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

Abstract

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 parasite's defense network against oxidative damage caused by H₂O₂. The future investigations will address more exactly the signaling pathways involved in redox homeostasis. Antioxid. Redox Signal. 19, 746–754.

Introduction

L eishmaniasis is a disease caused by the trypanosomatid parasites belonging to the genus Leishmania spp. and is transmitted by the bite of certain species of sandfly (6). The burden of leishmaniasis is 2 million new cases annually and 14 million infected people worldwide (www.who.int/gb/ebwha/pdf_files/EB118/B118_4-en.pdf. Accessed on 19th December 2008). Generally, Leishmania species exist as flagellated extracellular promastigotes in the alimentary tract of the sandfly vector (39) and as aflagellar intracellular amastigotes within the phagolysosomes of the macrophages of their vertebrate hosts (11). In the gut of the female Phlebotomine sandflies, the promastigotes go through various morphological stages. During infection, the complement pathway triggers programmed cell death in non-infective procyclics, while the infective metacyclics escape the assault, get themselves attached to the surface of the macrophages, and are phagocytosed (62), whereupon they transform into aflagellated amastigotes. The procyclic to metacyclic differentiation is associated with increased infectivity to the mammalian host, where the parasite invades and multiplies in specialized phagocytic cells like macrophages and thus has to resist the microbiostatic and microbicidal mechanisms, for example, oxidative burst, for successful colonization. However, unlike most eukaryotes, Leishmania lack catalase and selenium-containing glutathione peroxidase (GPX), enzymes capable of rapidly metabolizing hydrogen peroxide (H₂O₂). Nevertheless, Leishmania are equipped with a robust trypanothione-dependent H₂O₂-detoxifying system (10). But, interestingly, detoxification of H₂O₂ by amastigotes is markedly attenuated by aminotriazole and sodium azide, which are well-known inhibitors of heme-containing enzymes like catalase or peroxidase (12). Throughout this review, the ascorbate peroxidase from Leishmania major (LmAPX) will be presented from a structure–function point of view, emphasizing on its contribution to the redox homeostasis of the parasite.

Redox Homeostasis in Leishmania

Reactive oxygen species (ROS) in Leishmania are generated by means of cellular metabolism, uncoupled electron transfer in mitochondria, drug metabolism, exogenous agents, etc. Right after phagocytosis, as the first line of defense, the macrophage produces ROS, like superoxide anion (O₂ ^•−) and H₂O₂, with the help of NADPH oxidase, a phenomenon known as oxidative burst. Once the infection is established, the proinflammatory cytokines like tumor necrosis factor-α (TNF-α) and interferon-γ activate the macrophage to produce reactive nitrogen species (RNS) by upregulating inducible nitric oxide synthase expression (25). Also, the production of hypohalide by myeloperoxidase or eosinophil peroxidase and peroxynitrite (RNS) as secondary oxidants is observed as a result of oxidative burst (21). Small (physiological) amounts of ROS are a cellular requirement, because they are involved in signaling pathways, inducing and regulating a variety of cellular activities for example, growth, differentiation, and gene expression (32). However, excessive ROS have the potential to induce significant biological damage and, hence, cells possess many antioxidant systems for maintaining the ROS threshold at physiological concentrations (Fig. 1). Oxidative stress can occur when ROS production is increased or when the mechanisms involved in maintaining the normal reductive cellular milieu are impaired. Redox active thiol groups in proteins, superoxide dismutase (SOD), catalase, and low-molecular mass compounds, such as ascorbic acid, tocopherol, thiols including glutathione, and uric acid play key roles as redox buffers that balance any disturbance of the intracellular redox state (43, 60). Depending on the level and extension of the oxidative stress, the cells may establish short- or long-term adaptive responses (40). The redox homeostasis in Leishmania parasites appears to be efficiently regulated since they can successfully withstand the oxidative burst during host infection and perfectly adapt to the different metabolic and environmental conditions imposed by their digenetic life cycle (40). The Leishmania are equipped with SOD, tryparedoxin (TXN), TXN-dependent peroxidases and ascorbate peroxidase (APX) to maintain cellular redox equilibrium (3, 8, 9, 24, 26, 27, 52, 63). In addition, Leishmania produces significant amounts of low-molecular mass thiols such as trypanothione, glutathione, and ovothiol A (2, 16, 24). Of these, only glutathione is present in cells of the host (30, 49). All the three thiols are directly or indirectly maintained in a reduced state by trypanothione reductase (TR) (64). Most of the glutathione content in Leishmania is found in the form of trypanothione (N1,N8-bis(glutathionyl) spermidine), a unique thiol consisting of two glutathione molecules joined by a spermidine linker that is produced by trypanothione synthetase (51). As in other organisms, in Leishmania also, the enzymes γ-glutamylcysteine synthetase and glutathione synthetase successively act for the synthesis of glutathione (49). The second order rate constants for reactions with H₂O₂ have proved ovothiol A to be functionally less efficient than trypanothione; however, in promastigote stages of L. major, L. Mexicana, and L. aethiopica it can be considered as the principal thiol, particularly in the late logarithmic and stationary phases of growth (2). Ovothiol A and trypanothione can reduce H₂O₂ nonenzymatically. The anti-oxidative armamentaria of Leishmania also include TXN and tryparedoxin peroxidases. TXNs belong to the thioredoxin-fold family but trypanosomatids lack thioredoxin reductase (58). Unlike typical thioredoxins, which are directly reduced by NADPH-dependent flavoreductases, Leishmania TXNs are oxidoreductases requiring trypanothione as the mediator to take up electrons from the flavoprotein TR. Two classes of peroxidases occur in trypanosomatids: the non-selenium peroxiredoxin and the cysteine GPX members, which reduce H₂O₂ using electrons from TXN (9). Leishmania can synthesize ascorbic acid, which is a powerful antioxidant (4, 68). Very recently, in L. major an ascorbate-dependent peroxidase has been detected as a part of the organism's antioxidant defense system. As biochemical and functional characterization of LmAPX is the subject matter of this review, a brief introduction about peroxidases is given below.

FIG. 1.

The key players involved in redox homeostasis of Leishmania life cycle.

Peroxidases: An Overview

Peroxidases hold a venerable position in enzymology. This is one of the most extensively studied group of enzymes and literature has been enriched with the reviews and a large number of basic research articles dating back to the early part of the 19th century. Peroxidases are mainly classified into heme- and non-heme-containing peroxidases. The native heme peroxidases contain a heme prosthetic group, usually ferriprotoporphyrin IX, with four pyrrole nitrogens bound to the Fe (III). The fifth coordination position on the proximal side of the heme is usually the imidazole side chain of a histidine residue. The sixth coordination position is vacant in the native enzyme on the distal side of the heme. The distal cavity is the region in which the peroxidase reactions occur. The peroxidative reaction cycle follows the initial formation of compound I followed by compound II formation and regeneration of native enzyme. Compound I and compound II are the two and one electron-oxidizing equivalents higher than native ferric state of peroxidase respectively (21). Hugo Theorell was awarded Nobel Prize in 1955 for the discovery of horseradish peroxidase (HRP) compound I. A major breakthrough was the determination of the X-ray crystal structure of yeast cytochrome c peroxidase (CCP) in the year 1980. These discoveries established both HRP and CCP as model peroxidases, based on which structure-function studies of other peroxidases are carried out. Heme peroxidases are distributed throughout plant and animal kingdom. All currently known heme-containing peroxidases can be divided in two main superfamilies (peroxidase–cyclooxygenase and peroxidase–catalase superfamily) and three families (di-heme peroxidase, dyp-type heme peroxidase, and haloperoxidase) (73). The peroxidase–catalase superfamily is further divided into three distinct classes. Class I peroxidases include intracellular peroxidases of prokaryotic origin-like plant chloroplast and cytosolic APX, yeast CCP, and gene duplicated catalase-peroxidase. Analyses of the amino acid sequences of catalase-peroxidase indicate that the double length of the bacterial peroxidases can be ascribed to gene duplication (66). Class II peroxidases comprise fungal secretory peroxidases like lignin and manganese peroxidases from Phanerochaete chrysosporium and the ink cap mushroom peroxidase from Coprinus cinereus (21). Examples of Class III peroxidases are endoplasmic reticulum mediated classical secretory plant peroxidases like HRP isoenzyme C. During 1940–1960s a large number of animal peroxidases were characterized and their physiological functions revealed. Plant ascorbate dependent peroxidase was discovered in 1979 (29). APX from pea is well characterized biochemically and the crystal structure has been solved in great detail in the year 1995 (55). In plants, different isoforms of APX are shown to localize in chloroplast, cytosol, and microbody where they eliminate photosynthetically generated H₂O₂ by glutathione/ascorbate cycle and control H₂O₂-mediated redox signaling (47).

GPXs and vanadium haloperoxidases lack heme in their active sites. GPXs share a common catalytic core formed by selenocysteine (Sec), tryptophan, and a glutamine residue. However, in some GPXs, a Cys replaces the Sec residue. Such substitution, characteristic of the so-called nonselenium GPX-like enzymes, confers decreased peroxidase activity to these proteins (33). Vanadium haloperoxidase contains a vanadate prosthetic group and utilize H₂O₂ to oxidize a halide ion into a reactive electrophilic intermediate (69).

Ascorbate and APX in Leishmania

l-Ascorbic acid or vitamin C acts as a pivotal antioxidant molecule. It is also used as a cofactor in various enzymatic reactions. Humans lack the ability to synthesize ascorbate, whereas a vast majority of plants and animals can synthesize ascorbic acid de novo. Although, presence of ascorbate has long been detected in trypanosomatids (15), only recently the biosynthetic pathway has been demonstrated in Trypanosoma brucei, T. cruzi, and L. donovani (4, 68). Biosynthesis of ascorbate in both the kinetoplastids takes place within glycosomal compartment. Detection of ascorbate-dependent peroxidase activity in T. cruzi epimastigote extracts dates back to 1980 (5) and in 2002, an endoplasmic reticulum-located unusual plant-like APX enzyme has been discovered in the organism (67). But, until recently, apart from the Leishmania genome database, which predicted the presence of the putative enzyme in the organism, nothing was known about the enzyme in Leishmania. Our laboratory has reported, for the first time, the presence of an ascorbate-dependent peroxidase from L. major in 2005. LmAPX is more related to T. cruzi APX than to plant APX. In contrast to plant APX, LmAPX lacks arginine 172 residue at the ascorbate-binding site.

Physical and Spectral Characteristics of LmAPX

LmAPX primary sequence has the high level of sequence identity with both yeast CCP (∼35%) and the pea APX (∼36%) (70). In silico analysis of LmAPX sequence (TargetP V1.0) predicts (22) that the N-terminal 12 amino acids constitute a signal sequence, which is followed by a stretch of 22 amino acids containing a hydrophobic region resembling a transmembrane domain (Fig. 2A). All the key residues on both distal and proximal sites of the heme are conserved among LmAPX, yeast CCP, and plant APX. However, it lacks both the cytochrome c-binding residues (Asp34/Glu35, Tyr39, and Glu290 in yeast CCP) and critical ascorbate-binding residue (Arg 172 in pea APX) (7, 56, 59). The proximal cation (K⁺) binding loop of the LmAPX is more similar to pea APX, whereas the c-terminal insertion and proximal Trp radical stabilizing Met residues of LmAPX are more similar to CCP (35, 70). The gel filtration results indicate that Δ34 LmAPX (34 amino acids deleted from the N-terminus sequence of LmAPX) is a monomeric enzyme with a molecular weight of ∼33 kDa (1). The UV-visible spectra of ascorbate-free Δ34 LmAPX shows the Soret peak at 408 nm with charge transfer peaks at around 500 and 640 nm (Fig. 2B). The calculated purity number, Rz (A₄₀₈/A₂₈₀), for Δ34 LmAPX is 0.98 (Fig. 2B). Addition of an equimolar H₂O₂ to resting state of Δ34 LmAPX produces compound I* within 0.75 ms absorbing at 420 nm at Soret region with visible peaks at 532 and 560 nm. Extensive spectroscopic studies have demonstrated that the Soret band of compound I* is similar to an oxyferryl tryptophan cation radical of yeast CCP compound I species (23).

FIG. 2.

Physical characteristics of LmAPX. (A) Shows the difference between premature and mature protein. (B) Represents the UV-visible spectra of ascorbate-free LmAPX before (solid line) and after addition of equimolar H₂O₂ (dotted line), and after addition of 5 μM ascorbate (dashed line). H₂O₂, hydrogen peroxide; LmAPX, ascorbate peroxidase from Leishmania major.

Catalytic Mechanism of LmAPX

Based on previous yeast CCP, plant APX, and current LmAPX results (35, 54, 70, 72), a plausible mechanism for the redox reaction catalyzed by LmAPX may be suggested as shown in Figure 3. In the presence of low-molecular mass (micromolecule) electron donors like ascorbate or guaiacol (2-methoxyphenol), the reaction of H₂O₂ with the resting form of LmAPX (Fe^III,P,Trp) leads to the formation of CMPI (Fe^IV=O,P^•+,Trp), which contains an oxyferryl heme Fe^IV=O and a porphyrin π-cation radical. Low-molecular mass electron donor initially reduces π-cation radical of CMPI (Fe^IV=O,P^•+,Trp) to produce compound II (Fe^IV=O,P,Trp), which contains an oxyferryl heme Fe^IV=O. A second molecule of the electron donor then reduces the oxyferryl heme in CMPII (Fe^IV=O,P,Trp) to form the resting enzyme (Fe^III,P,Trp). However, in the presence of cytochrome c, the situation is altered. Indeed, one of the curious features of LmAPX is that, in contrast to plant APX, its CMPI (Fe^IV=O,P^•+,Trp) is most probably comparatively unstable and, it leads to the generation of another species CMPI* (Fe^IV=O,P,Trp^•+), which contains an oxyferryl heme Fe^IV=O and a Trp^•+ radical cation located on the indole ring of Trp-208. Ferrocytochrome c initially reduces the Trp-208 radical cation in CMPI* (Fe^IV=O,P,Trp^•+) to produce compound II (Fe^IV=O,P,Trp). By internal electron transfer in CMPII (Fe^IV=O,P,Trp) the Trp radical cation in CMPII* (Fe^III,P,Trp^•+) is regenerated, which is then reduced by cytochrome c to form the native enzyme (48).

FIG. 3.

Universal model for the macro- and micromolecule oxidation by heme peroxidase. A common hemeferryl protoporphyrin π-cation radical (^+•P Fe^IV=O Trp) intermediate can react with a micromolecule (RH). If the electron is transferred from the proximal tryptophan to the ferryl protoporphyrin π-cation radical, a tryptophan radical (P Fe^IV=O Trp^+•) is generated, that can react with the macromolecule ferrocytochrome c (Cyt C^II). This ensures the route of electron transfer from the ferrocytochrome c to heme iron via proximal tryptophan residue. RH, Fe^IV=O, P, Trp indicate low-molecular mass electron donors, ferryl heme iron, porphyrin, and proximal tryptophan residue respectively.

Pseudocatalase Activity of LmAPX

In addition to peroxidase activity of lactoperoxidase and thyroid peroxidase, several reports have shown that in case of iodide as electron donor, the intermediate enzyme with bound hypoiodous acid reacts with a second molecule of H₂O₂ in a catalase-like reaction, with liberation of O₂ (34, 44, 45). The pseudohalide, SCN⁻, cannot replace I⁻ as a promoter of pseudocatalase activity, even though SCN⁻ is readily oxidized by thyroid peroxidase or lactoperoxidase in presence of H₂O₂ (34, 44, 45). In this case, H₂O₂ is utilized solely for the oxidation of SCN⁻, in contrast to the catalytic cleavage of H₂O₂, which occurs in the presence of low I⁻ concentration (34, 44, 45). Like well known peroxidases, LmAPX catalyzes the degradation of H₂O₂ with concomitant formation of O₂ under low ascorbate concentration at physiological pH (18). The reaction is initiated by its peroxidase activity with the generation of ascorbate-free radicals via one-electron-transfer mechanism and then regeneration of ascorbate by reduction of ascorbate radical with H₂O₂. The requirement of enzyme, H₂O₂ and ascorbate indicates that monodehydroascorbate plays a vital role in free radical mediated H₂O₂ consumption, rather than the disproportionation reaction of the free radicals.

Structural Investigation of the Catalytic Domain of LmAPX

The structure of the distal pocket, proximal pocket, and the cation-binding loop in LmAPX, yeast CCP, and plant pea APX is shown in Figure 4. The 1.76 Å resolution of LmAPX structure demonstrates 10 alpha helical bundle folds, characteristic of peroxidases (35). The distal Arg-Trp-His and proximal His-Asp-Trp arrangements in LmAPX are typical of Class I peroxidases, which include CCP and APX. The electron density of LmAPX shows two cations, a proximal K⁺ that is visible up to 20 σ in the Fo-Fc difference map and a distal Ca²⁺ visible up to 26 σ in the same map (35). As expected from sequence alignments, LmAPX can be considered as a hybrid peroxidase that shares structural features with various classes of heme peroxidases. Like class III peroxidases such as HRP, LmAPX contains two cation-binding sites. The following structural features are similar between yeast CCP and LmAPX (35): (i) presence of the triple beta strand, (ii) the proximal Met residues (Met248 and Met249) that are responsible for formation of the stable proximal Trp208 radical (35, 72), and (iii) a stretch of acidic residues (D225, E226, and D227) before a c-terminal sequence insertion (228–240 aa), one of them (E226) is responsible for proper maintenance of active site conformation (71). Again, some plant APX-like structural elements, present in LmAPX are: (i) the insert region between A and B helices, which provides contacts with cytochrome c in the CCP–cytochrome c complex (56), is much shorter in LmAPX and forms part of the ascorbate binding pocket and (ii) the monovalent nature of the proximal cation (35). Analysis of the proximal K⁺ cation-binding loop in LmAPX indicates that three main-chain ligands, three side-chain ligands, and one water ligand are bonded to K⁺ (35). Those seven coordinations inside the loop are formed by residues 193 (164 in pea APX and 176 in yeast CCP) to 218 (189 in pea APX and 201 in yeast CCP) in LmAPX. In LmAPX, T193 (side chain OH, back bone CO), T209 (side chain OH), D211 (back bone CO), G214 (back bone CO), and S218 (side chain OH) amino acid residues, and H₂O ligand contribute to form coordinate bonds with K⁺. LmAPX and pea APX differ in coordination bond formation: (i) H₂O molecule and D211 residue in LmAPX form one coordinate bond each with K⁺, whereas the corresponding residue N182 in pea APX forms two coordinate bonds. (ii) One coordinate bond is formed by S218 in LmAPX but the corresponding residue S189 in pea APX does not form coordinate bond. Instead, D187 residue in pea APX takes part in coordinate bonding. However, LmAPX has Phe201 residue in the ascorbate-binding site instead of Arg residue (Arg172 in plant pea APX) (56, 59), which may account for the lower APX activity of LmAPX, 32 min⁻¹ compared with ∼250 s⁻¹ for plant APX (42). The main structural dissimilarity between LmAPX and CCP involves the presence of Cys197 in LmAPX, whereas the analogous residue in CCP is Thr180. Mutational and electron paramagnetic resonance studies suggest that this major structural difference plays a key role in LmAPX for stabilization of proximal Trp radical in the presence of K⁺ ion (35). Other reports reveal that in plant APX and LmAPX, the K⁺-binding residues function in maintaining the protein structure in the heme vicinity to favor the enzymatic activity by controlling the specific structural conformation of the proximal and distal histidines (14, 53). Recently, a model of the L. major APX–cytochrome c complex, using the yeast system as a guide, suggests that its complex is very close to the yeast CCP–cytochrome c crystal structure (36). K_M and the efficiency (k_cat /K_M) of LmAPX for L. major cytochrome c are 6 μM and 1.6×10⁸ M ⁻¹ s⁻¹, respectively, similar to those exhibited by CCP in the yeast system (36, 38). It has been suggested that the biological function of LmAPX is to act as a CCP (36).

FIG. 4.

X-ray crystal structure of LmAPX, pea APX, and yeast CCP. The PDB coordinates were taken from published LmAPX (PDB entry code: 3RIV), pea APX (PDB entry code: 1APX), and yeast CCP (PDB entry code: 2CCP). (A–C) Showed cartoon structures of LmAPX, yeast CCP, and pea APX respectively, where the heme group is superimposed. (D–F) Shows the active site residues of LmAPX, yeast CCP, and pea APX respectively. Structures were illustrated by using PyMOL (17) software. The alternative designation of LmAPX is LmP (peroxidase from L. major). APX, ascorbate peroxidase; CCP, cytochrome c peroxidase; PDB, protein data bank.

Localization and Regulation of LmAPX

Localization and regulation of a protein provides vital information for a group of many co-operating proteins, which are responsible for an integrated physiological function. Thus, exact information regarding the localization of LmAPX will be supportive for predicting the possible physiological function of this protein. TargetP 1.1 Server predicts that the N-terminal signal sequence of LmAPX is a mitochondrial targeting signal. Subcellular fractionation, Western-blot analysis, and confocal microscopy suggest that the enzyme is localized in the mitochondrion of Leishmania (19). Mitoplast (mitochondria without outer membrane) preparation by treatment of digitonin, sub-mitochondrial fractionation, and activity measurement of kynurenine hydroxylase (outer membrane marker), succinate dehydrogenase (inner membrane marker), and malate dehydrogenase (matrix marker) enzymes have revealed that LmAPX is localized in the inner membrane of the mitochondria with its catalytic domain in the intermembrane space (19), which is similar to the location of CCP in yeast (13, 28, 37). Therefore, LmAPX is more likely to be accessible to scavenge endogenous ROS produced in the mitochondrial inter membrane space. Although, chloroplasts and peroxisomes in plants are the main organelles in terms of both ROS formation and breakdown (50), yet intermembrane space of mitochondria is clearly the major site of ROS generation in yeast and animal systems. The expression of yeast CCP gene and plant APX gene was also increased on treatment with various oxidative stressors (41, 61). Similarly, upregulation of LmAPX gene expression by H₂O₂ suggests that the parasite sequentially uses this enzyme to overcome oxidative stress (19).

Physiological Function of LmAPX

In a wide variety of species, ranging from bacteria to higher mammals, the mechanisms of H₂O₂-detoxification are grossly distributed into variations of two mechanisms: mediated either by the heme proteins and/or by non-heme proteins like selenium-containing GPX. Detailed cell biological approaches have successfully established the biochemistry and physiological significance of LmAPX enzyme in Leishmania promastigotes (see working model on Fig. 5) (19, 20, 52). Overexpression of LmAPX in L. major protects promastigote cells against oxidative stress-induced apoptosis (19, 20). Clearly, knocking the gene out of the cell has thrown the parasite under a constant level of oxidative stress as evidenced by the higher intracellular H₂O₂ content (52). This basal level of oxidative stress acts as the driving force for the early onset of metacyclogenesis producing infective metacyclic promastigotes from non-infective procyclic ones. Unless favorable condition (high temperature and low pH, characteristic of the condition prevalent in the mammalian host) is reached, the metacyclics proceed toward apoptosis; in this respect, metacyclogenesis is comparable to terminal differentiation. The infectious parasite population in the midgut of infected sandfly or in vitro stationary phase cultures contain both metacyclic and apoptotic parasites and a combination of these two types of cells are needed for infectivity of the parasite within host macrophages (65). In accordance with this, deprived of LmAPX, the stationary phase culture contains a higher number of metacyclic and apoptotic parasites compared with both wild-type and LmAPXverexpressing cells (52). The apoptotic cells suppress macrophage activation by downregulating the inflammatory cytokines like TNF-α and upregulating the anti-inflammatory cytokines like transforming growth factor-β, while the virulent metacyclics proceed to invade macrophages and cause disease. These data establish vital contributions of LmAPX: (i) the enzyme has a direct role in effective detoxification of ROS, which is significant enough considering the absence of any other potent ROS scavenging heme enzyme in Leishmania. (ii) The indirect effect of the enzyme was revealed on creation of the knock out mutant, where its absence enhances cellular differentiation and virulence mediated by oxidative stress. Generally, attention is given to the genes whose loss leads to decreased virulence, pathogenicity, or infectivity. LmAPX represents a new class of genes whose deletion helps in the infectivity of the parasite. Thus, the enzyme possesses great evolutionary significance, being effectively utilized by the parasite at different conditions, which is not surprising, considering the high degree of adaptability of the phylum, protozoa. Hence, our research presents an ideal model system to study parasite virulence.

FIG. 5.

Physiological functions of LmAPX in Leishmania promastigotes. Ψ represents mitochondrial membrane potential.

Potential of the Defense System Involving LmAPX in Oxidative Stress

Like yeast CCP, one of the electron donors of LmAPX was found to be ferrocytochrome c (18), which is co-localized at intermembrane space side of the inner membrane. The fact that cytochrome c is a much better substrate than ascorbate, as observed from the k_cat values for the reaction of these molecules with LmAPX, suggests that the biological function of the enzyme is similar to yeast CCP (35, 36, 72). Since LmAPX can oxidize ferrocytochrome c in presence of endogenous H₂O₂, it can be considered as an excellent candidate for playing key functions in mitochondria-related redox control, such as elimination of H₂O₂ and O₂ ^−•, and resistance to mitochondrial ROS. Although the relative contribution of mitochondria to ROS production in green plants is very low (57) yet mitochondria is the major source of ROS in non-photosynthetic organisms (31). The plausible antioxidant role of mitochondrial LmAPX in Leishmania (catalase absent) is illustrated in Figure 6. The processes possibly involved in the LmAPX-linked defense mechanism are indicated in this model. LmAPX could detoxify H₂O₂ in the presence of reduced cytochrome c. The reduction of cytochrome c could occur either by NADH-cytochrome b₅ reductase-cytochrome b₅ pathway or O₂ ^−•, which was generated by complexes I and III of the respiratory chain. Thus, the cytochrome c-LmAPX system appears to play an important antioxidant role in L. major cells by scavenging both H₂O₂ and O₂ ^−•.

FIG. 6.

The predicted schematic presentation for the H₂O₂ and O₂ ^•− scavenging role of mitochondrial LmAPX in Leishmania promastigotes. Electrons move from NADH to O₂ via a series of redox reactions in the mitochondrial electron transport chain. Three proton pumps (I, III, and IV) create a proton gradient across the mitochondrial inner membrane for synthesizing ATP via ATP synthase. A small percentage of electrons directly leak to O₂ without completion of the whole series, resulting in the generation of O₂ ^−•. Fb₅, b₅, I, III, IV, Cytc³⁺, and Cytc²⁺ denoted cytochrome b5 reductase, cytochrome b₅, NADH-CoQ reductase, CoQH₂-cytochrome c reductase, cytochrome c oxidase, oxidized cytochrome c, and reduced cytochrome c, respectively.

Conclusion

Living cells require ROS for their normal growth and proliferation and at the same time must possess appropriate scavenging system to get rid of the excess ROS, which, otherwise, might be detrimental. So, there is a constant struggle for maintaining the balance, leading to a variety of adaptations, honed through evolution, which is evident from the redundancy of antioxidant systems as well as intelligent use of a single protein in diverse physiological processes. Single-celled trypanosomatids are adroit adaptors, which is not unexpected considering their digenetic life cycle, exposing them to very different environments. Structural and functional analysis of LmAPX has pointed to its novel evolutionary consequence as a hybrid of APX and CCP (35, 70, 72). LmAPX is located in the intermembrane space side of inner mitochondrial membrane, where cytochrome c is present (19). The high in vitro oxidation rate for cytochrome c (35, 36, 72) suggests that this molecule likely is the probable physiological substrate of the enzyme. Also, the enzyme has been shown to have pseudocatalase activity (18) and its role in protection of the cell against oxidative stress mediated cardiolipin oxidation, extensive protein damage, and apoptosis (19, 20, 52) has also been established. It is also possible that the loss of LmAPX activity has secondary effects on leishmanial gene expression caused by an alteration in the redox balance in the protozoa. As a consequence, modulation of surface lipophosphoglycan (LPG) of L. major (an approximate doubling in length of LPG and capping of majority of the galactose sugars with arabinose residues) and simultaneous differentiation of the promastigotes into infective metacyclic form may occur (46, 52). Thus, in L. major APX acts to protect the cells from ROS-mediated apoptosis and may have a major role in cellular differentiation. Future works will more precisely determine the signaling pathways involved in the processes.

Footnotes

Acknowledgments

This work was supported by the Council of Scientific and Industrial Research (CSIR) Network Project NWP 0038. Dr. Swati Pal is supported by an individual fellowship from the CSIR, Government of India.

Abbreviations Used

References

Adak

, Datta

. Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain. Biochem J, 390:465–474. 2005.

Ariyanayagam

, Fairlamb

. Ovothiol and trypanothione as antioxidants in trypanosomatids. Mol Biochem Parasitol, 115:189–198. 2001.

Barr

, Gedamu

. Cloning and characterization of three differentially expressed peroxidoxin genes from Leishmania chagasi. Evidence for an enzymatic detoxification of hydroxyl radicals. J Biol Chem, 276:34279–34287. 2001.

Biyani

, Madhubala

. Leishmania donovani encodes a functional enzyme involved in vitamin C biosynthesis: arabino-1,4-lactone oxidase. Mol Biochem Parasitol, 180:76–85. 2011.

Boveris

, Sies

, Martino

, Docampo

, Turrens

, Stoppani

. Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem J, 188:643–648. 1980.

Bray

. Leishmaniasis. Trop Dis Bull, 66:501–507. 1969.

Bursey

, Poulos

. Two substrate binding sites in ascorbate peroxidase: the role of arginine 172. Biochemistry, 39:7374–7379. 2000.

Castro

, Romao

, Carvalho

, Teixeira

, Sousa

, Tomas

. Mitochondrial redox metabolism in trypanosomatids is independent of tryparedoxin activity. PLoS One, 5:e12607. 2010.

Castro

, Sousa

, Novais

, Santos

, Budde

, Cordeiro-da-Silva

, Flohe

, Tomas

. Two linked genes of Leishmania infantum encode tryparedoxins localised to cytosol and mitochondrion. Mol Biochem Parasitol, 136:137–147. 2004.

10.

Castro

, Tomas

. Peroxidases of trypanosomatids. Antioxid Redox Signal, 10:1593–1606. 2008.

11.

Chang

. Leishmania donovani: promastigote—macrophage surface interactions in vitro. Exp Parasitol, 48:175–189. 1979.

12.

Channon

, Blackwell

. A study of the sensitivity of Leishmania donovani promastigotes and amastigotes to hydrogen peroxide. II. Possible mechanisms involved in protective H₂O₂ scavenging. Parasitology, 91,(Pt 2):207–217. 1985.

13.

Charizanis

, Juhnke

, Krems

, Entian

. The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7) Mol Gen Genet, 262:437–447. 1999.

14.

Cheek

, Mandelman

, Poulos

, Dawson

. A study of the K(+)-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side. J Biol Inorg Chem, 4:64–72. 1999.

15.

Clark

, Albrecht

, Arevalo

. Ascorbate variations and dehydroascorbate reductase activity in Trypanosoma cruzi epimastigotes and trypomastigotes. Mol Biochem Parasitol, 66:143–145. 1994.

16.

Colotti

, Ilari

. Polyamine metabolism in Leishmania: from arginine to trypanothione. Amino Acids, 40:269–285. 2011.

17.

DeLano

. 2002. The PyMOL molecular graphics system. www.pymol.org/. 2007 Nov 30.

18.

Dolai

, Yadav

, Datta

, Adak

. Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase. Biochim Biophys Acta, 1770:247–256. 2007.

19.

Dolai

, Yadav

, Pal

, Adak

. Leishmania major ascorbate peroxidase overexpression protects cells against reactive oxygen species-mediated cardiolipin oxidation. Free Radic Biol Med, 45:1520–1529. 2008.

20.

Dolai

, Yadav

, Pal

, Adak

. Overexpression of mitochondrial Leishmania major ascorbate peroxidase enhances tolerance to oxidative stress-induced programmed cell death and protein damage. Eukaryot Cell, 8:1721–1731. 2009.

21.

Dunford

. Heme Peroxidases. New York: John Wiley & Sons, Inc., 1999.

22.

Emanuelsson

, Nielsen

, Brunak

, von Heijne

. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol, 300:1005–1016. 2000.

23.

Erman

, Vitello

, Mauro

, Kraut

. Detection of an oxyferryl porphyrin pi-cation-radical intermediate in the reaction between hydrogen peroxide and a mutant yeast cytochrome c peroxidase. Evidence for tryptophan-191 involvement in the radical site of compound I. Biochemistry, 28:7992–7995. 1989.

24.

Fairlamb

, Blackburn

, Ulrich

, Chait

, Cerami

. Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science, 227:1485–1487. 1985.

25.

Gantt

, Goldman

, McCormick

, Miller

, Jeronimo

, Nascimento

, Britigan

, Wilson

. Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi. J Immunol, 167:893–901. 2001.

26.

Getachew

, Gedamu

. Leishmania donovani iron superoxide dismutase A is targeted to the mitochondria by its N-terminal positively charged amino acids. Mol Biochem Parasitol, 154:62–69. 2007.

27.

Ghosh

, Goswami

, Adhya

. Role of superoxide dismutase in survival of Leishmania within the macrophage. Biochem J, 369:447–452. 2003.

28.

Goltz

, Kaput

, Blobel

. Isolation of the yeast nuclear gene encoding the mitochondrial protein, cytochrome c peroxidase. J Biol Chem, 257:11186–11190. 1982.

29.

Groden

, Beck

. H₂O₂ destruction by ascorbate-dependent systems from chloroplasts. Biochim Biophys Acta, 546:426–435. 1979.

30.

Grondin

, Haimeur

, Mukhopadhyay

, Rosen

, Ouellette

. Co-amplification of the gamma-glutamylcysteine synthetase gene gsh1 and of the ABC transporter gene pgpA in arsenite-resistant Leishmania tarentolae. EMBO J, 16:3057–3065. 1997.

31.

Halliwell

, Gutteridge

JMC

. Free Radicals in Biology and Medicine, 2nd. Clarendon: Oxford, 1989.

32.

Hensley

, Robinson

, Gabbita

, Salsman

, Floyd

. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med, 28:1456–1462. 2000.

33.

Herbette

, Roeckel-Drevet

, Drevet

. Seleno-independent glutathione peroxidases. More than simple antioxidant scavengers. FEBS J, 274:2163–2180. 2007.

34.

Huwiler

, Kohler

. Pseudo-catalytic degradation of hydrogen peroxide in the lactoperoxidase/H₂O₂/iodide system. Eur J Biochem, 141:69–74. 1984.

35.

Jasion

, Polanco

, Meharenna

, Li

, Poulos

. Crystal structure of Leishmania major peroxidase and characterization of the compound I Tryptophan radical. J Biol Chem, 286:24608–24615. 2011.

36.

Jasion

, Poulos

. Leishmania major peroxidase is a cytochrome c peroxidase. Biochemistry, 51:2453–2460. 2012.

37.

Jiang

, English

. Phenotypic analysis of the ccp1Delta and ccp1Delta-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling. J Inorg Biochem, 100:1996–2008. 2006.

38.

Kang

, Ferguson-Miller

, Margoliash

. Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase. J Biol Chem, 252:919–926. 1977.

39.

Killick-Kendrick

, Molyneux

, Hommel

, Leaney

, Robertson

. Leishmania in phlebotomid sandflies. V. The nature and significance of infections of the pylorus and ileum of the sandfly by leishmaniae of the braziliensis complex. Proc R Soc Lond B Biol Sci, 198:191–199. 1977.

40.

Krauth-Siegel

, Comini

. Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim Biophys Acta, 1780:1236–1248. 2008.

41.

Kwon

, Chong

, Han

, Kim

. Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae. Biochim Biophys Acta, 1623:1–5. 2003.

42.

Lad

, Mewies

, Raven

. Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites. Biochemistry, 41:13774–13781. 2002.

43.

Lillig

, Holmgren

. Thioredoxin and related molecules—from biology to health and disease. Antioxid Redox Signal, 9:25–47. 2007.

44.

Magnusson

, Taurog

. Iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase. Biochem Biophys Res Commun, 112:475–481. 1983.

45.

Magnusson

, Taurog

, Dorris

. Mechanism of iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase. J Biol Chem, 259:197–205. 1984.

46.

McConville

, Turco

, Ferguson

, Sacks

. Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage. EMBO J, 11:3593–3600. 1992.

47.

Miller

, Suzuki

, Rizhsky

, Hegie

, Koussevitzky

, Mittler

. Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses. Plant Physiol, 144:1777–1785. 2007.

48.

Miller

, Vitello

, Erman

. Regulation of interprotein electron transfer by Trp 191 of cytochrome c peroxidase. Biochemistry, 34:12048–12058. 1995.

49.

Mukherjee

, Roy

, Guimond

, Ouellette

. The gamma-glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants. Mol Microbiol, 74:914–927. 2009.

50.

Noctor

, Veljovic-Jovanovic

, Foyer

. Peroxide processing in photosynthesis: antioxidant coupling and redox signalling. Philos Trans R Soc Lond B Biol Sci, 355:1465–1475. 2000.

51.

Oza

, Shaw

, Wyllie

, Fairlamb

. Trypanothione biosynthesis in Leishmania major. Mol Biochem Parasitol, 139:107–116. 2005.

52.

Pal

, Dolai

, Yadav

, Adak

. Ascorbate peroxidase from Leishmania major controls the virulence of infective stage of promastigotes by regulating oxidative stress. PLoS One, 5:e11271. 2010.

53.

Pal

, Yadav

, Adak

. Role of K+ binding residues in stabilization of heme spin state of Leishmania major peroxidase. Biochim Biophys Acta, 1824:1002–1007. 2012.

54.

Pappa

, Patterson

, Poulos

. The homologous tryptophan critical for cytochrome c peroxidase function is not essential for ascorbate peroxidase activity. J Biol Inorg Chem, 1:61–66. 1996.

55.

Patterson

, Poulos

. Crystal structure of recombinant pea cytosolic ascorbate peroxidase. Biochemistry, 34:4331–4341. 1995.

56.

Pelletier

, Kraut

. Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c. Science, 258:1748–1755. 1992.

57.

Purvis

. Role of the alternative oxidase in limiting superoxide production by plant mitochondria. Physiol Plant, 100:165–170. 1997.

58.

Romao

, Castro

, Sousa

, Carvalho

, Tomas

. The cytosolic tryparedoxin of Leishmania infantum is essential for parasite survival. Int J Parasitol, 39:703–711. 2009.

59.

Sharp

, Mewies

, Moody

, Raven

. Crystal structure of the ascorbate peroxidase-ascorbate complex. Nat Struct Biol, 10:303–307. 2003.

60.

Shelton

, Chock

, Mieyal

. Glutaredoxin: role in reversible protein s-glutathionylation and regulation of redox signal transduction and protein translocation. Antioxid Redox Signal, 7:348–366. 2005.

61.

Shigeoka

, Ishikawa

, Tamoi

, Miyagawa

, Takeda

, Yabuta

, Yoshimura

. Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot, 53:1305–1319. 2002.

62.

Spath

, Garraway

, Turco

, Beverley

. The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts. Proc Natl Acad Sci U S A, 100:9536–9541. 2003.

63.

Spies

, Steenkamp

. Thiols of intracellular pathogens. Identification of ovothiol A in Leishmania donovani and structural analysis of a novel thiol from Mycobacterium bovis. Eur J Biochem, 224:203–213. 1994.

64.

Taylor

, Kelly

, Fairlamb

, Miles

. The trypanothione reductase gene of Leishmania donovani. Biochem Soc Trans, 18:869–870. 1990.

65.

van Zandbergen

, Bollinger

, Wenzel

, Kamhawi

, Voll

, Klinger

, Muller

, Holscher

, Herrmann

, Sacks

, Solbach

, Laskay

. Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum. Proc Natl Acad Sci U S A, 103:13837–13842. 2006.

66.

Welinder

. Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily. Biochim Biophys Acta, 1080:215–220. 1991.

67.

Wilkinson

, Obado

, Mauricio

, Kelly

. Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum. Proc Natl Acad Sci U S A, 99:13453–13458. 2002.

68.

Wilkinson

, Prathalingam

, Taylor

, Horn

, Kelly

. Vitamin C biosynthesis in trypanosomes: a role for the glycosome. Proc Natl Acad Sci U S A, 102:11645–11650. 2005.

69.

Winter

, Moore

. Exploring the chemistry and biology of vanadium-dependent haloperoxidases. J Biol Chem, 284:18577–18581. 2009.

70.

Yadav

, Dolai

, Pal

, Adak

. Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme. Biochim Biophys Acta, 1784:863–871. 2008.

71.

Yadav

, Dolai

, Pal

, Adak

. Role of C-terminal acidic cluster in stabilization of heme spin state of ascorbate peroxidase from Leishmania major. Arch Biochem Biophys, 495:129–135. 2010.

72.

Yadav

, Pal

, Dolai

, Adak

. Role of proximal methionine residues in Leishmania major peroxidase. Arch Biochem Biophys, 515:21–27. 2011.

73.

Za'mocky

, Obinger

. Molecular phylogeny of heme peroxidases. Biocatalysis Based on Heme Peroxidases. Torres

, Ayala

Heidelberg: Springer-Verlag Berlin, 2010; 7–35. 2010.