Elevated ergosterol protects Leishmania parasites against antimony-generated stress

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1 The FASEB Journal Research Communication Elevated ergosterol protects Leishmania parasites against antimony-generated stress Radhika Mathur, Rajeev Patrick Das, Archana Ranjan, and Chandrima Shaha 1 Cell Death and Differentiation Research Laboratory, National Institute of Immunology, New Delhi, India ABSTRACT Parasite lipids can serve as signaling molecules, important membrane components, energy suppliers, and pathogenesis factors critical for survival. Functional roles of lipid changes in response to drug-generated stress in parasite survival remains unclear. To investigate this, Leishmania donovani parasites, the causative agents of kala-azar, were exposed to the antileishmanial agent potassium antimony tartrate (PAT) (half-maximal inhibitory concentration 284 mg/ml). Analysis of cell extracts using gas chromatography-mass spectrometry showed significant increases in very long-chain fatty acids (VLCFAs) prior to an increase in ergosterol in PATtreated parasites as compared with vehicle-treated controls. Ergosterol biosynthesis inhibition during PAT treatment decreased cell viability. VLCFA inhibition with specific inhibitors completely abrogated ergosterol upsurge followed by a reduction in cell viability. Following PAT-induced VLCFA increase, an upsurge in reactive oxygen species (ROS) occurred and inhibition of this ROS with antioxidants abrogated ergosterol increase. Genetically engineered parasites expressing low constitutive ergosterol levels showed more susceptibility to PAT as compared with wild-type control cells but ergosterol supplementation during PAT treatment increased cell viability. In conclusion, we propose that during antimony treatment, the susceptibility of parasites is determined by the levels of cellular ergosterol that are regulated by oxidative stress generated by VLCFAs. Mathur, R., Das, R. P., Ranjan, A., Shaha, C. Elevated ergosterol protects Leishmania parasites against antimony-generated stress. FASEB J. 29, (2015). Key Words: fatty acids cell death sterol reactive oxygen species The appearance of oxygen in the atmosphere drove the evolution of single-celled eukaryotes followed by development of more complex life forms that derive energy via aerobic metabolism (1). However, this development required modifications in the metabolic pathway for protection of organisms against oxidative damage. It is believed that formation of sterols such as cholesterol and Abbreviations: AAPH, 2, 2 -azobis-2-methyl-propanimidamide, dihydrochloride; BSTFA, N,O-bistrifluoroacetamide; EI, electron impact; FA, fatty acid; GC-MS, gas chromatography-mass spectrometry; LCFA, long-chain fatty acid; NAC, N-acetyl cysteine; RBC, red blood cell; ROS, reactive oxygen species; PAT, potassium antimony tartrate; SBP, sterol biosynthesis pathway; VLCFA, very long-chain fatty acid ergosterol from squalene coincided with the gradually increasing levels of atmospheric oxygen (2, 3). Various examples across eukaryotic life show that the levels of sterols increase or decrease as a function of oxidative stress (4, 5), which is very important in the context of pathogensastheyareexposedtoreactiveoxygenspecies (ROS) during host invasion. The host uses ROS to kill the invading pathogens and the success of the pathogens in creating a successful infection lies in their ability to protect themselves from the host s defensive arsenal. The Leishmania parasites are the causative agents of cutaneous, mucocutaneous, and the potentially fatal visceral leishmaniasis. They have a digenetic life cycle surviving alternatively within the insect and the mammalian host where they are exposed to a large amount of ROS. As a defense to host-generated antileishmanial activity, these parasites express a number of defensive enzymes like the cytosolic and the mitochondrial form of the tryparedoxin peroxidases (6, 7), antioxidant thiols like trypanothione and ovothiol, but lack catalase and the selenium-dependent glutathione peroxidases, which are the primary arsenal against the ROS and its products (8, 9). Nonetheless, these parasites are able to defend themselves well, create successful infections, and resist drugs. Although years of research have gone into studying the mechanisms by which these parasites survive in the harsh conditions of the host, a comprehensive understanding of the molecular changes that occur in response to oxidativestressislacking. The first line of defense against oxidative stress is the plasma membrane. Leishmania parasites are endowed with a lipophosphoglycan coat on the cell surface beneath which the plasma membrane is located (10). The plasma membrane is a complex structure consisting of numerous proteins and a large variety of different amphipathic lipids that regulate membrane fluidity, permeability, and cellular signaling (11 13). Because sterols and fatty acids (FAs) are located on the membrane, they are in a position to act as the first line of defense against oxidative stress. Trypanosoma, the group to which the Leishmania parasites belong, has certain unique features in their sterol and FA profile that separates them from other organisms and may give 1 Correspondence: Cell Death and Differentiation Research Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi , India. cshaha@nii.ac.in doi: /fj This article includes supplemental data. Please visit to obtain this information /15/ FASEB 4201

2 them an edge in surviving under unfavorable conditions (11, 14). The differences between lipid biosynthesis pathways of Leishmania parasites and mammals have also opened new avenues for exploration of novel targets for drug development. Studies have revealed that drugs such as amphotericin B and miltefosine can cause extensive changes in the lipid metabolism of Leishmania parasites, and these alterations may also lead to the development of resistance toward these drugs (13, 15). Even though antimony has been the longest prescribed and most effective drug against leishmaniasis, there are virtually no investigations into how the antimonials alter lipids, especially in view of available literature on changes in lipids induced by other antileishmanial agents. Preparations containing antimony remain primary drugs for the treatment of visceral leishmaniasis in Brazil and sub-saharan Africa, but in India, these compounds have lower efficacy, although they are still prescribed in field conditions (16). Because of its successful extensive use in field conditions, parasites exposed to antimony treatment serve as good models to study the association between lipid changes to cell survival. Our earlier studies have demonstrated that parasites with lower sterol content are more susceptible to antileishmanial drugs such as potassium antimony tartrate (PAT) and ROS (17). PAT, a trivalent antimonial (Sb III ), is the active form of pentavalent antimonial (Sb V ) sodium stibogluconate and is accumulated in parasites after Sb V treatment (18). The present study shows a new finding that the parasite responds to antimony treatment by creating alterations in FA levels and inducing an ergosterol upsurge. This ergosterol surge is necessary to prevent antimony cytotoxicity and susceptibility of the parasite toward oxidative stress. Therefore, we suggest that these changes could be a part of a primitive defense system in these parasites that belong to the most ancient eukaryotic lineages. formamide) for 15 min at 37 C, and OD at 570 nm was measured using a m-quant microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) with calibration against a standard curve with known number of viable cells (6). ROS levels To monitor the level of intracellular ROS, the cell-permeable probe CMH 2 DCFDA (Molecular Probes, Eugene, OR, USA) was used. Promastigotes were resuspended in phenol red free DMEM and labeled with CMH 2 DCFDA (4 mg/ml) for 20 min in the dark. Fluorimetric analyses were carried out at 507 nm excitation and 530 nm emission using a FluoStar Omega fluorescence reader (BMG Labtechnologies Incorporated, Offenburg, Germany). For all measurements, the basal fluorescence was subtracted (19). In situ labeling of DNA fragments Cells undergoing apoptosis generate abundant DNA fragments in their nuclei. In situ detection of DNA fragments by TUNEL was performed using an apoptosis detection system as described previously (20). Briefly, PAT-treated promastigotes were harvested at different times, fixed in 4% paraformaldehyde, and coated onto grease-free slides. Permeabilization was done with 0.2% (vol/vol) Triton X-100 in 13 PBS and equilibration buffer (200 mm potassium cacodylate, 25 mm Tris-HCl, 0.2 mm DTT, 0.25 mg/ml bovine serum albumin, 2.5 mm cobalt chloride) for 10 min at room temperature followed by incubation with terminal deoxynucleotidyl transferase buffer containing nucleotide mix (50 mm fluorescein-12-dutp, 100 mm datp, 10 mm Tris-HCl, 1 mm EDTA, ph 7.6) for 1 h at 37 C in dark. The samples were counterstained with 10 mg/ml propidium iodide in the presence of 25 mg/ml RNase and visualized under a TCS-SP5 II confocal microscope from Leica (Solms, Germany). At least 400 cells of 4 independent experiments were counted. Sterol profile MATERIALS AND METHODS Reagents and parasite culture All chemicals were from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. Trolox, ethanol, diethyl ether, chloroform, heptane, hexane, and methanol were obtained from Merck Specialties Private Ltd. (Mumbai, India). DeadEnd Fluorometric TUNEL System was obtained from Promega Corp. (Madison, WI, USA). Promastigotes of Leishmania donovani were cultured in M199 medium (ph 7.4) supplemented 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and maintained at 23 C. CYP5122HKO parasites were cultured in medium supplemented with hygromycin to prevent the loss of allelic replacement construct (17). Cell viability Briefly, cells from various treatment groups were incubated in media containing 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 250 mg/ml for 1 2 h at 23 C, following which cells were solubilized in lysis buffer (20% SDS in 50% dimethyl Sterols were isolated from the promastigotes as described previously, with slight modifications (17). Briefly, cells were suspended in 30% alcoholic KOH and incubated at 80 C (1 h) followed by cooling to room temperature. Sterols were extracted from the organic phase with heptane, dried under nitrogen gas, resuspended in n-hexane, and derivatized with BSTFA (N, O-bistrifluoroacetamide) containing 1% trimethylsilyl chloride at 75 C for 1 h. The derivative mixture was dried under nitrogen to remove excess BSTFA and subsequently redissolved in n-hexane prior to gas chromatography-mass spectrometry (GC-MS) analysis. Calibration curve was generated using n-hexane stock solutions of standard ergosterol dilutions (5 500 ng/ml) in duplicates and plotting the peak area vs. the concentration. FA profile FAs were isolated as described previously (12), with slight modifications. Briefly, cells were harvested and the resulting pellet was resuspended in chloroform-methanol (2:1 vol/vol) for 2 h at 4 C. Sonications were performed at 4 C with a Q700 sonicator (Qsonica, Newtown, CT, USA). After centrifugation at 3000 g for 5 min, the lower phase containing total lipids was collected and evaporated to dryness at room temperature 4202 Vol. 29 October 2015 The FASEB Journal x MATHUR ET AL.

3 under a constant flow of nitrogen gas. The residue was saponified with 30% KOH in alcohol at 80 C for 2 h. Sterols were extracted from the organic phases with heptane, and the lower aqueous phase of KOH ethanolic extracts was acidified with 6 N sulfuric acid to ph 3. FAs were extracted with hexane and transesterified with boron trifluoride-methanol at room temperature for 2 h. Methyl esters were extracted with hexane and redissolved in methanol-diethyl ether (1:1 vol/vol) prior to GC-MS analysis. GC-MS Gas-liquid chromatography was performed with an Agilent model 7890A gas chromatography instrument coupled to an Agilent 5975C mass spectrometer and Agilent ChemStation software (version G1701EA; Agilent Technologies, Palo Alto, CA, USA). An HP5 column (methyl:phenylsiloxane ratio: 95/5; dimensions: 30 m mm mm) was used for separation. Pure helium (grade I) at a flow rate of 1 ml/min wasusedasgascarrier. The condition for analysis for sterols was as follows. The column was kept at 100 C, the injector at 250 C, and the detector at 280 C. The oven program for methyl esters was as follows: 100 C for 5 min, then 20 C/min to 200 C for 10 min followed by 10 C/min to 250 C for 0 min, and finally 2.5 C/min to 320 C for 5 min. MS conditions were as follows. source temperature was 280 C, quad temperature was 150 C, and electron impact (EI) energy was 70 ev. The conditions for analysis of FAs was as follows. The column was kept at 100 C, the injector at 300 C, and the detector at 300 C. The oven program for methyl esters was as follows: 100 C for 1 min, then 8 C/min to 290 C for 25 min followed by 15 C/min to 330 C for 4 min. MS conditions were as follows: source temperature was 280 C, quad temperature was 150 C, and EI energy was 70 ev. In vitro hemolysis assay Antioxidant activity was estimated by hemolysis assay as described previously (21), with slight modifications. Blood was obtained from healthy volunteers in accordance with the standards specified by Institutional Human Ethics Committee of National Institute of Immunology (New Delhi, India; IHEC no.83/14).briefly, blood was collected from volunteers in tubes containing EDTA to prevent coagulation. Samples were centrifuged at 450 g for 10 min at 4 C. Plasma and buffy coat were removed and the red blood cells (RBCs) were washed and resuspended at 5% hematocrit in cold PBS. To evaluate the capacity of ergosterol to protect RBCs from AAPH (2, 29- azobis-2-methyl-propanimidamide, dihydrochloride) induced hemolysis; RBCs were pretreated with different doses of ergosterol for 15 min followed by addition of 50 mm AAPH. An aliquot of RBC suspension was taken every hour for a period of 8 h, diluted with 20 volumes of saline solution and centrifuged at 3200 g for 8 min. The absorption (A) ofsupernatant was read at 540 nm. To yield absorption after complete hemolysis (B), an identical aliquot of RBCs was diluted with ice cold water. Percentage hemolysis was then calculated as (A/B) Statistical analysis Unless mentioned in figure legends, at least 3 independent experimental repeats were performed for all experiments. P value was calculated by Student s t test. Graphs were plotted using SigmaPlot (Systat Software Inc., San Jose, CA, USA). RESULTS Treatment with antileishmanial drug PAT alters sterol and FA profiles In response to stress, unicellular organisms can alter their lipid composition (12, 13), and these changes may have important functional implications. Leishmania promastigotes were the ideal parasite forms to be studied because these are the infective forms exposed to most severe oxidative stress in the insect or the mammalian host (22, 23). The primary question addressed in this part of the investigation was to determine if antimony exposure brought about any changes in the lipid profile of the promastigotes. Trivalent antimonial PAT (24) was used at the dose of 100 mg/ml, because this dose precipitated lesser cell death as compared with other doses therefore distortion of data due to excessive cell death would be minimal (Fig 1A) (TUNEL-positive cells at 12 h at doses of 50 mg/ml: ;1 5%; 100 mg/ml: ;10 12%; 200 mg/ml: ;20 25%). Our studies demonstrate that PAT exposure globally alters the profileoffasinthepromastigotesgrownin vitro within 3 h of treatment (Fig. 1B). From the chromatogram obtained after GC-MS analysis of extracts of drug-exposed and vehicle-treated promastigotes, a totalof32fascouldbedetected.outofthese,saturated FA-like stearic acid (Table 1, no. 13) and unsaturated FA-like oleic acid (Table 1, no. 10) or vaccenic acid (9/11 octadecenoic acid) (Table 1, no. 11) and linoleic acid (Table 1, no. 9) represented ;70 77% of the total FAs. Interestingly, upon treatment with PAT there was a down-regulation of oleic acid (Table 1, no. 10) to insignificant levels with a major up-regulation of vaccenic acid (Table 1, no. 11) that was otherwise not present in the vehicle-treated cells. Analysis of FAs based on their carbon chain length showed an alteration in the ratio of long-chain FAs (LCFAs, C-12, C-14, and C-16) to very long-chain FAs (VLCFAs) (C-20, C-22, and C-24) with a prominent increase in the levels of VLCFAs (Fig. 1B; Table 2). Overall, the data presented above indicated that one of the cellular changes in response to PAT consisted of substantial alterations in the profile of FAs in comparison with vehicle-treated controls. GC-MS analysis of sterol extracts from vehicle-treated promastigotes showed ergosterol as the predominant sterol (Fig. 1C). This also confirmed prior reports that have identified ergosterol as the primary sterol in L. donovani (25). Peaks of cholesterol and ergosterol precursors such as squalene and ergostatetraenol could also be identified in the chromatogram (Fig. 1C). The peak of cholesterol was possibly the result of its uptake from the culture medium because Leishmania promastigotes do not synthesize cholesterol but are able to take it up from the surrounding medium (15). When sterol extracts from promastigotes exposed to different doses of PAT were analyzed and compared with vehicle-treated controls, a significant increase in the levels of ergosterol was observed in 50 and 100 mg/ml PAT-treated parasites within 12 h of exposure (Fig. 1C, D). Interestingly, the levels of squalene and ergostatetraenol also increased significantly suggesting that PAT upregulates sterol biosynthesis pathway (SBP) upstream ELEVATED ERGOSTEROL IS PROTECTIVE AGAINST ANTIMONY 4203

4 Figure 1. PAT induces changes in sterol and FA profile of L. donovani promastigotes. A) DNA fragmentation in L. donovani promastigotes with or without PAT treatment for 12 h as detected by TUNEL staining: (a) vehicle treatment only; (b) PAT (50 mg/ml); (c) PAT (100 mg/ml); (d) PAT (200 mg/ml). White arrows indicate parasites with damaged DNA. B) Overlay of GC chromatogram of FA extracts from log-phase vehicle-treated and PAT-treated (100 mg/ml for 3 h) promastigotes. Peaks of linoleic acid, vaccenic acid/oleic acid, and stearic acid are marked as a, b, and c, respectively. Table 2 shows percentage change in the FA composition based on carbon chain length when log-phase L. donovani promastigotes were treated with PAT (100 mg/ml) for 3 h. C) Overlay of GC chromatogram of sterol extracts from log phase promastigotes treated with vehicle or different doses of PAT ( mg/ml) for 12 h. Peaks of squalene, cholesterol, ergostatetraenol, and ergosterol are marked as a, b, c, and d, respectively. D) Bar graph showing changes in ergosterol levels over a period of 24 h when log-phase L. donovani promastigotes grown in vitro were treated with different concentrations of PAT. All n =3;*P, of squalene. A time-dependent analysis also revealed that the increase in ergosterol levels was transient becauseadeclinewasobservedafter24hoftreatment (Fig. 1D). In summary, this part of the data clearly indicated that PAT induces a transient increase in the level of sterols, which is preceded by alterations in the FA profile. FA and sterol changes are linked to cell survival The observed changes in FAs and sterols prompted us to ask about the functional relevance of such changes. Because PAT is an antileishmanial agent, the alterations recorded could herald either a survival or a death-inducing response. The ideal experiments to arrive at a conclusion were to inhibit VLCFA and sterol increase and examine effects on cell viability. Allidochlor and metazachlor, 2 specific plant VLCFA inhibitors (26, 27), were used because there are no known VLCFA inhibitors for trypanosomatids. Importantly, both the inhibitors were able to block PAT-induced VLCFA increase successfully in the promastigotes (Supplemental Table 1). Biochemical changes following VLCFA inhibition resulted in a decrease in cell viability (Fig. 2A) and a significant lowering of PAT-induced ergosterol increase (Fig. 2B). Arguably, if VLCFA inhibition was blocking ergosterol increase, administration of VLCFAs should increase ergosterol levels. Three VLCFAs that showed substantial increase after PAT exposure were selected and used to treat the parasites, following which sterols were measured. FAs 5,8,11,14-eicosatetraenoic acid (C-20) and 4,7,10,13,16,19-docosahexaenoic acid (C-22) at doses of 10 and 5 mm wereabletoincreasein the levels of ergosterol (Fig. 2C); however, vaccenic acid 4204 Vol. 29 October 2015 The FASEB Journal x MATHUR ET AL.

5 TABLE 1. Identification and quantification of FA methyl esters obtained from phospholipid saponification in total membranes from wild-type and PAT-treated (100 mg/ml) L. donovani promastigotes (n = 3) Serial no. Chain length FA Control PAT (3 h) 1 C-12 Dodecanoic acid, methyl ester (lauric acid) C-14 Tetradecanoic acid, methyl ester (myristic acid) C-16 9-Hexadecenoic acid, methyl ester (palmitoleic acid) C-16 9-Hexadecenoic acid, 9-methyl-, methyl ester ND C-16 Hexadecanoic acid, methyl ester (palmitic acid) C-16 Hexadecanoic acid, 14-methyl-, methyl ester ND 7 C-16 Hexadecanoic acid, 15-methyl-, methyl ester C-17 cis-10-heptadecenoic acid, methyl ester C-18 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (linoleic acid) C-18 9-Octadecenoic acid, methyl ester (oleic acid) ND 11 C Octadecenoic acid, methyl ester (vaccenic acid) ND C-18 9,12,15-Octadecatrienoic acid, 2-propyl methyl ester C-18 Octadecanoic acid, methyl ester (stearic acid) C-18 9,12,15-Octadecatrienoic acid, methyl ester (linolenic acid) C-18 7,10-Octadecadienoic acid, methyl ester ND C-18 Octadecanoic acid, 17-methyl-, methyl ester C-18 6,9,12-Octadecatrienoic acid, methyl ester ND C-18 8,11-Octadecadienoic acid, methyl ester ND 19 C-19 cis-10-nonadecenoic acid, methyl ester C-19 Nonadecanoic acid, methyl ester (nonadecyclic acid) C-20 7,10,13-Eicosatrienoic acid, methyl ester C-20 5,8,11,14-Eicosatetraenoic acid, methyl ester (arachidonic acid) C-20 5,8,11,14,17-Eicosapentaenoic acid, methyl ester C-20 8,11,14-Eicosatrienoic acid, methyl ester C-20 Methyl 8,11,14,17-eicosatetraenoate C-20 cis-11,14-eicosadienoic acid, methyl ester C-20 Eicosanoic acid, methyl ester (arachidic acid) C-22 Docosanoic acid, methyl ester (behenic acid) ND 29 C-22 cis-4,7,10,13,16,19-docosahexaenoic acid, methyl ester C-22 cis-7,10,13,16-docosatetraenoic acid, methyl ester ND C-22 7,10,13,16,19 Docosapentaenoic acid, methyl ester ND 32 C-24 Tetracosanoic acid, methyl ester (lignoceric acid) ND, nondetectable. (C-18) at the dose of 200 mm was unable to alter ergosterol (Fig. 2C). Also, as observed with PAT treatment, these FAs up-regulated the SBP upstream of squalene (data not shown). Combined, these results suggest that PATinduced increase in VLCFAs, such as eicosatetraenoic and docosahexaenoic acids, are responsible for up-regulation of the SBP. Next, we attempted to inhibit sterol biosynthesis and create a situation where cells were exposed to PAT but ergosterol was not allowed to increase. Two sterol biosynthesis inhibitors, amorolfine and simvastatin, the former acting on ERG24 and ERG2 genes and the latter operating on ERG13 gene of the ergosbp, were used (14, 28). Treatment of promastigotes with either amorolfine or simvastatin (10 mg/ml) in the presence or absence of PAT wasabletoreduceergosterollevelssignificantly within 12 h (Fig. 2D). Interestingly, both amorolfine and simvastatin by themselves did not kill cells when compared with vehicle-treated parasites. However, when they were given in the presence of PAT, the death-inducing property of PAT was increased significantly, resulting in reduced cell viability. A combination of both amorolfine and simvastatin during PAT treatment was most lethal (Fig. 2E). This suggested that lack of sterol biosynthesis during antimony exposure made the cells more susceptible to drug-induced death. Parasites with altered sterol levels respond differently to PAT The data indicate a correlation between sterol levels and parasite survival. To further confirm the link between ergosterol and cell death, we used Leishmania parasites expressing constitutively low ergosterol levels. These TABLE 2. Percentage difference in the FA composition based on carbon chain length when log-phase L. donovani promastigotes were treated with PAT (100 mg/ml)for3h Chain length % Change C C C C C C C ELEVATED ERGOSTEROL IS PROTECTIVE AGAINST ANTIMONY 4205

6 Figure 2. Changes in sterol biosynthesis can be linked to alterations in the VLCFA pool. A) Graph shows change in percentage of viable cells when wild-type Leishmania promastigotes were treated with allidochlor (50 mm) and metazachlor (200 mm) in presence or absence of PAT (100 mg/ml) for 24 h. B) Bar graph shows changes in the level of ergosterol when L. donovani parasites are treated with allidochlor (50 mm) and metazachlor (200 mm) in presence of PAT (100 mg/ml) for 24 h. C) Changes in the level of ergosterol when L. donovani parasites were treated with 200 mm VA, 10 mm EA, and 5 mm 4,7,10,13,16,19-docosahexaenoic acid (DA) for 3, 6, and 12 h. D) Ergosterol content in log-phase promastigotes treated with amorolfine (10 mg/ml) and simvastatin (10 mg/ml) with or without PAT (100 mg/ml) for 12 h. E) Bar graph shows decrease in percentage viability when log phase promastigotes were treated with amorolfine (10 mg/ml) and simvastatin (10 mg/ml) along with PAT (100 mg/ml) for 24 h. A, amorolfine; Al, allidochlor; C, control; EA, 5,8,11,14-eicosatetraenoic acid; M, metazachlor; P, PAT; S, simvastatin; VA, vaccenic acid. All n = 3;*P, parasites, previously generated in the laboratory, expressed the single allele of CYP5122A1, a cytochrome P450 of the L. donovani, resulting in low ergosterol levels (17). These parasitesweremoresensitivetopatascomparedwith control wild-type cells expressing both alleles of CYP5122A1 (Supplemental Fig. 1). Based on the above results, arguably therefore, if low ergosterol level was related to cell death, supplementation of ergosterol would make them better survivors. When ergosterol was supplemented in vitro to these parasites expressing low ergosterol during exposure to PAT, there was a small but significant increase in cell viability (Fig. 3A). Similarly, supplementation of cholesterol 4206 Vol. 29 October 2015 The FASEB Journal x MATHUR ET AL.

7 Figure 3. Parasites with altered ergosterol levels respond differently to PAT. A) Graph shows change in percentage viability when log-phase CYP5122A1 HKO parasites were cultured for 48 h in medium supplemented with either 5 mg/ml ergosterol or cholesterol and subsequently treated with PAT (100 and 200 mg/ml) for 24 h. B) Ergosterol levels of log-phase L. donovani wildtype, K39, GE1, and LDPGE1 promastigotes grown in the presence or absence of PAT (100 mg/ml) for 12 h. C) Bar graph shows ergosterol levels when log-phase K39 and GE1 promastigotes were treated with amorolfine (10 mg/ml) and simvastatin (10 mg/ml) with or without PAT treatment (100 mg/ml) for 12 h. D) Percentage viability when log-phase K39 and GE1 promastigotes were treated with combinations of amorolfine (10 mg/ml), simvastatin (10 mg/ml), and PAT (100 mg/ml) for 24 h. A, amorolfine; S, simvastatin; P, PAT. All n =3;*P, also increased cell viability during PAT treatment (Fig. 3A), suggesting a protective role of sterols during oxidative stress. To further confirm the role of ergosterol in deciding the vulnerability of parasites toward PAT, antimony-resistant strains (K39, GE1, and LDPGE1) were selected to check if they expressed higher sterol levels. K39 is a clinical isolate that is resistant to PAT (Sb III ) (29, 30), and GE1 is a laboratorygenerated strain resistant to sodium stibogluconate (Sb V ), the pentavalent antimony (30). LDPGE1 is a GE1 strain (with partial resistance to Sb III ) made fully resistant to Sb III by prolonged incubation with PAT in the laboratory. The comparative resistance of these parasites to PAT is shown in Supplemental Fig. 1. GC-MS analysis of ergosterol levels showed that K39 and LDPGE1 had much higher ergosterol levels than GE1 and the wild-type cells (Fig. 3B). Exposure to PAT could not increase ergosterol levels further in K39 and LDPGE1, but it could induce an increase in GE1 parasites expressing lower ergosterol in comparison with K39 and LDPGE1 parasites (Fig. 3B). When ergosterol biosynthesis was inhibited using sterol biosynthesis inhibitors amorolfine and simvastatin, the resistant parasites like K39 and GE1 showed lower sterol levels (Fig. 3C) and increased susceptibility to PAT (Fig. 3D), clearly suggesting that ergosterol increase correlated with higher cell survival and decreased ergosterol correlated with lower cell survival in the face of oxidative stress. Because our results with K39 and GE1 showed that increased sterol levels were associated with lesser susceptibility to PAT, we next wanted to check the changes in the levels of FAs. FAs were extracted from PAT-resistant K39 parasites, and the profile was compared with that of PATsensitive promastigotes. The results show that in the K39 parasites, a higher pool of VLCFAs was constitutively expressed (Table 3). Also, unlike PAT-sensitive promastigotes, K39 parasites expressed higher levels of vaccenic acid (data not shown). ROS determines changes in sterols but not in FAs PAT generates ROS within the first 20 min of exposure as was shown by us previously and further confirmed in this ELEVATED ERGOSTEROL IS PROTECTIVE AGAINST ANTIMONY 4207

8 TABLE 3. Percentage difference in the FA composition of PATsensitive and PAT-resistant L. donovani promastigotes (n = 3) Chain length K39 (% change) C C C C C C C study (Fig. 4A). Because ROS generation preceded the changes in FAs and sterols, we sought to investigate if ROS was linked to changes observed in sterol and FA profiles. ROS is particularly relevant for these parasites as they are exposed to a significant amount of ROS while infecting the macrophages and during residence in the secondary host gut (31). When PAT-induced ROS increase was scavenged with antioxidants like Trolox, a cell-permeable derivative of vitamin E, and N-acetyl cysteine (NAC), an antioxidant that raises the cellular pool of scavengers of free radicals, and vitamin C (Fig. 4A, B), ergosterol increase did not occur. Because ROS inhibition resulted in lowering of ergosterol levels, we sought to investigate if scavenging of ROS also inhibited the increase of VLCFA pool obtained with PAT treatment. Interestingly, treatment of parasites with vitamin C and Trolox did not inhibit accumulation of VLCFAs (Table 4). However, these antioxidants were able to inhibit PAT-induced vaccenic acid production and upregulated oleic acid production (data not shown). Treatment with antioxidants also promoted the accumulation of LCFAs (C-12, C-14, and C-16) in the parasites (Table 4). To confirm if the changes observed in lipid profiles could also be independently generated by ROS, we used H 2 O 2 as a direct source of ROS to treat the parasites. H 2 O 2 could induce an increase in the levels of various sterols including ergosterol, and the sterol profile was similar to the pattern generated by PAT (Fig. 4C). Like our results with PAT, this increase in ergosterol level was also transient and was inhibited in the presence of NAC and Trolox (Fig. 4D). Overall, the above observations show that direct application of ROS and drug-induced production of ROS both cause a transient increase in ergosterol content of the cell. Because the pattern of alteration in the sterol levels by H 2 O 2 was similar to PAT, we next sought to investigate the changes in FA profile after exposure to H 2 O 2. Leishmania promastigotes treated with H 2 O 2 showed an increased accumulation of VLCFAs (C-20, C-22, and C-24) and lowering of concentrations of LCFAs (Table 4). Also, similar to our observations with PAT, treatment with antioxidants along with H 2 O 2 did not inhibit the accumulation of VLCFAs (Table 4). To further confirm that changes in ergosterol and VLCFAs are indeed linked to increased ROS production, we used AAPH, a water-soluble peroxyl radical initiator (32), and tert-butylhydroperoxide, an organic hydroperoxide (33). Both of these compounds have been widely used to induce oxidative stress in a variety of cells (32 34). Treatment of Leishmania parasites with AAPH or tert-butylhydroperoxide increased ergosterol production (Fig. 4E) and elevated VLCFA levels (Table 5). Interestingly, similar to PAT-treated parasites, H 2 O 2, AAPH- or tert-butylhydroperoxide-treated parasites also accumulated vaccenic acid instead of oleic acid (data not shown). Inhibition of ROS increase reduces cell death Our results confirm that ROS induces an increase in the levels of sterols with ergosterol as the most prominent sterol. To further consolidate the link between ROS and ergosterol, we checked ROS production by K39 and GE1 parasites that bear less sensitivity to PAT. ROS production by these parasites in response to PAT was low as compared with the wild-type cells (Fig. 5A). Further, scavenging of ROS by antioxidants during PAT or H 2 O 2 treatment also resulted in increase in cell viability (data not shown), suggesting that oxidative stress promotes ergosterol upsurge for the protection of these parasites. Because ROS was promoting the up-regulation of ergosterol biosynthesis and this up-regulation was preceded by VLCFA increase, we wanted to put together the relationship between PAT-induced increase in ergosterol, VLCFAs, and ROS. Studies across various cell lines have shown that treatment with VLCFAs induces increased intracellular ROS levels and provokes apoptosis (35). To understand the effect of VLCFAs on ROS production in Leishmania parasites, we treated log-phase promastigotes with vaccenic acid, 5,8,11,14-eicosatetraenoic acid, and 4,7,10,13,16,19-docosahexaenoic acid. In accordance with our expectations, all 3 FAs increased the production of ROS with 5,8,11,14-eicosatetraenoic acid and 4,7,10,13,16,19-docosahexaenoic acid, generating significantly higher levels of ROS as compared with vaccenic acid (Fig. 5B). This suggested that intracellular ROS production could be dependent on the carbon chain length of the FAs and the degree of unsaturation. Our previous results have shown that VLCFA inhibitors abolish PAT-induced increase in ergosterol. To confirm if PAT-induced increased VLCFAs are responsible for high-intracellular ROS, parasites were administered with VLCFA inhibitors prior to treatment with PAT. Inhibition of VLCFA production significantly reduced PAT-induced ROS production (Fig. 5C), clearly indicating that VLCFAs were responsible for the ROS increase. Several data shown above indicate that ergosterol could have a protective role and the question was whether it could be acting as an antioxidant. A hemolysis assay with human RBCs was performed in the presence or absence of ergosterol. Cells treated with different concentrations of ergosterol (5 25 mg/ml) prior to treatment with 50 mm of AAPH, a water-soluble peroxyl radical initiator capable of inducing hemolysis of RBCs, demonstrated reduced hemolysis in the presence of ergosterol in a concentrationdependent manner (Fig. 5D). This showed that ergosterol could also be acting as a scavenger for ROS to reduce cell damage. DISCUSSION Antimonial compounds were the mainstay of antileishmanial chemotherapy for the last 6 decades and are the only chemotherapeutic alternative in some areas (36). Our 4208 Vol. 29 October 2015 The FASEB Journal x MATHUR ET AL.

9 Figure 4. Alteration in ergosterol synthesis is preceded by an increase in ROS. A) ROS levels were measured with fluorescent dye CMH 2 DCFDA preloaded onto cells after treatment with PAT (100 mg/ml). NAC (20 mm) and Trolox (5 mm) were used as antioxidants. B) Changes in ergosterol levels when log-phase promastigotes were pretreated with NAC (20 mm), Trolox (5 mm), or vitamin C (1 mm) followed by treatment with PAT for 12 h. C) Overlay of GC chromatogram of sterol extracts from log-phase promastigotes wild-type control cells and those treated with 100 mm of H 2 O 2 for 12 h. Peaks of squalene, cholesterol, ergostatetraenol, and ergosterol are marked as a, b, c, and d, respectively. Inset shows overlay of ergosterol peak (in single ion monitoring mode) of wild-type and H 2 O 2 -treated (100 mm) promastigotes. D) Bar graph shows changes in ergosterol levels when log-phase promastigotes were pretreated with NAC (20 mm) or Trolox (5 mm), followed by treatment with H 2 O 2 (100 mm) for 12 or 24 h. E) Bar graph shows changes in ergosterol levels when log-phase promastigotes were treated with AAPH (25 mm) or tert-butylhydroperoxide (50 mm) for 12 h. C, control; P, PAT; tbhp, tert-butylhydroperoxide. All n = 3;*P, ELEVATED ERGOSTEROL IS PROTECTIVE AGAINST ANTIMONY 4209

10 TABLE 4. Percentage change in the FA composition when log-phase L. donovani promastigotes were treated with PAT (100 mg/ml) or H 2 O 2 (100 mm) in the presence or absence of either Trolox (5 mm) or vitamin C (1 mm) for 3 h (n =3) Chain length PAT (% change) Vitamin C + PAT (% change) Trolox + PAT (% change) H 2 O 2 (% change) Vitamin C + H 2 O 2 (% change) Trolox + H 2 O 2 (% change) C C C C C C C queries on possible changes in lipid repertoire in the Leishmania parasite as a part of cellular drug response, originated out of observations showing extra sensitivity of Leishmania parasites with low lipid levels to Sb III (PAT) (17). This study projects a new finding of a negative correlation between the level of ergosterol in the parasite and its susceptibility toward PAT, suggesting an important role of ergosterol in modulation of the antimonial action. Importantly, this investigation lends credence to earlier literature suggesting lipid changes as important events. Leishmania parasites are protozoans from phylogenic branches of eukaryotes that are most ancient. With a digenetic life cycle, these parasites are exposed to high ROS during interactions with the primary and secondary host and therefore require a robust defense to create successful infections. Although expressing various defensive molecules, they lack catalase and selenium-dependent glutathione peroxidase (8, 20), the primary enzymes required for protection from ROS and its products. Antimonial compounds are known to generate ROS after conversion from the pentavalent form to the trivalent form. Extensive studies have revealed that this conversion can occur either in the host cell or in the parasite; however, major conversionoccursintheparasitegeneratingalethallevelof Sb III (37). Sb III hasbeenusedinthepromastigotemodel for studying mechanism of action in multiple studies, and this has contributed much to the knowledge on effect of this drug in the Leishmania parasite (38). Because our studies with the promastigotes clearly show a major change in FA and sterol profile initiated by Sb III and although amastigotes differ in the extent of expression of similar macromolecules, this study forms a basis of further studies in intracellular amastigote forms. The increase in both FAs and ergosterol in response to PAT raised the question of whether these changes were defensive in nature or a signal to initiate cell death. To answer this, ergosterol increase induced by PAT was blocked by sterol biosynthesis inhibitors, which resulted in a significant decrease in cell viability suggesting prosurvival action of the elevated ergosterol. Indirect correlation of ergosterol being protective was also obtained from PATresistant parasites that showed higher ergosterol levels, suggesting that increased ergosterol makes the parasites resistant to drug-induced death. This interpretation is consistent with the fact that in contrast to higher ergosterol containing drug resistant strains, the parasites expressing lower ergosterol were more susceptible to PAT. Therefore, theprotectiveroleofergosterolwasclearlyindicatedbythe above observations. The data of ergosterol increase by ROS is in consonance with reports showing ROS-induced stigmasterol increase in plants (39) and cholesterol in RBCs (40, 41). Ergosterol is a primary component of the plasma membrane regulating its rigidity and acting as the first line of defense against exogenous ROS. Accordingly, increased ergosterol at the membrane would be beneficial to the cell during oxidative stress. Various studies also suggest that sterolscanactasantioxidants(40).directevidenceofthe antioxidant function of ergosterol comes from the studies where ergosterol biosynthesis inhibition encourages generation of higher ROS (Supplemental Fig. 2). Although within the cell, the exact mechanism by which ergosterol lowers ROS levels remains unclear, the ability of ergosterol to prevent RBC hemolysis is strongly suggestive of its ability to directly scavenge ROS. Literature suggests that sterols evolved as life forms adapted to aerobic life (40), and as shown in our studies ergosterol may be part of a primitive defense system dealing with oxidative stress in these parasites. Experimental evidence across various cell lines show that sterols negatively feedback on one of its key inputs, O 2, by limiting its entry into the cell (40). Trypanosomatids are unique organisms as they express exceptionally LCFAs in significant amounts suggesting their importance in the regulation of cellular processes (11). The significant increase in the parasite s VLCFAs in response to PAT preceding a transient increase in ergosterol prompted us to investigate whether elevated VLCFA levels caused changes in ergosterol concentrations. As VLCFA inhibition resulted in down-regulation of PAT-induced TABLE 5. Percentage change in the FA composition when log-phase L. donovani promastigotes were treated with AAPH (25 mm) or tertbutylhydroperoxide (50 mm) for 3 h (n =3) Chain length AAPH (% change) tbhp (% change) C C C C C C C Vol. 29 October 2015 The FASEB Journal x MATHUR ET AL.

11 Figure 5. ROS-induced cell death can be linked to sterol biosynthesis. A) ROS levels were measured with fluorescent dye CMH 2 DCFDA preloaded onto WT, K39, and GE1 parasites prior to treatment with PAT (100 mg/ml). B) ROS levels were measured with fluorescent dye CMH 2 DCFDA preloaded onto WT cells followed by treatment with 50 mm VA, 5,8,11,14- eicosatetraenoic acid (EA), and 4,7,10,13,16,19-docosahexaenoic acid (DA). C) ROS levels were measured with fluorescent dye CMH 2 DCFDA in Leishmania promastigotes treated with allidochlor (50 mm) or metazachlor (200 mm) in presence or absence of PAT (100 mg/ml). D) Human RBCs at 5% hematocrit were treated with AAPH (50 mm) in the presence or absence of ergosterol (5 25 mg/ml). Percentage hemolysis was calculated at different time points (2 8 h). Al, allidochlor; M, metazachlor; VA, vaccenic acid; WT, wild-type. All n =3;*P, increase in the level of ergosterol, it was evident that VLCFAs mediated the rise in ergosterol levels. Arguably, therefore, if PAT-induced enhancement of VLCFAs was responsible for the transient increase in ergosterol, it was expected that addition of VLCFAs to untreated parasite cultures would increase ergosterol levels. Consistent with this, the ability of exogenously supplied VLCFAs like eicosatetraenoic acid and docosahexaenoic acid to induce a transient increase in the levels of ergosterol corroborated the involvement of VLCFAs in determining ergosterol levels. The complete disappearance of oleic acid and appearance of vaccenic acid is an interesting observation, but we do not have the data to suggest the functionality of this change. Although oleic acid has been shown to have aprodeathroleinhigherorganisms(42),furtherinvestigations are required to attribute a functional role to its down-regulation in PAT-treated parasites. The subsequent query was how PAT, ergosterol, and VLCFAs were interlinked mechanistically. Earlier studies from this and other laboratories have shown that there is an increase in the levels of ROS in response to antimonial compounds (38, 43, 44). To explore if PAT-generated ROS could lead to a change in the levels of ergosterol as well as VLCFAs, various antioxidants were used. Although antioxidants inhibited PAT/H 2 O 2 -induced up-regulation of ergosterol, addition of antioxidants did not interfere with VLCFA increase, suggesting that ROS might not be responsible for the up-regulation of VLCFAs. Interestingly, inhibition of VLCFAs led to a decrease in the levels of PATinduced intracellular ROS. Combined with the data of VLCFAs leading to an increase in the levels of intracellular ROS, this information points to VLCFAs being the cause of ROS increase rather than the effect. The ability of antioxidants to abolish ergosterol increase further strengthens the link between ROS and increase in ergosterol. Taken into account together, these observations can be interpreted as a cellular defense response to PAT through augmentation of VLCFAs and ROS, resulting in elevated ergosterol (Fig. 6). It has been reported in other organisms that increased levels of cholesterol and phospholipids with long carbon chains increase the rigidity of the plasma membrane, thus making it less permeable (41). The production of FAs with long carbon chains that eventually leads to increased ELEVATED ERGOSTEROL IS PROTECTIVE AGAINST ANTIMONY 4211

12 Figure 6. Schematic drawing showing possible interaction of the different pathways during treatment with PAT. Treatment with PAT leads to an increase in the levels of ROS, ergosterol, and VLCFAs (blue arrows). VLCFAs further increase ROS leading to up-regulation of ergosterol production (green arrows). Antioxidants inhibit ROS production by PAT and VLCFAs, thus inhibiting ergosterol increase (bar-headed lines marked in red). VLCFA inhibitors also abrogate ergosterol increase by inhibiting PAT induced VLCFA up-regulation and subsequent ROS increase (bar-headed lines marked in purple). Although sterol biosynthesis inhibitor (SBI) down-regulates ergosterol levels, it induces an increase in oxidative stress (marked in brown). Ergosterol can reinforce cell membrane or at an elevated level act as an antioxidant (orange arrows). Antioxidants ROS DNA damage Cell death PAT SBI Ergosterol VLCFA Antioxidant Action ELO Inhibitors Reinforcement of bilayer Cell membrane ergosterol production may be one of the ways by which these kinetoplastid parasites restrict membrane permeability to limit the entry of drug or ROS inside the cell for survival. The abundance of phosphatidylcholine with polyunsaturated FA chains in Leishmania is also believed to play a role in modulating membrane physiology and conferring resistance to host-derived oxidants (45). The prevalence of high levels of ergosterol and VLCFAs in the PATresistant parasite K39 shown in this study also corroborates this idea. Increased ergosterol levels during Sb III exposure could therefore have a dual role in Leishmania parasites. The first role could be the reinforcement of membranes so that the rigidity and fluidity of the structure is retained to combat stress, and the second role could be to act directly as an antioxidant. In all possibility, both functions appear to be active. Minute quantities of ergosterol peroxide, an oxysterol, were detected in parasites treated with PAT (data not shown). It is believed that sterols and efficient metabolism of oxysterols into sterols constitute an effective antioxidant system (40). Whether oxysterols are a part of the antioxidant system in the Leishmania parasites remains an open question. It is important to mention here that studies have indicated the involvement of macrophage lipids such as cholesterol,triglycerides,andsoonintheprocessofinfection (46, 47). Also, although host cell lipids have been reported to modulate the action of drugs such as amphotericin B (48), no studies have identified the role of macrophage lipids in modulating the action of antimonial compounds. Because our studies have established that Sb III affectsbothsterolandfasynthesisinleishmania parasites, it will be of interest to study the effect of this drug on host cell lipids as well. Such studies will increase our understanding of the mode of action of antimonial compounds in the host cell and may also provide insight into the mechanism of drug resistance. In summary, our results project a novel role of VLCFAs and ergosterol in parasite survival when treated with trivalent antimony. The importance of the observations presented here lies in the fact that sterols constitute an important defense mechanism for the Leishmania parasites and therefore interference with sterol biosynthesis would have an impact on its survival. As the SBP of the parasites is significantly different from the mammalian hosts, agents impacting leishmanial sterol biosynthesis could be evaluated as antileishmanial chemotherapeutic agents (14). This work was supported by grants from the Department of Biotechnology (New Delhi, India; asp) to the Indian National Institute of Immunology (Grant No. DBT.BT/PR11330/BRB/10/651/2008), Centre for Molecular Medicine (New Delhi, India; Grant BT/PR14549/ MED/14/1291/2010), and Life Science Research Board of Defense Research Development Organization (New Delhi, India; Grant DLS/81/48222/LSRB-169/1D/2008). R.M. was supported by a fellowship from the Council of Scientific and Industrial Research (India; Award 09/485(0219)/2011-EMR-I). The authors acknowledge the receipt of the resistant parasite strains from Dr. Syamal Roy and Dr. Chitra Mandal (Indian Institute of Chemical Biology, Kolkata, India). Technical assistance from Mr. G. S. Neelaram is also acknowledged. REFERENCES 1. Falkowski, P. G. (2006) Evolution. Tracing oxygen s imprint on earth s metabolic evolution. Science 311, Summons, R. E., Bradley, A. S., Jahnke, L. L., and Waldbauer, J. R. 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