Role of intracellular cAMP in differentiation-coupled induction of resistance against oxidative...

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Free Radical Biology & Medicine 44 (2008) 779–794www.elsevier.com/locate/freeradbiomed

Original Contribution

Role of intracellular cAMP in differentiation-coupled induction ofresistance against oxidative damage in Leishmania donovani

Arijit Bhattacharya, Arunima Biswas, Pijush K. Das ⁎

Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Kolkata 700032, India

Received 23 August 2007; revised 10 October 2007; accepted 31 October 2007Available online 21 November 2007

Abstract

Even though the human parasite Leishmania donovani encounters tremendous oxidative burst during macrophage invasion, a set of parasitessurvives and proliferates intracellularly, leading to transformation from promastigote to amastigote form and diseasemanifestation. The striking shifts intemperature (from 22°C in the insect gut to 37°C in the mammalian host) and pH (7.2 in the insect gut to 5.5 in the parasitophorous vacuole ofmacrophages) are the key environmental triggers for differentiation as these cause an arrest in the G1 stage of the cell cycle and initiate transformation.Using an established in vitro culture and differentiation system our study demonstrates that the differentiation-triggering environment induces resistanceto oxidative damage and consequently enhances infectivity. Differentiation conditions caused a three- to fourfold elevation in cAMP level as well ascAMP-dependent protein kinase activity. Similar to stress exposure, positive modulation of intracellular cAMP resulted in blockage of cell cycleprogression and induction of resistance against oxidative damage. Resistance against pro-oxidants from either stress or cAMP may be associated withupregulation of the expression of three major antioxidant genes, peroxidoxin 1, trypanothione reductase, and superoxide dismutase A. Positivemodulation of the intracellular cAMP response enables cells to resist the cytotoxic effects of pro-oxidants. In contrast, downregulation of intracellularcAMP by overexpression of cAMP phosphodiesterase A resulted in a decrease in resistance against oxidative damage and reduced infectivity towardactivatedmacrophages. This study for the first time reveals the importance of cAMP response in the life cycle and infectivity of the Leishmania parasite.© 2007 Elsevier Inc. All rights reserved.

Keywords: Leishmania donovani; Macrophage; Oxidative damage; cAMP; Free radicals

Protozoan parasites of the genus Leishmania cause a diversegroup of diseases collectively called leishmaniases, which range inseverity from spontaneously healing cutaneous ulcers in L. majorinfection to potentially fatal visceral disease in L. donovani infec-tion. The parasite is transmitted in an infective promastigote form

Abbreviations: pCPTcAMP, para-chlorophenylthio cAMP; DDA, dideox-yadenosine; H89, N-[2-(( p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfo-namide; PDE, phosphodiesterase; FCS, fetal calf serum; PKA, protein kinase A;IBMX, 3-isobutyl-1-methylxanthine; ORF, open reading frame; PBS, hosphate-buffered saline; DAPI, 4′,6-diamidino-2′-phenylindole dihydrochloride; RNI,reactive nitrogen intermediate; ROS, reactive oxygen species; H2DCFDA, 2′,7′-dihydrodichlorofluorescein diacetate; LdpdeA, L. donovani cAMP phospho-diesterase A; LdpkaC, L. donovani protein kinase A catalytic subunit; LdpkaR,L. donovani protein kinase A regulatory subunit; MnTBAP, , Mn(III) tetrakis(4-benzoic acid) porphyrin; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; MTT, 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide;ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid).⁎ Corresponding author. Fax: +91 033 2473 5197.E-mail address: [email protected] (P.K. Das).

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from the gut of its insect vector, female phlebotomine flies of thegenera Phlebotomus and Lutzomyia, to mammalian hosts. Pro-mastigotes get phagocytosed in the mammalian host by macro-phages and convert into the amastigote form, which is able tosurvive and replicate within the phagolysosome. Along with asubstantial alteration of its chemical environment the parasiteencounters several other rapid changes in the environment, inclu-ding increased temperature and decreased pH. These physicalconditions have proved indispensable for Leishmania differentia-tion and in vitro transformation protocols mimicking the physicalconditions encountered in the mammalian host are already in use[1,2]. After their phagocytosis bymacrophages in the initial stagesof infection the parasites suffer tremendous oxidative stress due toa respiratory burst ofmacrophages producing reactive oxygen andreactive nitrogen species [3,4]. In the face of this exposure to toxicpro-oxidants a subset of the parasites survives and subsequentlyconverts into intracellular amastigotes, finally leading to diseasemanifestation [5].

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780 A. Bhattacharya et al. / Free Radical Biology & Medicine 44 (2008) 779–794

The molecular mechanisms by which Leishmania circumventsthe toxic effects of these reactive species are not fully understood.SomeLeishmaniamolecules implicated in the antioxidant defenseagainst reactive oxygen species (ROS)1 and reactive nitrogenintermediates (RNI) include superoxide dismutase, peroxidoxin,and trypanothione reductase. Disruption of these genes or trans-fection with a trans-dominant inactive counterpart renders para-sites more susceptible to intracellular killing in macrophagescapable of generating reactive oxygen intermediates [6–10].Moreover, preexposure to environmental stress has been shown toinduce resistance against oxidative damage in this organism[3,11]. The ability of Leishmania parasites to resist oxidativedamage seems to be coupled with their transformation and theremay bemore than onemechanism of environmental sensing alongwith stress exposure, which finally trigger differentiation of theparasite. Cyclic AMP (cAMP) response has been implicated asone of the major environmental sensing machineries associatedwith stress response in many unicellular eukaryotes. Thus, theRas–cAMP pathway serves as a negative regulator of stress res-ponse via Msn2/4p in Saccharomyces cerevisiae [12,13]. In Dic-tyostelium YakA, cAMP, and protein kinase A (PKA) areimportant elements of the stress-induced growth arrest and deve-lopmental trigger [14]. cAMP is involved in the melatonin-dependent regulation of the Plasmodium falciparum cell cycle[15] and PKA inhibition results in suppression of its growth ininfected erythrocytes [16]. Encystation in Giardia lamblia hasbeen reported to be regulated by a cAMP-dependent PKAhomologue [17]. In Trypanosoma, also, numerous reports haveshown the involvement of cAMP in the parasitic differentiationfrom bloodstream to stumpy form [18,19]. Moreover, cAMPaffects the growth kinetics of Trypanosoma and causes growtharrest, which serves as differentiation trigger [20]. Toxoplasmagondii bradyzoite differentiation has also been shown to beregulated by cyclic nucleotide signaling [21]. cAMP acts as animportant chemotactic regulator in Paramecium, and a defect incold-sensitive response in calmodulin mutants of Parameciumhas been shown to be restored by cyclic nucleotides [22]. TheLeishmania genome project reveals that the parasite does containall the members of the cAMP cascade such as adenylate cyclase,phosphodiesterases, and catalytic and regulatory subunits of PKA[23]. However, even though Leishmania parasites suffer hugestress, which serves as a differentiation trigger, and the importanceof cAMP in differentiation has been established, very little isknown regarding the role of cAMP in Leishmania virulence andoxidative stress resistance. In this study we present evidence forthe cAMP-mediated response as a major regulator of the diffe-rentiation-coupled oxidative stress-resistant machinery. Further-more, we demonstrate cAMP to be the positive regulator of at leastthree major antioxidant genes, resulting in a direct correlationbetween cAMP response and oxidative stress resistance.

Experimental procedures

Parasites and cell line

The pathogenic strains of L. donovani AG83 (MHOM/IN/1983/AG83) and GE1 (MHOM/IN/89/GE1) were maintained

in susceptible BALB/c mice and cultured as promastigotesin medium 199 (M199; Invitrogen, Carlsbad, CA, USA) withHanks' salt containing Hepes (12 mM), L-glutamine (20 mM),10% heat-inactivated FCS, 50 U/ml penicillin, and 50 μg/mlstreptomycin. The promastigotes were obtained by culturingthe respective infected spleens in M199 for 5 days at 22°C.The adherent murine macrophage cell line RAW 264.7 wascultured at 37°C with 5% CO2 in RPMI 1640 (Invitrogen)supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

In vitro assay of L. donovani growth in macrophages

Promastigotes were used to infect cultures of adherent ma-crophages (activated by 500 U/ml IFN-γ) on glass coverslips(18 mm2; 5×105 macrophages/coverslip) in 0.5 ml of RPMI1640/10% FCS at a ratio of 10 parasites/macrophage. Infectionwas allowed to proceed for 4 h, unphagocytosed parasites wereremoved by washing with medium, and cells were resuspendedin RPMI 1640/10% FCS for 24 h at 37°C. Cells were then fixedin methanol and stained with propidium iodide for determina-tion of intracellular parasite numbers. Cells were visualized andquantified using a TCS-SP2 Leica confocal microscope.

Stress exposure and viability assay

Promastigotes in medium 199 were exposed to heat shock byincubation at 37°C. For acidic stress, parasites in the samemedium were titrated to pH 5.5 with 10 mM succinate–Tris andincubated at either 37 or 22°C. Viability assay was carried outaccording to Miller et al. [12]. Briefly, 2×106 stress-exposed orcontrol promastigotes in 100 μl of Hanks' balanced salt solution(HBSS) were exposed in triplicate to varying concentrations ofH2O2 (Merck) and ONOO− (Calbiochem, San Diego, CA,USA) in 96-well plates at 22°C. After 1 h the H2O2-exposedcells were treated with 10% heat-inactivated FCS and 500 U/mlcatalase, whereas ONOO−-exposed cells were washed threetimes with HBSS. Parasite viability was measured by incuba-tion in 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) for 3 h followed by addition of 100 μlof 0.04 N HCl in isopropyl alcohol. Living mitochondriaconvert MTT to dark blue formazan that is soluble in acidisopropyl alcohol. Formazan was detected on a microplatereader at 570 nm. The percentage viability was calculated fromthe ratio of OD readings in wells with H2O2 or ONOO

− versuswells without H2O2 or ONOO

−×100.

Intracellular cAMP and PKA activity

The intracellular cAMP concentrations were determined usingthe cAMP EIA assay kit (Sigma–Aldrich, St. Louis, MO, USA)according to the instructions of the manufacturer. PKA activitywas assayed in parasite supernatant as described by Beraldo et al.[15] by 32P phosphorylation of Kemptide (Sigma–Aldrich). Atypical assay was performed in a total volume of 25 μl containing50 mM Mops, pH 7.0, 40 μg/ml BSA, 0.5 mM MgCl2, 300 μMKemptide, 300 μM [γ-32P]ATP (200–500 cpm/pmol), 30 μg/ml

781A. Bhattacharya et al. / Free Radical Biology & Medicine 44 (2008) 779–794

bestatin, and 12.5μg of parasite protein. After 20min at 30°C, 32Pincorporation was measured by spotting 10 μl of the reactionmixture onto P81 phosphocellulose filters (Whatman, Middlesex,UK). After air-drying, the filters were washed three times for5 min in 1% phosphoric acid and once for 5 min in acetone anddried before scintillation counting. Specificity of the reaction wasassessed by performing the assay in the presence of 10 μM H89(PKA-specific inhibitor).

Immunoblotting and immunoprecipitation

Anti-phospho-Ser and phospho-Thr antibodies were fromQiagen (Germantown, MD, USA), anti-phospho-Tyr antibodywas from Cell Signaling, whereas antibodies against phospho-diesterase A (LdpdeA), PKA catalytic subunit (LdpkaC), andPKA regulatory subunit (LdpkaR) were raised in rabbits using therespective recombinant proteins expressed in Escherichia coli.Antibodies against peroxidoxin 1 (Ldpxn1), trypanothione reduc-tase (Ldtr), and superoxide dismutase A (LdsodA) were raised inrabbits using peptides comprising the C-terminal sequences of therespective proteins (ANWKKGDPGLKVDHNK for Ldpxn1,AYFYESGKRVEKLSSNL for Ldtr, and QIVDWEFVCQMYE-KATK for LdsodA) and purified by Aminolink column (Pierce,Rockford, IL, USA) according to the manufacturer's protocol.Western blot analyses were performed as described previously[24]. The primary antibodies were used at dilutions of 1:1000 andsecondary antibodies (alkaline phosphatase conjugated) wereused at a dilution of 1:10,000. For immunoprecipitation, ProteinA/G Plus agarose beads (Santa Cruz Biotechnology, Santa Cruz,CA, USA) were washed twice with TBS–Triton (50 mMTris, pH7.5, 150 mMNaCl, 0.5% Triton X-100) and dispensed into 20 μlaliquots in microcentrifuge tubes. Thirty microliters of eithernormal rabbit serum or rabbit antiserum raised against recombi-nant LdpkaRwas added to 20 μl of beads and incubated on ice for2 h. L. donovani lysate was prepared by solubilizing parasites inTBS–Triton. Insoluble material was removed by centrifugation at18,000g for 10 min. One hundred twenty microliters of parasitelysate, representing 1.5×107 parasites, was added to 20 μl ofantibody-coated beads and incubated on ice for 2 h. The beadswere washed three times in TBS before the bound enzyme wasanalyzed by Western blotting with LdpkaC antibody.

RT-PCR analysis

Total cell RNAwas isolated with the RNAeasy kit (Qiagen).Five micrograms of total RNA was used to synthesize the first-strand cDNAwith the SuperScript reverse transcriptase (Invitro-gen). cDNAs were PCR amplified with gene-specific primers asfollows: LdpdeA, 5′-TTTCTGCAAAAATTCAAGATT-3′ (for-ward), 5′-AAATGTCGGCCATTTTCAGA-3′ (reverse); Ldpxn1,5′-ATGTCCTGCGGTGACGCC-3′ (forward), 5′-TTACT-TATTGTGATCGACCTTCAGGCC-3′ (reverse); Ldtr, 5′-ACG-CGGCCGTCACGCACAA-3′ (forward), 5′-TTATTCCGTTCGCCCCCA-3′ (reverse); LdsodA, 5′-GCTCGGCTTCAAC-TACAA-3′ (forward), 5′-TTACGTGGCCTTTTCA-3′ (reverse);Ldhprt, 5′-ATGAGCAACTCGGCCAAGT-3′ (forward), 5′-CTA-CACCTTGCTCTCCGGCTT-3′ (reverse).

Cell cycle analysis

Flow cytometry for cell cycle analysis was performed asfollows: For each assay, 5 ml of cell culture (106–107 cells/ml)was aliquoted, washed twice with PBS, and suspended in 90%ice-cold methanol for fixation. These cells were kept at −20°Cfor further use. Before analysis, the cells were treated with20 mg/ml RNase for 1 h at 37°C. Subsequently, DNA wasstained using propidium iodide and analyzed for DNA contentusing FACSCalibur (Becton–Dickinson, Rockville, MD, USA).In each assay, 50,000 cells were counted. The distribution of G1,S, and G2/M phases in each experiment was calculated fromeach histogram using the CellQuest software (BD Biosciences).

[3H]Thymidine incorporation

In order to evaluate the rate of [3H]thymidine incorporation,cells were seeded in 24-well plates for various time periods.[3H]Thymidine (1 μCi) was added to each well and incubatedfor 2 h before the level of incorporated radioactivity was read.

DAPI staining

Parasites were harvested at 1000g for 10 min at 4°C, washedonce in PBS, and fixed on slides with methanol. The slides wereoverlaid with DAPI (4′,6-diamidino-2-phenylindole) (1 μg/ml)in PBS plus 10 μg/ml RNase A (Sigma–Aldrich) and analyzedimmediately. Images were captured using an Olympus BX61microscope (at a magnification of 1000) and an Olympus DP71digital camera and were processed using ImagePro Plus (MediaCybernetics).

Preparation of cytoplasmic extract

L. donovani promastigotes in lysis buffer (25 mM Hepes, pH7.2, 100 mM NaCl, 10% glycerol, 0.01% Triton X-100, 1 mMDTT, 1 μM pepstatin, 1 μM leupeptin, 1 mM PMSF) weredisrupted using a 26-gauge needle and freeze–thawing threetimes. Extracts were precleared (17000g, 15 min, 4°C) and thealiquots were quick-frozen and stored at −80°C. Antioxidantassays were performed with this extract.

Pyrogallol red bleaching assay for ONOO− scavenging

The reaction mixture contained 100 mM phosphate buffer,pH7.0,1μMDTE,50μMpyrogallolred(ϵ =2.4×104/mol/L/cm)and 100 μg protein at 25°C. The reagent peroxynitrite wasadded at 20 μM to the reaction for 5 min after which theabsorbance at 542 nm was measured.

ONOO−-induced DNA nicking assay

The reagent ONOO− was added at 50 μM to the reactionmixture containing 50 mM phosphate, pH 7.0, 10 mM NaCl,0.1 mM diethylenetriaminepentaacetic acid, 0.5 μg of intactpTEX plasmid DNA (a kind gift fromDr.Martin Taylor, LondonSchool of Hygiene and Tropical Medicine, London, UK), and


250 μg protein according to Barr et al. [25] and incubated atroom temperature for 5 min. The DNA was separated on a 1%agarose gel containing 0.2 μg/ml ethidium bromide at 100 V.

UNO detoxification assay

Sodium nitroprusside (100 mM) and 50 μg protein wereincubated in PBS for 5 min. 2,2′-Azino-bis(3-ethylbenzthiazo-line-6-sulfonic acid) (ABTS) was then added to the reactionmixture (5 mM final concentration), and the ·NO-induced oxi-dation of ABTS to ABTS+ was measured by monitoring thechange in absorbance at 420 nm according to Barr et al. [25].

Deoxyribose degradation assay for UOH scavenging

The production of UOH and the UOH-induced damage of2-deoxy-D-ribose were measured according to Gedamu and Barr[25]. Ferrous ammonium sulfate (21 μM) was added to a 50-μlreaction mixture containing 10 mM phosphate buffer, pH 7.4,63mMNaCl, 0.8mM2-deoxy-D-ribose, 0.2mMDTE, and 50μgprotein (preincubated in 0.2 mM DTE for 30 min at 37°C) andwas incubated at 37°C for 15 min. One hundred microliters ofthiobarbituric acid (1% w/v) and 100 μl of trichloroacetic acid(2.8% w/v) were then added and boiled for 10 min and fluore-scence was measured using an LS55 luminospectrometer(Perkin–Elmer), with six reads per sample (excitation at 532nm; emission at 553 nm).

UOH-induced DNA nicking assay

FeCl3 (3 μM), 0.1 mMEDTA, and 10mMDTEwere allowedto react for 10 min at 37°C to generate ·OH as previouslydescribed [25]. Two hundred fifty micrograms of protein(preincubated with 0.2 mM DTE for 30 min at 37°C) was thenadded to the mixture and incubated at 37°C for 30 min. Twomicrograms of pTEX plasmid was then added to each tube, andthe mixture was incubated at 37°C for 4 h. The DNA wasseparated on a 1% agarose gel containing 0.2 μg/ml ethidiumbromide at 100 V.

Detection of protein carbonylation by slot-blotting

L. donovani promastigotes were first treated with 400 ìMH2O2. After 15 min, cells were centrifuged, resuspended in lysisbuffer (200 mM phosphate buffer, pH 6.5, containing 1% SDS),vortexed vigorously, and boiled for 5 min. Total protein (100 μg/ml) of the cell extract was mixed with 1 volume of 12% SDS.Ten-microliter samples were derivatized with 2,4-dinitrophe-nylhydrazine and neutralized according to the methods of Le-vine et al. [26] and slot-blotted onto polyvinylidene difluoridemembranes. The membranes were blocked in 5% nonfat milkpowder in TBST (Tris-buffered saline, pH 7.4, plus 0.05%Tween 20) and then incubated for 1 h in 2% nonfat milk powderin TBST containing a 1:500 dilution of goat anti-2,4-dinitrophenol or 2,4-dinitrophenyl antibody (Bethyl, Montgom-ery, TX, USA), washed in TBST, and incubated as before with a1:12,000 dilution of anti-goat horseradish peroxidase secondary

antibody (Santa Cruz) for immunoblot analysis using an ECLWestern blotting analysis system (Amersham, Piscataway, NJ,USA) according to the manufacturer's protocol.

Electron microscopy

After various treatments, promastigotes were washed twicewith PBS, fixed in 5% (w/v) glutaraldehyde in PBS, incubatedwith 2.5% (w/v) OsO4 for 1 h, gradually dehydrated in ethanol[30, 50, 70, 90, and 100% (v/v); 30 min each] and propyleneoxide (1 h), embedded in SPRLV (Sigma), and observed using aTechnai Spirit electron microscope (FEI).

Flow cytometry for measurement of intracellular peroxidedetoxification

Promastigotes were exposed to 400 μM H2O2, washed withPBS three times, and then incubated for 15 min with the H2O2-activated green fluorescent dye 2′,7′-dihydrodichlorofluores-cein diacetate (H2DCFDA) (Molecular Probes, Eugene, OR,USA). The fluorescence levels (excitation 488 nm and emission530 nm) of 50,000 cells were then counted under each conditionusing a FACSCalibur cytometer (BD Biosciences). CellQuestsoftware (BD Biosciences) was used for data analysis and ge-neration of histograms.

LdpdeA-overexpressing promastigotes

From L. donovani genomic DNA the LdpdeA ORF wasamplified using the primers pdeA pc3.1, 5′-CACCATGCTC-GACTTTCTTGAGCAG-3′ (forward) and pdeA pc3.1, 5′-CTACGAGTCGTCGTGGTTG-3′(reverse), and cloned in thepcDNA 3.1 directional TOPO expression vector (Invitrogen).The pdeA gene was then subcloned into the BamHI- andEcoRV-digested pTEX vector [27]. Promastigotes were trans-fected with ∼20 μg of either vector alone (pTEX) or pTEX withthe pdeA gene in the correct orientation (pTEXpdeA) byelectroporation with a Gene Pulser (Bio-Rad, Hercules, CA,USA) under the conditions described earlier [9]. Transfectantswere allowed to recover in drug-free medium for 24 h and thenwere selected for resistance to G418 at 25 and 50 μg/ml.

Results

Stress-induced resistance to hydrogen peroxide and peroxynitrite

At the onset of mammalian infection parasites are exposedto a temperature shift from the ambient 22°C in the insect gutto 37°C in the mammalian host, and at the beginning of itsintracellular phase it encounters an acidic shock in theparasitophorous vacuole from a pH of 7.2 to 5.5. Uponphagocytosis by macrophage they are exposed to hydrogenperoxide, superoxide, and peroxynitrite, products of macro-phage oxidative burst. In order to reproduce the same con-ditions in vitro, L. donovani promastigotes were exposed to37°C and pH 5.5. Exposure either to temperature or to tem-perature and pH rendered the promastigotes more resistant to

Fig. 1. Stress-induced resistance of promastigotes to H2O2- and ONOO−-mediated toxicity. Promastigotes were preexposed overnight to (A) 37°C or (B) both 37°Cand pH 5.5. They were suspended in M199 and exposed to the indicated concentrations of H2O2 for 1 h. (C) Promastigotes that were preexposed either to 37°C or to37°C and pH 5.5 for various time periods were exposed to a fixed concentration of H2O2 (200 μM) for 1 h. (D) Both heat- and acid-exposed promastigotes were alsoexposed to ONOO−. Promastigote viability was measured according to their conversion of the dyeMTT to formazan, a function that depends on mitochondrial activity.Data show the mean viability in triplicate wells compared with wells with no H2O2 or ONOO

−. (E) Activated macrophages (pretreated with 500 U/ml IFN-γ for 24 h)were infected with L. donovani promastigotes and the number of intracellular promastigotes was determined after propidium iodide staining. Stress denotes parasitespreexposed to 37°C and pH 5.5 for 6 h. In separate experiments, macrophages were treated with either MnTBAP (50 μM) or TEMPOL (200 μM) 1 h before infection.(F) Representative confocal microscopic images of macrophages infected with normal or stress-exposed promastigotes and treated with various regimens. Phase-contrast images of each field are shown separately. Values are means±SD (n=3). ⁎⁎⁎pb0.001, ⁎⁎pb0.01, ⁎pb0.05 compared to control (A to C) and as shown(D and E).


H2O2 (Figs. 1A, B, and C) and peroxynitrite (Fig. 1D). Whenused to infect IFN-γ-activated macrophages, stress-exposedparasites were found to have enhanced intramacrophagesurvival compared to unexposed parasites (Figs. 1E and F).Moreover, increased intracellular survival of unexposedpromastigotes in the presence of the free radical scaven-

gers MnTBAP (cell-permeable superoxide dismutase mimeticand peroxynitrite scavenger) and TEMPOL (stable, cell-permeable nitroxide that acts as a free radical scavenger andnitric oxide spin trap) suggests that exposure to 37°C and pH5.5 might have resulted in induced resistance to H2O2 andperoxynitrite.


Role of cAMP in stress-induced resistance to H2O2 andperoxynitrite

Because cAMP plays a significant role in the differentiationof kinetoplastida like Trypanosoma and Leishmania, we wanted

Fig. 2. Role of cAMP in stress-induced resistance to oxidative damage. (A) Intracelpreexposed promastigotes at the indicated time intervals. Data represent means±SD (and stressed promastigotes was calculated from duplicated data from three different1 μM cAMP. Open and hatched bars denote PKA activity in the absence and presencusing the anti-PKA regulatory subunit antibody as described under Experimental prmembrane, proteins in both the immunoprecipitate (IP) and the supernatant (Sup) wResults are representative of duplicate samples of three separate experiments and the dvs control. α-Tubulin was used as indigenous control. (D) Western blot analysis oregimens by probing with anti-phosphoserine, anti-phosphothreonine, and anti-phospassay after various treatments. Values are means±SD (n=3). ⁎⁎⁎pb0.001, ⁎⁎pb0.0

to determine the role of cAMP in the stress-induced resistanceagainst oxidative damage by measuring the intracellular cAMPlevel in L. donovani promastigotes. The intracellular cAMPlevel was significantly elevated, with a maximum level (3.5times) observed after 60 min of stress exposure (Fig. 2A). PKA

lular cAMP concentrations were determined in either normal or heat- and acid-n=3). ⁎⁎⁎pb0.001, ⁎⁎pb0.01 compared to control. (B) PKA activity in normalexperiments, as a percentage of total PKA activity obtained with an additionale of H89, respectively. (C) Stressed cells were lysed and immunoprecipitated byocedures. After separation by 10% SDS–PAGE and transfer to a nitrocelluloseere probed with the anti-PKA catalytic subunit antibody for Western blotting.ensitometric evaluations (C1) are means of three separate experiments. ⁎pb0.05f the lysates of L. donovani promastigotes subjected to treatment with varioushotyrosine antibodies. (E and F) Promastigote viability was measured by MTT1, ⁎pb0.05.


activity was also found to be enhanced, with maximum activityobserved at 4 h after stress exposure (Fig. 2B). Because acti-vation of PKA is represented by the dissociation of the catalyticsubunit from the regulatory subunit, PKA complex was firstimmunoprecipitated with anti-regulatory subunit antibody andthen both the precipitate and the supernatant were immuno-blotted with anti-catalytic subunit antibody. PKA activation wasinitiated after 2 h with the maximum dissociation of the catalyticsubunit at 4 h after stress exposure (Fig. 2C). Unlike highereukaryotes, intracellular substrates for PKA have not yet beenidentified in Leishmania. Because PKA is known to undergomainly serine and threonine phosphorylation, these two phos-phorylation profiles were studied in L. donovani promastigotesas a result of stress exposure using anti-phosphoserine and anti-phosphothreonine antibodies. Tyrosine phosphorylation wasused as an internal control. Both serine and threonine phos-phorylation was altered by stress exposure, which was reversedwhen dideoxyadenosine (DDA; an inhibitor of adenylate cyc-lase) and H89 (an inhibitor of PKA) were used along withstress, suggesting thereby the occurrence of cAMP-mediatedevents after stress exposure (Fig. 2D). Furthermore, similar tostress exposure, viability was increased when promastigotes weretreated with pCPTcAMP, a cell-permeable cAMP analogue,whereas increased viability after stress exposure was reversed byDDA and H89 (Fig. 2E). Treatment with various cAMP-specificphosphodiesterase (PDE) inhibitors, IBMX (20 μM), etazolate(20 μM), rolipram (25 μM), and zardeverine (25 μM), resulted inincreased viability against both H2O2 and ONOO−, whereasthe cGMP-specific PDE inhibitor zaprinast (25 μM) showedlittle effect (Fig. 2F). All these results suggest that cAMP-mediated events are triggered after exposure to 37°C and pH 5.5,whichmay have some role in inducing parasitic resistance againstoxidative stress.

cAMP can trigger growth arrest and cell cycle arrest in G1phase—the initial event in differentiation of Leishmania

Morphological transformation of promastigotes to amasti-gotes by exposure to 37°C and pH 5.5 occurs during cell cyclearrest at G1 phase [28]. Nutritional starvation also triggersgrowth arrest in the promastigotes [29] and leads to metacy-clogenesis through PKA [30]. Because resistance againstoxidative damage and transformation are coupled and becausecell cycle arrest initiates differentiation, the effects of cAMPmodulation on the proliferation of log-phase promastigoteswere studied by measuring [3H]thymidine incorporation innuclear DNA. Intracellular cAMP concentration was modulatedby treating the cells with agents like pCPTcAMP, DDA, or H89.pCPTcAMP (500 μM) caused a significant decrease in [3H]thymidine incorporation, whereas exposure to 37°C resulted inan increase in [3H]thymidine incorporation at 4 h, whichdecreased at 12 h. However, stress exposure along with DDA(10 μM) or H89 (10 μM) treatment significantly enhanced the[3H]thymidine incorporation at 12 h (Fig. 3A). Introduction ofG1-phase-synchronized promastigotes into fresh mediumresulted in a decrease in the G1 population from 62.3±5.5%to 52.4±5.1 and 49.9±4.2% after 4 and 12 h, respectively, as

revealed by cellular DNA content estimation by propidiumiodide-based FACS analysis. However, pCPTcAMP treatmentcaused a smaller decrease in the G1 population (56.2±5.3 and55.1±4.7% from 60.3±5.8% after 4 and 12 h, respectively)(Fig. 3B). The kinetics of growth arrest was further studied bycytologically scoring 1 K/1 N (1 kinetoplast/1 nucleus repre-senting the G0/G1 population), 2 K/1 N, and 2 K/2 N (repre-senting the S and G2/M populations, respectively) after labelingwith DAPI, a DNA-binding fluorophore (Fig. 3C). The increasein intracellular cAMP by treatment with pCPTcAMP resulted ina marked increase in the 1 K/1 N population, from 54.0±3.2 to86.0±6.3% after 12 h, associated with a concomitant decreasein the 2 K/2 N and 2 K/1 N populations. Exposure to 37°C andpH 5.5 resulted in a slight decrease in the 1 K/1 N population at4 h (from 56.0±4.4 to 48±4.7%), which increased to 84.0±6.1% after 12 h with an equivalent decrease in the 2 K/2 N or2 K/1 N population. However, stress exposure coupled withDDA (10 μM) or H89 (10 μM) treatment showed K/N profilescomparable to that of the untreated cells (Fig. 3D). All theseresults suggest the involvement of cAMP in the G1 arrest ofLeishmania promastigotes during differentiation.

Stress-associated upregulation of antioxidant genes is acAMP-modulated response

Though Leishmania lacks functional catalase and glu-tathione peroxidase, antioxidant activities are carried out mainlyby Ldpxn, Ldsod, and Ldtr. To examine whether stress- anddifferentiation condition-induced resistance to oxidativedamage involves upregulation of these major antioxidantgenes and whether cAMP modulation has any effect in thisregard, transcript levels of these genes were measured bysemiquantitative RT-PCR analysis. mRNA expression of allthree genes was found to be upregulated when cells wereexposed to 37°C and pH 5.5 for 6 h, of which Ldpxn1 wasmaximally elevated, and this upregulation was reversed in thepresence of DDA and H89 (Fig. 4A). mRNA levels of all threeantioxidant genes were upregulated when the cells werepretreated with pCPTcAMP. Moreover, similar upregulationpatterns both by stress and by pCPTcAMP treatment were alsoobserved at the protein level (Fig. 4B). To further ascertainthe involvement of the cAMP-mediated response in this eventboth mRNA expression and protein levels of the genes wereassessed after treating the cells with the cAMP-specific PDEinhibitors IBMX (20 μM), rolipram (25 μM), and etazolate(20 μM), which considerably enhanced the expression of allthree genes. However, zardeverine (25 μM), another cAMP-specific PDE inhibitor, enhanced LdsodA expression butshowed little effect on Ldpxn1 or Ldtr expression. Zaprinast(25 μM), a cGMP-specific PDE inhibitor, on the other hand,showed little effect on the expression of these three genes(Figs. 4C and D). SYBR green-based quantitative real-timePCR analysis was also carried out to analyze the expression ofthe genes, and the results correlated with the semiquantitativeanalysis (data not shown). All these results indicate the activeinvolvement of cAMP-triggered events in developing resistanceagainst oxidative damage.

Fig. 3. Role of cAMP in L. donovani differentiation. (A) Time course of DNA synthesis of differentiating promastigotes as indicated by thymidine incorporation intonucleus after treatment with modulators of intracellular cAMP. Values are means±SD (n=6). ⁎⁎⁎pb0.001, ⁎⁎pb0.01. (B) Time course of flow cytometry analysis forDNA content by staining with propidium iodide. G1-phase-synchronized (4 mM hydroxy urea, 12 h) promastigotes were washed with PBS and suspended in M199 for30 min to remove residual intracellular hydroxyurea. Histograms showing cell cycle progression at 0, 4, and 12 h for untreated cells (a, b, and c) and pCPTcAMP-treated cells (d, e, and f ). (C) Cells were stained with DAPI and examined by fluorescence microscopy. Arrowheads indicate dividing cells. (D) Quantification of cellswith different numbers of nuclei (N) and kinetoplasts (K). Data are presented as the mean percentages±SD of the total populations counted (∼200 cells in each of threeindependent experiments).


Protection from H2O2-mediated cytotoxicity by positiveregulation of cAMP

Because the antioxidant activity of the cytosolic fraction doesnot directly indicate protection against oxidative damage, theeffect of cAMPmodulation on the extent of intracellular damage

by H2O2 was examined. Exposure of cells to 400 μM H2O2 for1 h causes DNA fragmentation in L. donovani promastigotes,which was significantly reduced when cells were either treatedwith pCPTcAMP or exposed to 37°C and pH 5.5 (stress), andthis was reversed upon treatment with DDA or H89 (Fig. 5A). Tofurther examine the levels of intracellular stress when cells were

Fig. 4. Role of cAMP in the expression of antioxidant genes. The expression of various antioxidant genes was determined by (A) RT-PCR analysis of the mRNA transcriptand by (B) immunoblot analysis of the protein levels using antipeptide antibodies comprising the C-terminal sequences of the respective proteins. L. donovanipromastigotes: untreated (lane 1), pCPTcAMP (500 μM)-treated (lane 2), stressed (37°C and pH 5.5) (lane 3), stressed plus DDA (10 μM)-treated (lane 4), and stressed plusH89 (10 μM)-treated (lane 5). (C) RT-PCR analysis and (D) immunoblot analysis of antioxidant proteins in L. donovani promastigotes treatedwith IBMX (20 μM) (lane 2),etazolate (20 μM) (lane 3), rolipram (25 μM) (lane 4), zardeverine (25 μM) (lane 5), and zaprinast (25 μM) (lane 6). Lane 1 represents untreated promastigotes. Bandintensitieswere analyzed by densitometry (A1, B1, C1, andD1). Hypoxanthine–guanine phosphoribosyltransferase (HPRT) expression levels were used as controls for RNAcontent and integrity, whereas α-tubulin was used as indigenous control for Western blotting. Results are representative of duplicate samples of three separate experimentsand the densitometric evaluations are the means of three independent experiments. ⁎⁎pb0.01, ⁎pb0.05 (A1 and B1) and ⁎⁎pb0.01, ⁎pb0.05 vs control (C1 and D1).


challenged with H2O2, two separate methods were employed.The first of these were FACS cell counting, using the H2O2-activated green fluorescent dye H2DCFDA. Log-phase promas-tigotes were grown for 1 h in H2O2-amended or control medium,washed in PBS, and incubated for 15 min in M199 containingH2DCFDA, and then the fluorescence levels of 50,000 cellswere counted. A gate (M2) was established that delineated theupper 5% of fluorescent cells. The percentage of gated cells washigher in normal cells exposed to H2O2 (45.0±2.5%) than instressed (32.6±1.6%) and pCPTcAMP-treated cells (38.8±2.8%)exposed to H2O2. When stressed cells were treated with DDA orH89 before exposure to H2O2, the percentage of gated cellsincreased (54.8±3.1 and 50.4±2.7%, respectively) (Fig. 5B). Thepercentage of cells within the M2 gate under each condition isdepicted in Fig. 5C. These results indicate that levels of in-tracellular H2O2 stress were significantly reduced when cellswere exposed to 37°C and pH 5.5 or treated with pCPTcAMP. Ifso, this should be reflected by the protein carbonylation levels.Carbonyl groups are introduced into protein side chains by site-specific oxidative modifications and carbonyl quantification isbelieved to provide an accurate estimate of the oxidation statusof proteins [26]. After exposure of cells to 400 μM H2O2 for15 min, the extent of protein carbonylation was measured byderivatizing the crude cell extract with the carbonyl reagent

2,4-dinitrophenylhydrazine and oxidatively modified proteinswere detected with anti-2,4-dinitrophenol antibody. As for theFACS data, quantification of the immunoassay bands revealedan almost twofold reduction in protein carbonylation afterstress exposure or pCPTcAMP treatment compared tountreated cells. The extent of protein carbonylation was againenhanced when stressed cells were treated with DDA or H89(Fig. 5D). To detect the effects of cAMP on the morphologicalchanges in promastigotes by oxidative damage, transmissionelectron microscopy experiments were carried out on promas-tigotes after incubation with 400 μM H2O2. As illustrated inFig. 5E, a severe disruption of the membrane structure,accompanied by a loss of electron-dense intracytoplasmicmaterial, was observed in promastigotes exposed to H2O2 (400μM, 1 h), whereas the disruption was much less inpromastigotes which were treated with pCPTcAMP. All theseresults indicate the role of cAMP-mediated events in protectingthe parasites from the cytotoxic effects of H2O2.

Stress-induced free radical scavenging capacity of L. donovaniparasites is a cAMP-dependent event

In order to further ascertain the role of cAMP in regulating freeradical scavenging capacity, cytosolic fractions were prepared

Fig. 5. Role of cAMP in stress-induced H2O2-mediated cytotoxicity. (A) DNA fragmentation profile of H2O2 (400 μM, 1 h)-exposed promastigotes was carried outwith an apoptotic DNA ladder kit (Roche) and agarose gel electrophoresis was done according to Sudhandiran et al. [59]. H2O2-exposed promastigotes were alsoexposed to stress (37°C and pH 5.5) and various modulators of intracellular cAMP. A typical result of three independent experiments is shown (M, 1 kb marker).(B) (a–j) Representative histograms plotting the fluorescence of 50,000 cells treated with 20 μMH2DCFDA. The lower boundary of the M1 gate defines the cutoff foran event to be registered as cellular fluorescence, whereas the M2 gate was established to measure population shifts and delineate approximately the upper 5% of thefluorescence boundary of normal untreated cells. a, Untreated cells, M2=27.5±2.5%; b, H2O2 (400 μM)-exposed cells, M2=45.0±2.5%; c, pCPTcAMP (500 μM)-treated cells, M2=30.0±2.7%; d, H2O2-exposed and pCPTcAMP (500 μM)-treated cells, M2=38.8±2.8%; e, stressed cells (37 °C and pH 5.5), M2=29.5±1.7%;f, stressed cells exposed to H2O2, M2=32.6±1.6%; g, stressed cells treated with DDA (10 μM), M2=38.7±3.3%; h, stressed cells treated with DDA (10 μM) andexposed to H2O2, M2=54.8±3.1%; i, stressed cells treated with H89 (10 μM), M2=42.3±3.6%; j, stressed cells treated with H89 (10 μM) and exposed to H2O2,M2=50.4±2.7%. (C) Bar graph summarizing the percentage of cells within the M2 gate under each condition. FACS data were collected in triplicate. Error bars aremeans±SD. ⁎⁎⁎pb0.001, ⁎⁎pb0.01. (D) Slot-blot analysis of carbonyl levels in unexposed (T=0) and H2O2-exposed (400 μM, T=15 min) promastigotes asdescribed under Experimental procedures. Representative blot indicating accumulation of protein carbonyl groups in H2O2-exposed cells under various conditions.(D1) Bar graph quantifying relative band intensities from cells under various conditions at 15 min; the data are the standardized [against the intensity of unexposed(T=0 min) cells] mean values from three separate experiments. ⁎pb0.05. (E) Electron microscopy of L. donovani promastigotes under various conditions. a, Normaluntreated cells; b, cells exposed to H2O2 (400 μM, 1 h); c, pCPTcAMP (500 ìM)-treated cells; d, pCPTcAMP (500 ìM)-treated cells exposed to H2O2. Membranedisruption, membrane blebbing, and breakages as well as depletion of electron-dense cytoplasmic material can be observed in H2O2-exposed cells.



from log-phase promastigotes either exposed to 37°C and pH5.5 or treated with agents that increase intracellular cAMPlevel. Pyrogallol red (PR) was earlier shown to be bleachedby ONOO− but not by decomposed ONOO−, nitrate, or nitrite[31]. When added to the reaction mixture the cytosolic fractionfrom both stressed and pCPTcAMP-treated cells and theONOO− scavenger Trolox significantly ( pb0.001) protectedPR from bleaching compared with untreated cells and BSAcontrols (Fig. 6A). DNA has also been shown to be a target ofONOO−, which converts supercoiled DNA into a slower mi-grating nicked DNA [32]. Similar to the PR study, the cytosolicfractions from both stressed and pCPTcAMP-treated cells,as well as Trolox, were able to protect pTEX from ONOO−-induced nicking, whereas untreated cells and BSA were much

Fig. 6. Role of cAMP in free radical scavenging capacity of L. donovani promastigottheir ability to detoxify various RNS and ROS. (A) Activity was assessed by the aONOO−-induced bleaching and to protect supercoiled pTEX plasmid (s) from ONOprotecting 5 mM ABTS from ·NO-induced damage. (C) Ability to protect 2-deoxynicking (n) (top). Data represent means ±SD (n=3). ⁎⁎⁎pb0.001, ⁎⁎pb0.01.

less protective (Fig. 6A, top). The increased activity of stressedcells to either protect PR from bleaching or protect super-coiled DNA from nicking was significantly diminished whentreated with DDA or H89 (Fig. 6A, top). The ability of the pro-mastigotes to detoxify ·NO was further assayed using ABTS,which is converted to the strongly absorbing ABTS+ complex by·NO-mediated oxidation [33]. When added to the reaction mix-ture, the cytosolic fractions from both stressed and pCPTcAMP-treated cells and the ·NO scavenger PTIO significantly (pb0.001)protected ABTS from oxidation compared with untreatedcells (Fig. 6B). Degradation of 2-deoxyribose is a good in-dicator of ·OH activity [25]. When added to the reactionmixture, the cytosolic fractions from stressed and pCPTcAMP-treated cells and the ·OH scavenger mannitol significantly

es. Cytosolic fractions of promastigotes under various conditions were tested forbility of promastigote cytosolic proteins to protect 50 μM pyrogallol red fromO− attack on the slower migrating nicked band (n) (top). (B) Activity towardribose from degradation and supecoiled (s) pTEX plasmid from ·OH-induced


( pb0.001) protected 2-deoxyribose from degradation com-pared with untreated cells and BSA controls (Fig. 6C). Similarto the 2-deoxyribose degradation study, the cytosolic frac-tions from both stressed and pCPTcAMP-treated cells, as wellas mannitol, were able to protect pTEX from ·OH-induced

Fig. 7. Effects of overexpression of LdpdeA on resistance against oxidative stress. Thevector showed a significant increase in the expression levels of the enzyme at both (Arespectively. RT-PCRproducts were visualized by ethidium bromide staining and hypoxcontrols for RNA content and integrity, whereas immunoblot analysis was donewith antwas used as loading control for immunoblotting. L. donovani transfected with vector alused as controls for RNA content and integrity, whereas α-tubulin was used as indigenthree separate experiments and the densitometric evaluations (A1 and B1) are the meanconcentration inL. donovaniwith overexpressedLdpdeA andwith vector only. ⁎⁎⁎pb0.RT-PCR analysis of the mRNA transcript and (E) by immunoblot analysis of the protein(lane 1), treated with stress (lane 2), treated with stress plus IBMX (lane 3), transfected(lane 5). Results are representative of duplicate samples of three separate experimentsexperiments. ⁎⁎pb0.01, ⁎pb0.05. (F) Cell viability was measured by MTTassay and (staining of intracellular amastigotes in promastigotes expressing LdpdeA and in control pstress (37 °C and pH 5.5) exposure. Values are means±SD (n=6). ⁎⁎⁎pb0.001, ⁎⁎pb

nicking, whereas untreated cells and BSA were much lessprotective (Fig. 6C, top). The increased activity of stressed cellsto either protect 2-deoxyribose from degradation or protectsupercoiled DNA from nicking was significantly diminishedwhen treated with DDA or H89 (Fig. 6C, top).

episomal integration of LdpdeA by transfection with a cloned gene in the pTEX) the mRNA and (B) the protein level as analyzed by RT-PCR and immunoblot,anthine–guanine phosphoribosyltransferase (HPRT) expression levels were used asibody raised in rabbits against recombinant LdpdeA expressed inE. coli.α-Tubulinone (lane 1) and vector containing LdpdeA (lane 2). HPRT expression levels wereous control for Western blotting. Results are representative of duplicate samples ofs of three independent experiments. ⁎pb0.05 vs control. (C) Intracellular cAMP001, ⁎⁎pb0.01. (D) The expression of various antioxidant geneswas determined bylevels as described earlier. L. donovani transfected with vector containing LdpdeAwith vector alone (lane 4), and transfected with vector alone and treated with stressand the densitometric evaluations (D1 and E1) are the means of three independentG) infectivity in IFN-γ-activated macrophages was measured by propidium iodidearasites transfectedwith empty vector alone. Both these parasites were subjected to0.01, ⁎pb0.05.


Downregulation of intracellular cAMP blocks induction ofresistance against oxidative damage

In order to ascertain the role of intracellular cAMP response, astrain of L. donovani promastigotes bearing the phosphodiester-ase A gene in the pTEX plasmid (a trypanosome-specific shut-tle vector) was generated. A 3- to 3.5-fold increase in LdpdeAwas observed in overexpressing cells compared to wild typeat both the mRNA and the protein level as revealed by semi-quantitative RT-PCR and Western blot analysis, respectively(Figs. 7A and B). Further, the intracellular cAMP level wasmarkedly less in LdpdeA-overexpressing promastigotes com-pared to wild-type cells (Fig. 7C). There was also less upre-gulation of the antioxidant genes Ldpxn1, Ldtr, and LdsodAat both the transcript level and the protein level in LdpdeA-overexpressing cells compared to wild-type carrying vectoralone as revealed by semiquantitative RT-PCR and Western blotanalysis (Figs. 7D and E). The transcript levels of all threeantioxidant genes were, however, upregulated when LdpdeA-overexpressing cells were exposed to stress along with IMBXtreatment (Figs. 7D and E).When these LdpdeA-overexpressingcells were subjected to stress (37°C and pH 5.5 for 6 h) andsubsequently treated with either 250 μM H2O2 or 500 μMONOO−, they showed significantly less resistance to oxidativedamage compared to cells bearing vector alone (Fig. 7F). How-ever, this effect was reversed when stress-exposed LdpdeA cellswere treated with 20 μM IBMX. Finally, when assessed for theability of intracellular survival in IFN-γ-activated macrophagesafter exposure to 37°C and pH 5.5, LdpdeA-overexpressing cellswere more susceptible to intracellular killing compared to L.donovani promastigotes bearing vector alone (Fig. 7G).All these results strongly indicate a major role for intracellularcAMP in the differentiation signal-coupled resistance to oxi-dative damage in L. donovani promastigotes.

Discussion

For Leishmania parasites the mechanism underlying theinduction of resistance against oxidative damage seems to beassociated with differentiation, and cAMP might act as an envi-ronmental sensor in the process. At the initial stages of macro-phage invasion, ROS and RNI, generated by oxidative burst, areactively engaged in restricting the disease progression due to theirmicrobicidal activity [34,35]. Establishment of an in vitro dif-ferentiation system revealed that exposure of promastigotes to37°C and pH 5.5 caused growth arrest and cell cycle blockage,which act as a prelude to transformation to amastigotes [28].Mining of the Leishmania genome revealed the presence of anumber of genes, members of the environmental sensing pathwaythat might play important roles in the differentiation process ofLeishmania [23]. However, the defined participation of any ofthose pathways in environmental sensing is yet to be determinedfor Leishmania. Results of the current study show that stress-induced elevation of the intracellular cAMP response enablespromastigotes to detoxify ROS and RNI encountered duringinvasion of activated macrophages. Notably the results also showthat direct free radical neutralizing capacity of promastigotes can

be modulated by chemically or genetically regulating the intra-cellular cAMP response.

Mimicking the physical environment promastigotes can betransformed to axenic amastigotes that are biochemically equi-valent to in vivo amastigotes [36]. There is increasing evidencefor the association of stress with the differentiation of Leish-mania [37,38]. Our current study also illustrates that heat andacidic stress exposure of promastigotes for a restricted periodof time enables the cells to become more resistant to oxidativedamage, which in turn results in greater survival of parasitesin IFN-γ-activated macrophages. IFN-γ-induced indoleamine2,3-dioxygenase-mediated depletion of the L-tryptophan pool inmacrophages was earlier suggested to be one of the oxidant-independent killing mechanisms against intracellular parasites[39], which was later found to be restricted only to humanmonocyte-derived macrophages [40]. However, recent studiessuggest a major role for IFN-γ in inducing macrophageactivation for killing of the parasites through oxidants likesuperoxide and nitric oxide [41], and therefore, the currentstudy is limited to the resistance of the parasite to oxidant-mediated killing. In addition to the stress-induced chaperoneactivity, some of the environmental sensing pathway [42] mightplay determining roles in the differentiation process of theparasite. In this regard the cAMP response could be one of themajor determinants of stress-induced differentiation as understress conditions both the intracellular cAMP level and the PKAactivity were found to be elevated. However, the exact mole-cular events leading to this elevation are not clear. In Myco-bacteria an adenylate cyclase is known to function as a pHsensor [43], whereas Trypanosoma differentiation occurs by adensity-sensing mechanism based on the release of stumpyinducing factor (SIF), which accumulates in the conditionedmedium, and SIF activity results in an immediate elevation ofintracellular cAMP in the slender form of the parasite [19].Similar effects might play a major role for Leishmania, asimmediately after stress exposure the proliferation rate of thepromastigotes increases rapidly.

The correlation between the ability to resist oxidative stressand the intracellular cAMP response has been shown in variousways by modulating intracellular cAMP either chemically orgenetically and monitoring the effects of H2O2 and ONOO− onparasite viability. However, in addition to the induction ofthe resistance to oxidative damage cAMP may have a role inparasitic multiplication. Thus, cell cycle arrest is known to be aprerequisite for the differentiation of the parasite from pro-mastigote to amastigote [28], and elevation of intracellular cAMPplays a regulatory role in cell cycle progression as observed byFACS analysis and K/N ratio determination. Positive modulationof intracellular cAMP has been shown to block cell cycleprogression as well as parasitic replication. However, [3H]thy-midine incorporation was significantly reduced at both 4 and 12 hcompared to the FACS-based cell cycle analysis in which thepopulation was found to be blocked to a lesser extent at G1. Dueto less G1 synchronization at T=0 in both normal andpCPTcAMP-treated parasites, the effect of pCPTcAMP on theG1 block seemed not that statistically pronounced (decrease of4.09 and 5.16%, respectively, at 4 and 12 h compared to 9.90 and


12.40% in normal cells). The effect of cAMP on parasiteproliferation was, therefore, further confirmed by K/N scoring,which suggested that cAMP could cause blockage at G1. This isnot surprising as PKA activity has been shown to be associatedwith metacyclogenesis in L. amazonensis [30] and nutritionalstarvation-induced events such as autophagy play an importantfunction in the transformation of L. major promastigotes tometacyclic promastigotes and to amastigotes [44,45]. However,in mouse embryonic fibroblasts and in S. cerevisiae autophagy isnegatively regulated by PKA activity [46,47]. An in-depthvisualization of cAMP dynamics, therefore, is needed to ascertainthe role of cAMP in regulating cell cycle and differentiation inLeishmania.

In various systems four enzymes have been implicated inantioxidant defense: catalase, glutathione peroxidase, SOD, andperoxidoxins. The first enzyme has not been found in Leishmaniaand, instead of glutathione, Leishmania possesses trypanothione,a unique redox cycling glutathione–spermidine conjugate which,in concert with trypanothione reductase, maintains the intracel-lular reducing environment and resistance to ROS. At least twoSOD isoforms are present in Leishmania, whereas three separateisoforms of peroxidoxin, pxn1, 2, and 3, are found, of which pxn1is differentially expressed and active against bothROS andRNI.Adirect correlation between the expression of these antioxidantgenes and the intracellular cAMP response was suggested, as bothmRNA and protein levels of these antioxidant genes, namelyLdpxn1, LdsodA, and Ldtr, were elevated by positive modulationof cAMP response. In contrast, chemical or genetic down-regulation of intracellular cAMP showed reduction of bothmRNAand protein levels of these three genes. Protein-coding genes inLeishmania are transcribed as polycistronic RNAs with tens-to-hundreds of adjacent genes on the same DNA strand [48–50] andmature mRNAs are subsequently obtained from coordinatedpolyadenylation and trans-splicing [51,52]. As a consequence ofthis unusual gene expression machinery, Leishmania geneexpression does not seem to be regulated at the level of tran-scription, and therefore expression of these three genes located atdifferent loci seems unlikely to be regulated by a single effectorlike cAMP. However, the stage-specific expression of a number ofgenes has been shown to be regulated via mRNA stability [53,54]and this in turn causes an ordered progression of transient andpermanent up- or downregulation of several hundred genes duringdifferentiation [55]. Transcription of a single gene (TC26) can beinduced in Trypanosoma epimastigotes by incubation with cAMPand cAMP analogues, but it is inhibited by activators of cAMP-dependent phosphodiesterases [56]. Because in Leishmania,S-phase-expressed genes have recently been identified [57]and cAMP can regulate cell cycle progression, a cell-cycle-specific global regulatory event might be associated with theupregulation of the antioxidant genes. It may be mentioned thatthioredoxin reductase has not been reported in Leishmania, butin P. falciparum, this enzyme-based antioxidant defense wasextensively studied by Krnajski et al. [58] and proved essentialfor parasite infectivity.

Intracellular pathogen survival and establishment of infec-tion depend upon the efficiency of host-defense mechanismslike the respiratory burst [4,34] that involves the generation of

large amounts of ROS and RNI. Positive cAMP modulationpotentiates the parasitic cytosol to detoxify H2O2, ONOO

−,·OH, and ·NO as observed in a number of in vitro assays.Furthermore, when cAMP level was positively modulated in-tracellular oxidative damage was also reduced in promastigotesas observed by DNA fragmentation, protein carbonylation le-vel, and ultrastructural analysis. It may be mentioned in thisregard that pentavalent antimonials, the mainstay of leishmanialchemotherapy, are supposed to exert leishmanicidal activitythrough oxidative damage by induction of apoptosis like death[59,60]. However, development of resistance against pentava-lent antimonials [Sb(V)] such as sodium stibogluconate seems tobe a major problem and a model for drug resistance has beenproposed in which Sb(V)/As(V)-containing compounds arereduced intracellularly to Sb(III)/As(III), conjugated to trypa-nothione, and extruded by the As-thiol pump [61]. cAMP-mediated events gain importance as induction of resistanceagainst oxidative damage is necessary for antimonial resistance.The importance of cAMP in parasite infectivity was furtherstrengthened by the observation that promastigotes having astable overexpressed phosphodiesterase gene showed reducedability to infect IFN-γ-activated macrophages compared to nor-mal cells.

Whereas most of the cyclic nucleotide studies in the parasiteare confined to cloning and characterizing the enzymesinvolved in cAMP metabolism, like receptor adenylate cyclase,phosphodiesterase, etc., we for the first time identified a defini-tive environmental response in the parasite, which has directinterrelation with the differentiation and infectivity of the para-site. The cAMP response is probably one of the many envi-ronmental sensing machineries associated with Leishmaniadifferentiation and many other biochemical events may work inconcert with cAMP in the differentiation process leading totransformation of the parasites. Earlier we reported a cross talkbetween Ca2+ and guanylyl cyclase in parasitic survival [62]and there might be many such cross talks, which warrant furtherinvestigation.

Acknowledgments

We thank Dr. M. Chatterjee of the Department ofPharmacology, Institute of Post Graduate Medical Educationand Research, Kolkata, India, for assistance in flow-cytometricstudies. We thank Mr. Sailen Dey for help with electronmicroscopy and Dr. Gayatri Tripathi for help with confocalmicroscopy. This work was supported by the Department ofScience and Technology and Network Project grants (NWP0038) of the Council of Scientific and Industrial Research(Government of India).

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