ABSTRACT
The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in Leishmania showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.
INTRODUCTION
The success or failure of an infection depends on how well the pathogens can protect themselves from oxidative stress generated by the host, because they are exposed to a high level of host-generated reactive oxygen species (ROS) as they invade cells (1, 2). For protection from the host arsenal of ROS, efficiency of the pathogen's cellular defense system is crucial. It is the critical balance between the competence of the host defense mechanisms and the pathogen protection system that determines the outcome of an infective event. One of the trypanosomatid parasites, Leishmania donovani, causes visceral leishmaniasis (VL), a potentially fatal disease prevalent in the poorer sections of the population. It is endemic in some countries and is responsible for considerable morbidity and mortality (3, 4). There is no vaccine for the treatment of VL, and only a few drugs are used for treatment, which is complicated by emerging drug resistance (5). Therefore, understanding the defense mechanisms of these parasites in order to develop drugs remains an important issue. The Leishmania parasites express a unique system of enzymes, including tryparedoxin peroxidase and trypanothione reductase, in which trypanothione, a small thiol unique to these organisms, is used as an electron donor to run a system of peroxide detoxification (6, 7). As this detoxification system is unique to these parasites, it has been proposed as a possible drug target (8, 9). L. donovani expresses two tryparedoxin peroxidases, a cytosolic form (cTXNPx) localized to the cytoplasm, which is encoded by a multicopy gene family, and a mitochondrial tryparedoxin peroxidase (mTXNPx) found only in the mitochondria, encoded by a single-copy gene (10, 11, 12). The level of similarity between the two enzymes at the DNA level is 61% and at the protein level is about 50%, although the three-dimensional structures show very close similarity (11). The enzymes are found in other trypanosomatid parasites and are highly conserved within the genus Leishmania (11). Since the trypanosomatids are deficient in selenium-dependent glutathione peroxidase and catalase, the tryparedoxin peroxidases are very important for their survival. There are multiple studies on these enzymes; however, their comparative responses to selective stress are not well defined, leaving an opportunity to investigate the responses of the parasites to exogenous and endogenous stress as demanded by their interactions with the host or drugs (6, 11).
The Leishmania parasite has a digenetic life cycle, surviving as free-swimming promastigotes in the digestive tract of the sandfly and as amastigotes in the host macrophages. The oxidative burst of the host cells consists of ROS and reactive nitrogen species (RNS) that the parasites are exposed to while invading (2, 13). Our earlier studies provided evidence for the susceptibility of the Leishmania parasites to both ROS and RNS during their life cycle as promastigotes, as well as amastigotes within macrophages (14, 15). We have shown responses of the parasite cTXNPx to hydrogen peroxide (H2O2)- and NO-induced stress, where cTXNPx provides protection against the combined stresses of the two reactive species (12). These enzymes are essential to detoxify drug-induced stress. The relevant drugs in VL are potassium antimony tartrate (PAT), miltefosine, paromomycin, and amphotericin B (16). Studies have shown upregulation of cTXNPx in amphotericin B-resistant isolates or in potassium antimony-resistant isolates of Leishmania spp., suggesting a possible role of cTXNPx in drug resistance (8, 17). Other reports demonstrated increased levels of both cTXNPx and mTXNPx in antimony-resistant field isolates of L. donovani (18, 19).
This study shows a new finding of the ability of mTXNPx to regulate oxidative stress generated by mitochondrial toxins more efficiently than cTXNPx, whereas cTXNPx was more competent in dealing with exogenous oxidative stress than mTXNPx. Further, the findings show an increase of early infection rates when cells were equipped with higher amounts of cTXNPx than of mTXNPx. Importantly, antileishmanial drugs like PAT and miltefosine showed different efficacies with increased quantities of the enzymes, where the presence of excess mTXNPx made the parasites less sensitive to miltefosine while high cTXNPx levels produced resistance to PAT.
RESULTS
Expression of cTXNPx and mTXNPx increases in response to stress inducers.Our initial aim was to make a comparative assessment of the expression of cTXNPx and mTXNPx when parasites were exposed to exogenous or endogenous oxidative stress. We used both mitochondrial and cytosolic stress inducers to determine the responses of the enzymes. For mitochondrial stress generation, mitochondrial respiratory chain inhibitors like rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, inhibitors of respiratory complexes I, II, and III, respectively, were used (20). In wild-type (WT) parasites, mitochondrial complex I inhibition with rotenone resulted in a dose-dependent increase in expression of both cTXNPx and mTXNPx (Fig. 1A). Complex II inhibition with TTFA at the same dose significantly increased mTXNPx expression, with cTXNPx remaining unaltered compared to controls (Fig. 1B). Antimycin A, a complex III inhibitor, showed a significant increase in the expression of both mTXNPx and cTXNPx compared to controls; however, the increase in mTXNPx was greater than that of cTXNPx (Fig. 1C). To create exogenous stress, H2O2 was used, and a greater increase of cTXNPx expression than of mTXNPx was observed, although mTXNPx levels did show an increase compared to vehicle-treated controls (Fig. 1D). Our previous reports demonstrated the dependence of the parasites on cTXNPx for the regulation of oxidative stress; however, a comparative study with mTXNPx using a variety of stress inducers was not carried out (12). This part of the study demonstrated a greater increase of mTXNPx levels than of cTXNPx levels when parasites were challenged with mitochondrial toxins, whereas parasites exposed to H2O2 showed a significant increase in cTXNPx expression, suggesting differential increases in the two enzymes depending on the source of the stress.
Differential expression profiles of defensive enzymes (cTXNPx and mTXNPx) of L. donovani (WT) on exposure to mitochondrial respiratory chain complex inhibitors for 6 h. (A) Rotenone (50 μM, 100 μM, and 150 μM). (B) TTFA (50 μM, 100 μM, and 200 μM). (C) Antimycin A (50 μM, 100 μM, and 150 μM). (D) H2O2 (80 μM, 100 μM, and 150 μM). The blots are representative of three independent experiments. The corresponding bar graphs represent the relative fold expression of the proteins. The data are the means and SE from three independent experiments performed with three independent lysate preparations. β-Tubulin was used as a loading control. The asterisks denote significant differences between cTXNPx and mTXNPx with reference to their WT expression. ***, P < 0.001; **, P < 0.01; ns, not significant.
cTXNPx/mTXNPx overexpression influences cell fate on exposure to mitochondrial and cytosolic stresses.The above-mentioned data established differential increases of cTXNPx and mTXNPx on exposure to oxidative stresses from different sources. Based on these observations, we explored if the observed increase of enzyme levels on stress exposure provided a survival advantage to parasites. For this, we overexpressed both active and inactive cTXNPx and mTXNPx proteins through the episomal expression system in the Leishmania promastigotes (data not shown). Arguably, functional correlation could be made based on the response of parasites with increased quantities of different enzymes exposed to oxidative stress. When the transfected parasites were exposed to mitochondrial respiratory chain complex inhibitors, the parasites overexpressing active mTXNPx (mTXNPx-OE) showed significantly lower parasite death rates than WT cells, parasites overexpressing cTXNPx (cTXNPx-OE), and parasites overexpressing the mitochondrial targeting signal only (MTS-OE) or expressing the inactive mTXNPx enzyme mutated at the active site [mTXNPx (Δ81)] (Fig. 2A to C). This clearly suggested a requirement for an active mTXNPx enzyme for survival under mitochondrial stress in comparison to active cTXNPx. On the other hand, when H2O2 was used as an exogenous stress inducer, cTXNPx overexpression was able to reduce the death percentage more efficiently than mTXNPx (Fig. 2D). The parasites overexpressing mTXNPx showed better survival at lower doses of H2O2, but at higher doses, their vulnerability was comparable to that of controls (Fig. 2D). This suggested that under higher exogenous stress, the utility of mTXNPx as a protective enzyme was limited. The cTXNPx-overexpressing parasites showed higher survival at both the higher and lower doses of H2O2 (Fig. 2D). In summary, the location of the enzymes appears to be crucial in dealing with stress generated at selective cellular locations.
Effects of the mitochondrial respiratory chain complex inhibitors and H2O2 on parasite viability at 24 h with rotenone (50 μM and 100 μM) (A), TTFA (150 μM and 200 μM) (B), antimycin A (100 μM and 200 μM) (C), and H2O2 (100 μM, 200 μM, and 500 μM) (D). The percentage of dead cells was calculated as the ratio of the fluorescence intensity of dead cells to that of total cells. The plots are representative of three independent experiments, with the results shown as means and SE. ***, P < 0.001.
Location-specific TXNPx quenches reactive oxygen species generated in its vicinity.The next question was whether the elimination of ROS and their products generated at a particular location is dependent on the presence of effective TXNPx in their vicinity. Tryparedoxin peroxidases primarily act as antioxidants by metabolizing hydrogen peroxide into oxygen and water molecules (1, 12). The mitochondrial respiratory chain inhibitors generate significant amounts of intracellular ROS that primarily accumulate in the mitochondria, although this ROS is gradually distributed to other parts of the cell, as well. Parasites overexpressing active mTXNPx showed lower total 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA)-sensitive ROS accumulation when respiratory chain inhibitors were used than cells expressing inactive enzymes, wild-type cells, or cells overexpressing cTXNPx (Fig. 3A). This observation suggested that the presence of high levels of mTXNPx was able to suppress the total ROS levels generated by the mitochondrial respiratory chain inhibitors. On the other hand, when H2O2 was used to raise intracellular ROS levels, parasites with higher levels of cTXNPx showed lower ROS levels than parasites overexpressing mTXNPx or wild-type parasites, suggesting that higher cTXNPx levels were able to reduce the ROS levels in the cytosol (Fig. 3A). The data on total ROS production prompted us to measure mitochondrial superoxide, as it is the site of stress generated with the mitochondrial toxins. Observations agreed with the above-mentioned results, as antimycin A, rotenone, and TTFA generated high mitochondrial superoxide levels in cells overexpressing the inactive enzymes and cTXNPx and in WT cells; however, mTXNPx-overexpressing cells showed lower mitochondrial superoxide levels, again suggesting a protective role of mTXNPx (Fig. 3B). During H2O2-induced stress, the level of mitochondrial superoxide generated was lower in cTXNPx-overexpressing cells than in mTXNPX-overexpressing cells and other controls (Fig. 3B).
(A) Total intracellular ROS production by parasites after treatment with rotenone (50 μM), TTFA (50 μM), antimycin A (100 μM), and H2O2 (80 μM), as detected by H2DCFDA fluorescence. Trolox (1 mM) and MitoTempo (50 μM) were used as cellular and mitochondrion-specific ROS scavengers. For Trolox and MitoTempo, parasites were pretreated with the required amount of scavenger for 1 h (MitoTempo) or 2 h (Trolox), washed, and then treated. The data are means ± SE; n = 3. (B) Changes in mitochondrial superoxide generation in log-phase L. donovani promastigotes (WT, MTS-OE, Δ81-OE, mTXNPx-OE, pXG-GFP+2-OE, Δ52-OE, and cTXNPx-OE) with MitoSOX at different time points with 50 μM rotenone, 50 μM TTFA, 100 μM antimycin A, and 80 μM H2O2. The data are means ± SE; n = 3. (C) Changes in mitochondrial membrane potentials of log-phase L. donovani promastigotes (WT, MTS-OE, Δ81-OE, mTXNPx-OE, pXG-GFP+2-OE, Δ52-OE, and cTXNPx-OE) at different time points with 50 μM rotenone, 50 μM TTFA, 100 μM antimycin A, and 80 μM H2O2. The data are means ± SE; n = 3.
Oxidative stress frequently disrupts mitochondrial potential, leading to loss of energy and eventual cell death. To investigate whether higher levels of active enzymes were able to provide protection against disruption of the mitochondrial potential, we evaluated the mitochondrial potentials of various groups as a functional output after exposure to the mitochondrial respiratory chain inhibitors and H2O2. All three mitochondrial respiratory chain inhibitors were able to disrupt mitochondrial potential in wild-type cells and cells overexpressing the inactive mTXNPx or cTXNPx (Fig. 3C). Cells with mTXNPx overexpression showed a smaller loss of mitochondrial potential than cells overexpressing cTXNPx, WT cells, or cells expressing inactive enzymes (Fig. 3C). Cells overexpressing cTXNPx were better protected against mitochondrial potential loss than cells overexpressing mTXNPx, WT cells, or cells expressing inactive enzymes when H2O2 was used to raise intracellular ROS levels (Fig. 3C). These data clearly suggested that overexpression of mTXNPx in the mitochondria made cells more efficient in protecting the organelle than overexpression of cTXNPx. Full enzyme activity was required for this protection, as the cells overexpressing inactive enzyme showed a greater loss of mitochondrial potential than the cells overexpressing active enzymes. This is in consonance with our earlier studies in the laboratory showing a loss of mitochondrial potential when Leishmania parasites were treated with H2O2 (15, 21). Taken together, the data suggest that the location of the mitochondrial enzyme in the mitochondria was important in providing protection against mitochondria-generated oxidants through prevention of loss of membrane potential and by preventing accumulation of ROS in the organelle. Similarly, cTXNPx overexpression provided protection against a greater amount of H2O2 in the cytosol that eventually affected the mitochondria.
Overexpression of cTXNPx provides an advantage during infection.Parasites are exposed to high levels of ROS generated by the host NADPH oxidase during host cell entry, and at this juncture, the efficiency of active defensive enzymes is of paramount importance for the parasites to retain their viability and mount a successful infection (22, 23). In this context, it was of interest to investigate the relative contributions of both cTXNPx and mTXNPx to creating a favorable environment for the parasite to succeed in invading the host. First, we checked for changes in the status of defensive enzymes in wild-type cells during infection of J774A.1 macrophages to assess changes in enzyme expression, if any (Fig. 4A, a). While cTXNPx expression showed an increase at 4 h postinfection, mTXNPx did not show any early increase, but at 12 h there was a significant increase in mTXNPx expression. This suggested a possible requirement for increase in both TXNPxs during invasion of the host. Up to 6 h, the cTXNPx-overexpressing parasites showed higher infection than parasites overexpressing mTXNPx; however, at later time points of 24 and 48 h, the infection rates seemed to be comparable between the groups (Fig. 4A, b). Having shown the effects of overexpression of cTXNPx and mTXNPx during infection in J774A.1 mouse macrophages, we attempted to corroborate the effects on infection in primary mouse macrophages derived from peritoneal exudates. Figure 4B, a, demonstrates increases in cTXNPx and mTXNPx levels in the parasites during infection of mouse primary macrophages as estimated by Western blotting. During the initial phase of infection until 6 h, parasites overexpressing cTXNPx were able to have a larger number of parasites surviving within macrophages than the parasites overexpressing mTXNPx (Fig. 4B, b), suggesting an important role of cTXNPx during early infection. At 24 h and 48 h, the parasite burdens were comparable for parasites overexpressing either cTXNPx or mTXNPx and were significantly higher than in WT cells or cells overexpressing inactive enzymes (Fig. 4B, b). These observations suggested that functionally active enzymes were necessary for protection during infection and that cTXNPx was essential in the early period. Arguably, the parasite cytosol would be the first to be exposed to host-generated ROS during invasion, and an active enzyme located in the cytosol would help to counter the oxidative stress. At early times, although mTXNPx-overexpressing parasites did not show increased invasion, at 24 h and 48 h the parasites that were successful in invading the cells proliferated and the numbers of parasites overexpressing either mTXNPx or cTXNPx were similar in J774A.1 macrophages and the primary macrophages derived from mice (Fig. 4A, b, and B, b).
(A) (a) Western blots of L. donovani probed with anti-mTXNPx and anti-cTXNPx showing expression profiles of the enzymes in the parasites on exposure to J774A.1 macrophages at 0 h, 4 h, and 12 h. β-Tubulin expression was used as a loading control. A corresponding densitometric analysis for comparative fold expression of cTXNPx and mTXNPx is shown in the bar graph. The blots are representative of three independent experiments. ***, P < 0.001; ns, no significant difference. (b) Photomicrographs showing infection of J774A.1 macrophages with WT parasites and transfected parasites (MTS, Δ81, mTXNPx, pXG-GFP+2, Δ52, and cTXNPx) for 6, 24, and 48 h. The corresponding bar graphs show the parasite burdens of J774A.1 at 6, 24, and 48 h postinfection. The parasite burden was calculated as the number of promastigotes/amastigotes per 100 macrophages. The results are the means and SE of three independent experiments. **, P < 0.01; ***, P < 0.001. (B) (a) Western blots of L. donovani probed with anti-mTXNPx and anti-cTXNPx showing expression profiles of the enzymes in the parasites on exposure to mouse peritoneal macrophages at 0, 4, and 12 h. β-Tubulin expression was used as a loading control. The corresponding densitometric analysis for comparative fold expression of cTXNPx and mTXNPx is shown in the bar graph. The blots are representative of three independent experiments. ***, P < 0.001; ns, no significant difference. (b) Infection of mouse peritoneal macrophages with the WT and transfectants (MTS, Δ81, mTXNPx, pXG-GFP+2, Δ52, and cTXNPx) for 6, 24, and 48 h. The bar graphs represent the parasite burdens of primary mouse macrophages for 6, 24, and 48 h. The results are the means and SE from three independent experiments. **, P < 0.01; ***, P < 0.001.
Overexpression of defensive enzymes resulted in reduced sensitivity to specific antileishmanial drugs.Drug exposure is a prominent stress factor for parasites, and it is important to understand this, because the inefficacy of many of these drugs could be related to the status of the defensive enzymes in the parasites. PAT and miltefosine are two antileishmanial drugs currently used in patients (alone or in combination therapy), and both drugs are known to generate ROS (24, 25). We used these drugs to investigate relative parasite survival in the presence of higher levels of active enzymes. First, we used WT parasites to check if there was any difference in the levels of the enzymes upon exposure to the drugs. A prominent increase in cTXNPx upon exposure to both PAT and miltefosine was observed, and the response was greater with PAT treatment than with miltefosine treatment (Fig. 5A and B). mTXNPx also increased after exposure to both drugs at 12 h, but at an earlier time point of 6 h, the mTXNPx level was higher with miltefosine and remained unaltered with PAT. Both PAT and miltefosine exposure affected the mitochondrial potential of WT parasites, with the data showing a fall in potential with the drugs (Fig. 5C). The total intracellular ROS generation was higher with PAT treatment (Fig. 5D). These data suggested that indeed, the response of the parasites to these drugs included an increase in the tryparedoxin peroxidase levels, indicating a possible functional role. Therefore, to explore if an increase in the enzymes was actually responsible for better survival during the drug treatments, we exposed the various transfectants, along with their respective mutants, to PAT/miltefosine and evaluated their survival. With lower doses of PAT, both enzymes were able to provide protection, but at higher doses, cTXNPx was capable of reducing cell death more effectively than mTXNPx (Fig. 6A). At lower doses of miltefosine, higher levels of both enzymes provided protection from death; however, at higher doses, an elevated level of mTXNPx afforded better protection (Fig. 6B). The data suggested that the two enzymes were differentially used by the parasite to handle specific drugs with different mechanisms of action. Interestingly, the above-mentioned observations correlated with ROS levels generated by the drugs (Fig. 6C and D). While PAT-generated ROS accumulated less in cTXNPx-overexpressing cells, miltefosine-generated ROS was lower in mTXNPx-overexpressing cells (Fig. 6C and D). A lesser presence of ROS in cTXNPx-overexpressing cells during PAT treatment also favored sustained mitochondrial potential better than parasites with inactive cTXNPx or overexpressing mTXNPx, suggesting differential behavior of the enzymes during treatment with different drugs (Fig. 6E). Also, higher mitochondrial potential was retained in mTXNPx-overexpressing cells than in cells overexpressing cTXNPx or the inactive mTXNPx when exposed to miltefosine (Fig. 6F). This is important, as it can be linked to possible refractoriness to specific drugs when either of the enzymes is overexpressed.
(A and B) Western blot analysis of wild-type parasites on exposure to PAT (25 μg/ml) and miltefosine (0.5 μg/ml) for 6 h (A) and 12 h (B) probed with anti-cTXNPx and anti-mTXNPx antibodies. P, PAT; M, miltefosine; C, vehicle only. The corresponding bar graphs show the results of densitometric analysis of the blots at 6 h and 12 h. The blots are representative of three independent experiments; the data are means and SE. **, P < 0.01; ***, P < 0.001; ns, no significant difference. (C) Changes in mitochondrial potentials of WT parasites at different time points represented as fluorescence ratios at 590 and 530 nm. (D) Total ROS generation in WT parasites upon exposure to PAT and miltefosine. The data are means ± SE.
Effects of different doses of PAT (A) and miltefosine (B) on the viability of the WT and transfectants (MTS-OE, Δ81-OE, mTXNPx-OE, pXG-GFP+2-OE, Δ52-OE, and cTXNPx-OE). The data are means and SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C and D) Intracellular ROS generation in response to PAT (C) and miltefosine (D). (E and F) Changes in mitochondrial membrane potentials of transfectants (MTS-OE, Δ81-OE, mTXNPx-OE, pXG-GFP+2-OE, Δ52-OE, and cTXNPx-OE) on exposure to PAT (100 μg/ml) (E) and miltefosine (2.5 μg/ml) (F).
Elevated expression of cTXNPx and mTXNPx occurs in PAT- and miltefosine-resistant parasites.Amplification of a particular stress-combating strategy of Leishmania parasites can lead to specific drug resistance (26, 27, 28). In order to understand whether overexpression of a particular defensive enzyme could be selected by the parasite as a mode of drug resistance, the expression profiles of these enzymes were checked in PAT- and miltefosine-resistant parasites. In this context, three PAT-resistant clinical isolates (29, 30, 31) and two miltefosine-resistant parasites were used. Miltefosine-resistant parasites were generated in the laboratory by gradual exposure to increased concentrations of miltefosine and examined for the levels of both cTXNPx and mTXNPx. Compared to controls, the expression of these enzymes was significantly higher than in the corresponding WT cells (Fig. 7A and B), suggesting that higher levels of both cTXNPx and mTXNPx were associated with lowered response to the two antileishmanial drugs. Although it is known that PAT-resistant isolates of L. infantum, L. braziliensis, and L. amazonensis express larger amounts of cTXNPx, the expression of mTXNPx has not been characterized for PAT-resistant isolates (8, 18). Our observations showed that overexpression of the two enzymes provided higher survival rates than for parasites overexpressing inactive forms of the enzymes.
(A) Expression profiles of cTXNPx and mTXNPx in several PAT- and miltefosine-resistant parasites. (B) Relative fold expression of the enzymes measured by densitometry. The blots and plots are representative of three independent experiments. The data are means and SE. *, P < 0.05; ***, P < 0.001.
DISCUSSION
Antioxidant defense systems involving tryparedoxin peroxidases in trypanosomatid parasites remain the primary target for developing chemotherapeutic approaches because host cells do not express these enzymes and the components are unique to the parasites. The enzymes are crucial for parasite survival (6, 11) because they require a robust antioxidant defense, as they are exposed to varying amounts of ROS during their life cycle in the gut of the insect vector, as well as during invasion of mammalian host cells (2). The superoxide radical can be formed within the parasite from its own electron transport chain or through redox cycling of drugs and can kill the parasites if they are unable to detoxify the products. Both the cytosolic and the mitochondrial tryparedoxin peroxidases typically carry out reduction of a broad range of peroxide substrates through a conserved cysteine residue and form the primary defensive arsenal of the Leishmania parasites, which lack catalase, the enzyme required for decomposition of hydrogen peroxide (32). Although several studies have shown the localization of these enzymes and their respective enzymatic activities (6, 7, 33, 34), their relative functional contributions in disparate situations during the parasite's life are not fully understood. This is important, because distinct microenvironments are maintained within the cell due to the presence of membrane-bound organelles that allow the cells to physically contain damage by preventing aberrant molecules from traveling to other parts of the cell. Knowledge of the relative importance of enzymes as a function of their location is important in regard to the extent of utility of the enzymes in a protective role. This may provide further clues to successful design of drugs targeting the dual antioxidant systems of parasites. This study presents a new finding of a differential response to specific kinds of stress that determines the functional outcome.
It is understood that correct subcellular localization of proteins is critical, as it defines the physiological context of the protein function. In that setting, mitochondrial localization of a peroxidase is essential, as mitochondria are the cradles of ROS generation. The extents of ROS production upon respiratory chain inhibition are different for complexes I, II, III, and IV (20). Slight inhibition of complex I is sufficient to increase ROS production, whereas about 70% inhibition could be tolerated by complexes III and IV without an increase in ROS formation. Our previous studies have shown that inhibition of complexes I, II, and III generates sufficient ROS to induce loss of mitochondrial function in Leishmania, culminating in cell death (20). The higher expression of mTXNPx than of cTXNPx in WT cells when the respiratory chain was inhibited suggested a location-specific defensive response of the cell to combat peroxides and their products. This was supported by observations of improved ability to withstand mitochondrial stress by parasites overexpressing mTXNPx compared to WT and cTXNPx-overexpressing cells. This was clear evidence of a mitochondrion-centric stress response when mitochondrial toxins disrupted the functioning of the organelle. The data on cell survival indicated better endurance post-mitochondrial stress in parasites overexpressing mTXNPx than in those overexpressing cTXNPx, substantiating the fact that appropriate localization favorably protected the parasite. Since mitochondria are hubs of energy supply and are closely related to the survival of cells, the higher capacity to survive mitochondrial stress in the parasites overexpressing mTXNPx supported the functional relevance of the presence of mTXNPx in the mitochondria. Higher death rates in parasites expressing inactive enzymes connected the enzymatic activity to the functional output, ruling out the involvement of any chaperone-like role (9).
When parasites are exposed to exogenous H2O2 or superoxide (during macrophage invasion), the antioxidant enzymes located in the cytosol become the first line of defense. The results of our studies with exogenous H2O2 (applied as a source of oxidative stress simulating conditions within macrophages) confirm a positive response by cTXNPx, as its constitutive expression level became higher than that of mTXNPx. During the early period of infection in both J774A.1 macrophages and the primary macrophages derived from mice, the parasites overexpressing cTXNPx appeared to be better protected from death than parasites overexpressing mTXNPx, suggesting the importance of an early cytosolic response to exogenous stress. At later phases of infection, when parasites are proliferating, the numbers of parasites that the macrophages were harboring were comparable between the mTXNPx- and the cTXNPx-overexpressing parasites. This was probably due to less exposure of mitochondria to the exogenous oxidative stress due to a cellular gradient of the oxidants possibly created by membrane permeability, as was shown in Saccharomyces cerevisiae (35). Therefore, it appears that increase of cTXNPx activity in the cytosol is followed by mTXNPx activity to provide an efficient ROS-eliminating strategy during infection, favoring parasite survival.
Parasites are exposed to oxidative stress, not only during host invasion, but also during exposure to drugs (25, 29). The antileishmanial agents are known to exert their actions through the generation of ROS, resulting in further generation of toxic by-products, creating severe oxidative stress. The fact that treatment with antileishmanial agents like PAT and miltefosine resulted in differential increases in enzyme levels suggested organelle-specific generation of ROS. However, notably, with both the loss of mitochondrial potential and cell death, overexpression of mTXNPx prevented membrane potential loss and protected cells from death induced by miltefosine with higher efficiency than cTXNPx-overexpressing cells. This could be due to the effect of miltefosine on the inhibition of mitochondrial proteins like cytochrome c, and therefore, the presence of mTXNPx in larger amounts could have helped (36, 37). This requires analysis using both overexpressing and resistant parasites. Arguably, if these defensive systems are strong enough, drug resistance can occur, as shown by other groups (8, 17, 18). Amphotericin B-resistant isolates express more cTXNPx, suggesting overexpression of cTXNPx as a possible cause of resistance (17). Our data for PAT- and miltefosine-resistant parasites showed comparable increases in both TXNPxs. The comparable expression of these enzymes in PAT- and miltefosine-resistant isolates/laboratory strains of parasites negates the drug-specific selection of the enzymes for the emergence of specific drug resistance. The results indicate that the selective specificity of the enzymes is the early event, which is followed by the involvement of both enzyme systems in collectively defending the parasite in the drug-dominated environment, possibly resulting in drug tolerance. The finding again strengthens the complementarity of the TXNPx system despite the different selective focuses on exogenous and endogenous stress inducers during infection, as well as responses to drugs.
In summary, the above-described data demonstrate the relative importance of the two enzymes in combating oxidative stress generated by variable stress conditions faced by the parasites. Importantly, both cTXNPx and mTXNPx have positive functions within the host. Since the two enzymes complement each other, the findings of the study provide a basis for using both enzymes as therapeutic targets.
MATERIALS AND METHODS
Animals.The mice used were from inbred BALB/c strain and were of either sex at 6 to 10 weeks of age, bred and maintained at the Experimental Animal Facilities of the National Institute of Immunology, New Delhi, India. The Institutional Animal Ethics Committee of the National Institute of Immunology approved all animal experiments (IAEC/AQ/2015/134; serial no. IAEC#372/15). All animal procedures were followed as prescribed by the Institutional Animal Ethics Committee.
Cells. (i) Parasites.L. donovani strain BHU1260 (Ag83) was derived from splenic aspirate from a VL patient at the Kala Azar Medical Centre of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. The other strain used in the study was UR6 (MHOM/IN/1978/UR6), used for generating miltefosine-resistant parasites. Episomal expression of proteins was done in Ag83 (WT) late-log-phase promastigotes. K39 (pR1), derived from sodium antimony gluconate (SAG)-unresponsive VL patients, was used as a SAG-resistant isolate of L. donovani. GE1 (pR2) and LDPGE1 (pR3) were used as PAT-resistant laboratory-generated strains (29), and mR1 and mR2 were used as miltefosine-resistant laboratory-generated strains in the present study. Laboratory generation of mR1 and mR2 was achieved by prolonged incubation (60 days) of UR6 (for mR1) and Ag83 (for mR2), with stepwise increases in drug pressure from 3 μM to 15 μM miltefosine, followed by the selection of parasites on blood agar slants containing 15 μM miltefosine.
(ii) Macrophages (J774A.1).The murine macrophage cell line J774A.1 (ATCC TIB-67) was maintained at 37°C in an atmosphere of air and 5% CO2 (12) in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO) containing 1.5 g sodium bicarbonate liter−1, 4.5 g glucose liter−1, and 10% fetal calf serum (FCS).
(iii) Mouse peritoneal macrophages.Mouse peritoneal macrophage monolayers were prepared as described previously (38). Briefly, peritoneal-exudate cells were harvested from the peritoneal cavities of BALB/c mice and lavaged using chilled serum-free RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO). Media recovered from several mice were pooled, and cells were collected by centrifugation and incubated for 2 h in culture flasks. Nonadherent cells were removed, and the adherent cells were further incubated in complete RPMI 1640 medium to form a macrophage monolayer. All experiments were performed after 12 h of macrophage monolayer formation.
Infections.For infection experiments, J774A.1 cells (5 ×105 cells/ml) or macrophages harvested from mouse peritoneum (5 ×105 cells/ml) were seeded on coverslips and incubated overnight using DMEM containing 10% FCS (Biological Industries, Kibbutz Beit-Haemek, Israel). After 24 h, the cells were incubated with stationary-phase L. donovani promastigotes at a ratio of 10 to 15 parasites/macrophage in serum-free medium for 2 to 6 h. Subsequently, the macrophages were washed several times with serum free medium to remove nonphagocytosed promastigotes. The infected cells were then maintained in DMEM containing 10% FCS for different time at 37°C with 5% CO2 and air (21).
Antibodies, drugs, and other chemicals.L. donovani-specific anti-cTXNPx and anti-mTXNPx were raised and characterized as described previously (10, 12). β-Tubulin (Invitrogen, Rockford, IL) was used to normalize the level-of-expression profiles of proteins. Anti-green fluorescent protein (GFP) antibody was purchased from Santa Cruz, San Diego, CA. The potentiometric probe JC-1 (5,5′6,6′-tetrachloro-1,1′3,3′-tetraethylbenzimidazolyl carbocyanine iodide) was used for measuring the mitochondrial potential, and H2DCFDA was used for evaluating total intracellular ROS. Mitochondrion-specific superoxide was estimated with MitoSOX Red, a mitochondrial superoxide indicator. JC-1, H2DCFDA, MitoSOX, Mitotracker Red, and Hoechst dye were obtained from Molecular Probes (Eugene, OR). Various stress inducers used were rotenone, TTFA, antimycin A, PAT (Sigma-Aldrich, St. Louis, MO), H2O2 (Merck, India), and miltefosine (Cayman Chemical Company, Ann Arbor, MI). Propidium iodide (PI) and 1,4-diazabicyclo[2.2.2]octane (DABCO) were purchased from Sigma-Aldrich (St. Louis, MO).
Preparation of cell lysates and antibody dilutions.Promastigotes were lysed in Laemmli buffer (20 mM Tris-HCl at pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 1 M leupeptin), and the protein content was quantified using a CBX protein assay kit (G-Biosciences, St. Louis, MO). SDS-PAGE and Western blot assays were carried out as described previously (10, 39). Transblotted proteins were probed with mouse anti-GFP antibody at 1:7,500, rabbit anti-β-tubulin at 1:10,000, rabbit anti-mTXNPx at 1:500, and rabbit anti-cTXNPx at 1:10,000 dilutions in 1× PBS containing 0.1% Tween 20. Goat anti-rabbit and anti-mouse horseradish peroxidase-conjugated immunoglobulin G secondary antibody (1:10,000) was used to detect reactivity of the blots to the primary antibody with enhanced chemiluminescence (ECL) using the FemtoLucent ECL kit (G-Biosciences, St. Louis, MO). β-Tubulin was used as a protein-loading control wherever required.
Microscopy.For selective staining of mitochondria, parasites immobilized on poly-l-lysine-coated coverslips were incubated with MitoTracker Red, a mitochondrial marker dye, in M199 (Sigma-Aldrich, St. Louis, MO) with 10% FCS for 10 min, followed by washes with serum-free M199. The stained cells were further incubated in serum-free M199 with Hoechst 33342 for nuclear staining, mounted with 2.5% DABCO, and imaged using a Leica TCS SP5 II (Leica Microsystems, Wetzlar, Germany) (10).
Flow cytometry and spectrophotometry.For detecting GFP expression in cells and measuring cellular viability, a BD Calibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15-mV, 488-nm air-cooled argon ion laser was used. Analyses were performed on 10,000 gated events, and numeric data were processed using FlowJo 8.7.1 software (Tree Star Inc., Ashland, OR, USA). For determination of intracellular ROS, mitochondrial membrane potential, and mitochondrial superoxide generation, steady-state fluorescence spectroscopy was carried out in a Clariostar spectrofluorometer (BMG Labtech GmbH, Ortenburg, Germany).
Expression profiles of defensive enzymes of L. donovani during stress.The stress inducers used in the present study were in the context of endogenous or exogenous ROS inducers. The endogenous stress inducers were mitochondrial respiratory chain inhibitors, rotenone (complex I inhibitor; 50, 100, and 150 μM), TTFA (complex II inhibitor; 50, 100, and 150 μM), and antimycin A (complex III inhibitor; 80, 100, and 150 μM). H2O2 at various doses was used in this study as an exogenous stress inducer. About 5 × 106 L. donovani parasites in the log phase of growth were exposed to different concentrations of exogenous and endogenous stress inducers for 6 h, and whole-cell lysates (WCL) of the parasites prepared at 6 h were subjected to Western blot analysis with anti-mTXNPx and anti-cTXNPx antibodies. The immunoreactivities of the proteins were captured in GeneSys (Cambridge, United Kingdom) G:Box Chemi-XX8. The fold expression of the respective proteins in all experimental groups was estimated through densitometry using GeneTools software (Syngene; Synoptic Ltd., Cambridge, United Kingdom) and compared to those of the respective WT parasites.
Overexpression of defensive enzymes in L. donovani.The mTXNPx gene (cloned in pXG-GFP+′) and the cTXNPx gene (cloned in pXG-GFP+2), along with genes coding for inactive enzymes, mTXNPx (Δ81) and cTXNPx (Δ52), were overexpressed in parasites. For the control groups, pXG-GFP+2, pXG-GFP+′, and mitochondrial targeting signal (MTS) were cloned in pXG-GFP+′ (data not shown) (10, 12, 39, 40). The overexpression of the proteins was confirmed by Western blotting with the respective antibody, as well as anti-GFP antibody. Successful transfection was further confirmed by measuring GFP expression in a BD Calibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15-mV, 488-nm air-cooled argon ion laser. The localization of the overexpressed proteins was further checked by imaging of transfectants using a confocal microscope (10, 39).
Cell viability by propidium iodide exclusion.The membrane permeability assay used as a viability test was carried out according to a protocol described previously (42). About 5 × 106 promastigotes were harvested after treatment (with different drugs) by centrifugation at 1,100 × g for 5 min, followed by resuspension in 1× phosphate-buffered saline (PBS), pH 7.4. PI was added at a final concentration of 4 μg/ml and incubated for 5 min. The cells were analyzed for nuclear DNA staining by PI (excitation and emission maxima of 493 and 636 nm) using a flow cytometer (BD Calibur flow cytometer; Becton Dickinson, San Jose, CA).
Measurement of total intracellular ROS.To estimate total intracellular ROS, the cell-permeable probe chloromethyl derivative of 2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was used. In the presence of proper oxidant, dichlorodihydrofluorescein is oxidized to the highly fluorescent 2,7-dichlorofluorescein. For ROS estimation, 5 × 107 cells were suspended in M199 and incubated with 2 μg ml−1 CM-H2DCFDA for 15 min in the dark; subsequent treatments were carried out, followed by fluorescence monitoring at an excitation wavelength of 480 nm and an emission wavelength of 520 nm (29). For each set of experiments, fluorometric measurements were performed in triplicate and expressed as fluorescence intensity units (FIU). For total intracellular ROS measurement, 50 μM rotenone, 50 μM TTFA, 100 μM antimycin A and 80 μM H2O2 were taken, as at these lower doses, significant differences in the expression profiles of TXNPxs were observed in WT parasites compared to controls.
Measurement of mitochondrial membrane potential.JC-1 dye (Molecular Probes; Eugene, OR) was used as a probe for the measurement of mitochondrial membrane potential as described previously (20). Briefly, 5 × 106 log-phase promastigotes were resuspended in 1 ml 1× PBS containing 10 μM JC-1 probe and incubated for 20 min at 26°C. Parasites were further suspended in 1 ml phenol-free DMEM with 10% FCS, followed by the different drug treatments (with drug doses similar to those used in intracellular-ROS measurement). Continuous changes in mitochondrial membrane potential were captured in a fluorometer with 530-nm excitation and 590-nm emission filters for green (monomeric form) and red (J-aggregate formation) fluorescence. The ratio of the reading at 590 nm to the reading at 530 nm was considered the relative change in mitochondrial membrane potential (20).
Superoxide assay.As the major source of oxidants in eukaryotes is the mitochondria, we used the MitoSOX dye (mitochondrial superoxide indicator) to measure the mitochondrial accumulation of superoxide, a measure indicative of mitochondrial ROS generation (41). Parasites (2 × 107 promastigotes) were preincubated with 0.5 M the red cell-permeable fluorogenic probe MitoSOX Red for 30 min at 28°C in Hanks' balanced salt solution with calcium and magnesium and then treated with different doses of drugs for 0, 10, 20, 30, 40, and 60 min. Fluorescence emission at 580 nm was measured with a fluorometer with an excitation wavelength of 488 nm and expressed as arbitrary fluorescence units.
Statistical analysis.The data were statistically analyzed by the one-way analysis of variance (ANOVA) test and a Tukey's post hoc test and are presented as means and standard errors (SE) of three determinations from at least three or four independent experiments. The statistical analysis was performed using GraphPad Prism version 5.01 (GraphPad, San Diego, CA). A P value of less than 0.05 was considered significant.
ACKNOWLEDGMENTS
The present work was supported by a Centre for Molecular Medicine grant from the Department of Biotechnology (http://dbtindia.nic.in), New Delhi, India (grant no. BT/PR/14549/MED/14/1291). C.S. is the recipient of a J.C. Bose Fellowship from the Department of Science and Technology (award no. SR/S2/JCB-12/2008). S.D. acknowledges the Department of Science and Technology for a DST Inspire Faculty Award (Award Sanction no. DST/INSPIRE/04/2015/002785). S.G. was supported by an NII fellowship.
FOOTNOTES
- Received 20 April 2017.
- Returned for modification 14 June 2017.
- Accepted 18 October 2017.
- Accepted manuscript posted online 23 October 2017.
- Copyright © 2017 American Society for Microbiology.
