international travel centers.     

Common HIV-1 peptide variants mediate differential binding of KIR3DL1 to HLA-Bw4 molecules

Epidemiological studies have shown the protective effect of KIR3DL1/HLA-Bw4 genotypes in human immunodeficiency virus type 1 (HIV-1) infection; however, the functional correlates for the protective effect remain unknown. We investigated whether human leukocyte antigen (HLA)-Bw4-presented HIV-1 peptides could affect the interaction between the inhibitory natural killer (NK) cell receptor KIR3DL1 and its ligand HLA-Bw4. Distinct HIV-1 epitopes differentially modulated the binding of KIR3DL1 to HLA-Bw4. Furthermore, cytotoxic T lymphocyte (CTL) escape mutations within the immunodominant HLA-B57 (Bw4)-restricted Gag epitope TSTLQEQIGW abrogated KIR3DL1 binding to HLA-B57, suggesting that sensing of CTL escape variants by NK cells can contribute to the protective effect of the KIR3DL1/HLA-Bw4 compound genotype.     

The Cry Toxin Operon of Clostridium bifermentans subsp. malaysia Is Highly Toxic to Aedes Larval Mosquitoes

The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity. We show here that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though C. bifermentans subsp. malaysia is more toxic to Anopheles. Hence, toxins that target Anopheles are different from those expressed by the Cry operon.