Changes in the proteome and infectivity of Leishmania infantum induced by in vitro exposure to a nitric oxide donor

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International Journal of Medical Microbiology 299 (2009) 221–232 www.elsevier.de/ijmm

Changes in the proteome and infectivity of Leishmania infantum induced by in vitro exposure to a nitric oxide donor Maria Auxiliadora Dea-Ayuelaa, Lara Ordon˜ez-Gutierrezb, Francisco Bola´s-Ferna´ndeza, a

Departamento de Parasitologı´a, Facultad de Farmacia, Universidad Complutense, Plaza de Ramo´n y Cajal s/n, Ciudad Universitaria, 28040 Madrid, Spain b Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Avda Puerta de Hierro s/n, Ciudad Universitaria, 28040 Madrid, Spain Received 2 September 2007; received in revised form 12 June 2008; accepted 6 July 2008

Abstract Leishmania species are protozoan parasites that exhibit an intracellular amastigote form within mammalian macrophages and an extracellular promastigote form inside the sandfly vector. The generation of nitric oxide (NO) upon activation of macrophages is surely the principal killing effector of intracellular amastigotes but little is known about the potential action of NO against the promastigote phase during its multiplication inside the digestive tract of the sandfly vector. Therefore, we have approached this issue by using an in vitro model to study the effect of an NO donor, 3-morpholinosydnonimine (SIN-1), on the proteome and infectivity of promastigotes of Leishmania infantum. Exposure of promastigotes to SIN-1 during its logarithmic growth phase caused a dramatic effect on parasite protein expression and viability, consequently killing about 60–70% of the promastigotes. The significant changes in the proteome included the over-expression of enolase, peroxidoxin precursors, and heat-shock protein 70 (HSP70), underexpression of 20S proteasome alpha 5 unit, and phosphomannomutase and induced expression of 3-hydroxy3-methyglutaryl-CoA (HMG-CoA) synthase and prostaglandine f2-alpha (PGD2) synthase. Interestingly, promastigotes that resisted treatment showed enhanced infectivity to J774 macrophages in comparison to the controls. This finding together with the appearance of the PGD2S and an over-expression of HSP70 isoforms in treated promastigotes led us to speculate the existence of NO-mediated programmed cell death (PCD) events as a potential mechanism of population regulation and selection of properly infecting forms that predominantly operate on the promastigote stage. r 2008 Elsevier GmbH. All rights reserved. Keywords: Leishmania infantum; Promastigotes; SIN-1; Nitric oxide; Proteome; Infectivity

Introduction The species of the genus Leishmania are obligate protozoan parasites with a heteroxenous life cycle which Corresponding author. Tel.: +34 91 3941818; fax: +34 91 3941815.

E-mail address: [email protected] (F. Bola´s-Ferna´ndez). 1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2008.07.006

involves a mammalian vertebrate and an invertebrate sandfly vector. The parasite is dimorphic exhibiting an intracellular amastigote form within mammalian macrophages and a flagellated extracellular promastigote form inside the midgut of the vector. In the macrophages, the amastigotes survive and replicate inside the phagolysosomes and, depending on the location of the

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macrophages, it can lead to clinical manifestations that range from cutaneous or mucocutaneous ulcers to a fatal visceral disease. Visceral leishmaniasis is usually caused by the species L. donovani, L. infantum, and L. chagasi (Pearson and Souza, 1996). The generation of nitric oxide (NO) from arginine by the inducible nitric oxide synthase (iNOs) upon activation of macrophages by interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha is surely the principal effector in killing intracellular amastigotes (Liew et al., 1990). iNOs have been shown to be expressed by murine and human macrophages during phagocytosis by L. chagasi (Gantt et al., 2001) and during human cutaneous leishmaniasis by L. tropica (Gamze and Atik, 2005). Moreover, it was required to control visceralization of L. donovani in mice (Murray and Nathan, 1999). Several cellular targets may be subject to NO toxicity in Leishmania parasites, including enzymes of glycolysis and respiratory metabolism as well as trans-membrane transport systems (Maue¨l and Ransijn, 1997). NO may limit parasite development not only in vertebrate but also in invertebrate intermediate hosts (Ascenzi and Gradoni, 2002). Recently, apoptopsis-like death effects have been observed in amastigotes of L. infantum from dog macrophages and extracellular amastigotes from L. amazonensis induced by NO (Holzmuller et al., 2002, 2005) as well as in promastigotes of L. donovani induced by H2O2 (Das et al., 2001) and in promastigotes of L. infantum by heat stress (Alzate et al., 2007). In addition, new evidence shows that NO is also important in defending invertebrates against parasites (Rivero, 2006). In Leishmania, proteomics have proved to be a useful tool for identifying developmentally regulated proteins or putative virulence and drug resistance factors in various species (Acestor et al., 2002; El Fakhry et al., 2002; Bente et al., 2003; Drummelsmith et al., 2003; Nugent et al., 2004; Walker et al., 2006). Indeed, comparative proteomics offers a complementary approach to microarrays in global expression profiles with the advantage of revealing protein post-translational modifications (Banks et al., 2000; Walker et al., 2006). Although the effect of NO on Leishmania and other protozoan parasites has been well documented, few molecular targets have been identified (Salvati et al., 2001; Bocedi et al., 2004). Therefore, in the present paper a proteomic approach was applied to prove changes in the proteome of L. infantum promastigotes induced under in vitro exposure to a nitric oxide donor and to assess the effect of these changes on the in vitro infectivity of these promastigotes. This model is being assayed as an approach to assess the promastigote stage as target for NO as well, either before undergoing transformation after macrophage invasion and/or during its multiplication inside the digestive tract of the sandfly vector.

Materials and methods Cell culture of parasites Promastigotes of the L. infantum strain MCAN/ES/ 92/BCN83 were grown in the medium Schneider modified to a final concentration of 0.4 g/l NaHCO3, 4 g/l HEPES, 100 mg/l penicillin, and 100 mg/l streptomycin and 10% fetal bovine serum (Gibco), pH 6.8 and 26 1C. Axenic extracellular amastigotes of the L. infantum strain MCAN/ES/89/IPZ229/1/89 were grown at pH 5.4 and 37 1C in M-199 medium supplemented with aminoacids and 10% heat-inactivated fetal calf serum (Gibco). This medium was modified from that of Armson et al. (1999). Serial passages were made every 2 days for maintenance of the culture.

NO donor and assessment of their effect on parasite growth As NO donor the 3-morpholinosydnonimine (SIN-1) (Sigma) was used. It is stable in the solid form when stored dry at 4 1C and protected from light and highly water-soluble and stable in acidic solution. The aqueous stock solutions of SIN-1 made up in distilled water and adjusted to pH 5 can be used throughout the day. At physiological pH, and more so at alkaline pH, SIN-1 undergoes rapid non-enzymatic hydrolysis into the open ring form SIN-1A, which chemically is a nitrosamine. For experimental purposes, logarithmic promastigotes were seeded in multi-well plates at 2  106 promastigotes/well. To assay the effect on promastigote growth, increased concentrations (10–3–10–6 mM) of SIN-1 were prepared from the stock solution and added in quadruplicate to tubes with the culture medium. Then, the kinesis of NO production was measured by quantifying nitrates and nitrites formed by reaction of NO with oxygen according to the Griess reaction. Also the effect on promastigote growth was assessed at various times by microscopical estimation of total and alive parasites by formaldehyde fixation and trypan blue dye, respectively.

Promastigotes infection ‘in vitro assay’ A total of 104 cells/well of the macrophage J774 line were cultured in 8-well Labteks chambers (NUNC) following the method described by Mendez et al. (1999). Once adhered, 105 stationary-phase Leishmania promastigotes (SIN-1 treated and untreated) were added and maintained at 33 1C, 5% CO2 overnight. Non-internalized promastigotes were eliminated by several washings. The prepared slides were fixed and stained with Giemsa, and the number of amastigotes/100 cells was

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determined at 24 and 48 h by microscopical examination. Every sample was cultured at least in triplicate.

Sample preparation for 2-DE SIN-1 treated and untreated viable Log-promastigotes were recovered by collecting supernatants containing motile forms (dead forms settle in the bottom of the flask) followed by centrifugation at 700g for 10 min and at 4 1C. The resulting pellet was washed 5 times with Tris–HCl pH 7.8 and resuspended in 0.1 ml of the same buffer. The sample was sonicated for 10 s in a Virsonic 5 (Virtis, NY, USA) set at 70% output power on ice bath. The homogenate was extracted in 5 mM Tris–HCl buffer pH 7.8, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor, at 4 1C overnight and subsequently centrifuged at 10,000g for 1 h at 4 1C (Biofuge 17RS: Heraeus Sepatech, Gmb, Osterode, Denmark). Proteins in promastigote crude extracts were precipitated using 20% trichloroacetic acid (TCA) in acetone with 20 mM dithithreitol (DTT) for 1 h at 20 1C followed by centrifugation to eliminate contaminating substances. The pellet was washed with cold acetone containing 20 mM DTT. Thereafter, the protein pellet was resuspended in 50 ml of pre-treatment solution (7 M urea, 2 M thiourea, 4% Chaps, 5 mM CO3K2, 2% IPG buffer pH 3–10, and Destreak reagent (Amersham Biosciences, Uppsala, Sweden) at room temperature for 30 min.

Two-dimensional electrophoresis (2-DE) Four hundred mg of protein was diluted in 340 ml of rehydration buffer (7 M urea, 2 M thiourea, 2% Chaps, 0.75% IPG buffer pH 3–10, bromophenol blue), then adsorbed onto 18 cm immobilized pH 4–7 gradient (IPG) strips (Amersham Biosciences) at 20 1C for 12 h and 50 V, and then focused on an IPGphor IEF unit (Amersham Biosciences) using the following program: 2 h at 150 V; 1 h at 500 V; 1 h at 1000 V; 2 h at 2000 V and 6 h at 8000 V. The strips were then incubated in 10 ml equilibrating buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 10 mg/ml DTT for 15 min. This was followed by another equilibration step for 25 min with equilibrating buffer containing 25 mg/ml iodoacetamide. For separation in the second dimension, the IPG strips were placed onto 12.5% SDSPAGE gels and run at 15 mA/gel. After separation, proteins were visualized by silver staining.

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Gharahdaghi et al. (1999). Briefly, gel spots were incubated in 100 mM sodium thiosulfate and 30 mM potassium ferricyanide, rinsed twice in 25 mM ammonium bicarbonate (AmBic) and once in water, shrunk with 100% acetonitrile (ACN) for 15 min, and dried in a Savant SpeedVac for 20–30 min. Then, the spots were reduced with 10 mM dithioerythritol in 25 mM AmBic for 30 min at 56 1C and subsequently alkylated with 55 mM iodoacetamide in 25 mM AmBic for 20 min in the dark. Gel pieces were alternately washed with 25 mM AmBic and ACN, and dried under a vacuum. Gel pieces were incubated with 12.5 ng/ml sequencing grade trypsin (Roche Molecular Biochemicals) in 25 mM AmBic overnight at 37 1C. After digestion, the supernatants were separated. Peptides were extracted from the gel pieces first into 50% ACN, then into 1% trifluoroacetic acid and into 100% ACN. Then, 1 ml of each sample and 0.4 ml of 3 mg/ml a-cyano-4-hydroxycinnamic acid matrix (Sigma) in 50% CAN, 0.01% trifluoroacetic acid were spotted onto a matrix-assisted laser digestion ionization (MALDI) target. MALDITOF MS analyses were performed on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA). The following parameters were used: cysteine as s-carbamidomethyl derivative and methionine in oxidized form. Spectra were acquired over the m/z range of 700–4500 Da. Tryptic, monoisotopic peptide mass lists were generated and exploited for database searching. The peptide mass was searched against the Swiss-Prot/TrEMBL non-redundant protein database (www.expasy.ch/sprot) and L. major protein database available from Sanger Institute (www.sanger. ac.uk) and using Mascot (www.matrixscience.com) software program. MS/MS sequencing analysis were carried out using the MALDI-tandem time-of-flight mass spectrometer 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Mass spectrometry was performed at the Proteome Facility of Complutense University of Madrid, Spain.

Statistics The results were expressed as mean values7standard deviation (S.D.) for each group of data. Statistical analysis was made by Mann–Whitney test (SPSS 12.0 for Windowss, Chicago, USA) and p-values o0.05 were considered significant.

Results

Mass spectrometric analysis and database searching

Adaptation of the NO donor system to the in vitro Leishmania model

Spots of interest were manually excised from silverstained 2-DE gels after being distained as described by

Initially, a series of experiments were carried out, in order to optimize the production/effects of NO in the in

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vitro model system seeking to mimic in vivo host– parasite conditions. The kinesis of NO donor released to culture medium was assessed by adding SIN-1 at 4 different concentrations (10–3, 10–4, 10–5, and 10–6 mM) to the Schneider medium used for in vitro growth of Leishmania promastigotes. Samples were collected at 2, 4, 6, and 8 h following incubation at 26 1C, and the NO production was measured by determination of the nitrates and nitrites formed (data not shown). The following step was to check the effect of NO donor on the growth of Leishmania promastigotes. The reported four SIN-1 concentrations were tested on promastigotes seed at a concentration of 2  106 cells/ml following incubation for 24 and 48 h at 26 1C. As shown in Fig. 1A, the mean data from 3 independent experiments clearly indicate that the first 3 concentrations (10–3, 10–4, and 10–5 mM) significantly inhibited the growth of promastigotes at 24 and 48 h with regard to untreated controls. However, with the lowest concentration (10–6 mM), although growth was significantly inhibited by 24 h, the surviving parasites were able to recover thus surpassing their growth rate with no significant difference from untreated controls by 48 h. Afterwards, the viability of treated promastigotes was assessed at 2, 4, 6, 8, and 24 h of incubation with SIN-1 at 26 1C and at the above-indicated concentrations by using the vital trypan blue dye. The results expressed as mean values from 3 independent experiments are plotted in Fig. 1B. The highest toxicity is achieved at 4 h with SIN-1 concentration of 10–3 mM with 80% of death. At the SIN-1 concentrations of 10–4 and 10–5 mM, the maximum death rate was achieved by 6 h (60–80%). It is important to note that live promastigotes exhibited apparent higher mobility (by microscopical observation) than untreated controls. Treatment at 10–6 mM SIN-1 concentration significantly increased the growth rate of promastigotes with regard to controls at 8 and 24 h. According to these results, we established working conditions for NO production at SIN-1 concentration of 10–5 mM for 6 h at 26 1C as they provided a cytocidal effect similar to those achieved by activated macrophages under physiological conditions (Ridnour et al., 2004).

Proteomic analysis of NO donor-treated and untreated L. infantum promastigotes For protein analysis mass cultures were established from 2-day growing promastigotes (logarithmic stage) in 500 bottle final volume at the concentration of 2  106 promastigotes/ml. Treatment with NO donor was carried out at conditions previously established (10–5 mM SIN-1 concentration for 6 h). Image analysis of at least 4 representative 2DE gels obtained from 3 independent experiments following silver staining

Number of promastigotes (×106) 24 h 48 h

3.5 3 2.5

*

*

2

*

* *

1.5 *

1 0.5 0 C

10-3mM

10-4 mM

10-5 mM

10-6 mM

Concentration SIN-1 Alive promastigotes (% of non-treated control)

160 140 120 100 80 60 40 20 0 2

4

6

8

24

Time (h)

Fig. 1. (A)Assessment of the effect of the NO donor on parasite growth. SIN-1 was added at the concentrations of 10–3, 10–4, 10–5, 10–6 mM to promastigotes at the logarithmic stage. Total and alive parasites were estimated after 24 and 48 h incubation by formaldehyde fixation and trypan blue dye, respectively. Results are expressed as mean values plus standard deviation of 4 replicates. *Statistically different from untreated controls at po0.05. (B) Optimization of the concentration/time conditions for the NO generating system. SIN-1 was added at concentrations of 10–3 (&), 10–4 (’), 10–5 (n), 10–6 (E) mM to promastigotes at logarithmic stage of growth. Life parasites were estimated at 2, 4, 6, 8, and 24 h incubation by the trypan blue dye exclusion. Results are expressed as mean values from 3 independent experiments.

showed highly reproducible maps with more that 300 protein spots resolved in gels from both control and treated samples (Fig. 2A and B). Nevertheless, the number of resolved proteins in this study carried out on logarithmic stages was much lower than that usually reached with stationary stages. Thirty-eight spots were submitted to in-gel digestion and mass spectrometry analysis. Few of them were selected as landmark proteins according to previously established reference map, whereas the majority was spots that were differentially expressed in treated and untreated samples. Protein identification is summarized in Table 1. Twenty-two spots could be identified either by mass spectrometry (MS) or MS/MS analysis. Five spots

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(Nos 1, 2, 19, 25, and 31) were clearly up-regulated in treated samples. Spots 1 and 2 were unambiguously identified as enolase (mowse score of 87), whereas spots 19, 25, and 31 matched the probable P28 protein, 20S proteasome beta 7 subunit, and hypothetical protein of unknown function, respectively (mowse scores of 98, 75, and 62, respectively) in the L. major database in combination with Swiss-Prot/TrEMLB search using Mascot. By contrast, 13 spots (Nos 6, 7, 8, 12, 13, 14, 15, 16, 21, 22, 26, 30, and 32) were clearly downregulated in treated samples with regard to untreated controls. Only 4 of them (8, 12, 15, and 16) could be

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identified as hypothetically predicted protein, heatshock 70 protein mitochondrial precursor fragment, 20S proteasome alpha 5 unit and probable phosphomannomutase, respectively (mowse scores of 56, 57, 130, and 90, respectively). Treatment did not modify the expression of proteins corresponding to spots Nos 18, 20, 23, 24, 33, 34, and 35, among them spot No 18 matched with 20S proteasome alpha 7 unit, spot No 20 matched with elongation factor 2 (EF2) and spot Nos 33, 34, and 35 were identified as beta tubulin, probable thermostable carboxypetidase, and trypanothione reductase, respectively (mowse scores of 75, 98, 169, 59, and 88, respectively). Six spots (Nos 3, 4, 9, 17, 27, and 28) identified as possible 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) synthase, prostaglandine f2-alpha (PGD2) synthase, probable tubulin alfa chain, and peroxidoxin precursor, respectively (mowse scores of 32, 66, 126, 61, 154, and 200, respectively) were only present in treated samples. By contrast, 3 unidentified spots (Nos 36, 37, and 38) were present only in untreated samples. In treated samples there were several spots of doubtful identification or expression. On the one hand, spot Nos 10 and 11 exhibited a clear differential expression patterns in treated samples and matched with probable tubulin alpha chain. However, as its molecular weights are lower than predicted, they may correspond to fragments of this protein. On the other hand, for spot Nos 5 and 29 differences in expression patterns in control and treated samples were not clearly manifested. Interestingly, extracellular amastigotes undergoing the same treatment as the promastigotes did not show any significant change in the expression protein pattern in comparison to untreated samples as shown in the 2D maps (Fig. 3A and B). Moreover protein profiles of these extracellular amastigote forms are scarcely comparable to those corresponding to the logarithm promastigote stages (Fig. 2A and B).

The effect of NO donor treatment on the in vitro infectivity of promastigotes to mouse macrophages

Fig. 2. Two-DE analysis of proteins from untreated (A) or NO donor-treated (B) logarithmic L. infantum promastigotes. First dimension (IEF) was performed with 400 mg of proteins using 4–7 pH range IPG strips. Second dimension (SDS-PAGE) was performed on 12.5% polyacrylamide gels and stained with silver. The numbers refer to the spot identity used in the tables and text. This figure is a reliable representation of at least 4 gel runs. Encircled areas show the absence of spots in the gel from untreated control samples.

SIN-1-treated and untreated promastigotes were used to infect macrophages of the murine line J774. The results from 2 independent experiments are summarized in Table 2. The mean values obtained from 4 replicates clearly show that treatment with SIN-1 significantly increased (po0.05) the infectivity of promastigotes to macrophages after 24 and 48 h of culture when compared to untreated controls.

Discussion In applying the in vitro extracellular assay to assess the effect of NO metabolites on the promastigote stage of L. infantum, our primary objective was to select a

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Identification by MALDI-TOF and MALDI-TOF/TOF of proteins differentially expressed by NO-treated promastigotes of L. infantum Sequence coverage (%)

Matched peptides (total)

Differences

MW exp (  103)

pI exp

NEAAFQDVGIEYYR

166 87 32

47 3 2

16 1 1

m m A

50.5 50.5 54

5.25 5.50 6.50

NEAAFQDVGIEYYR

66

2

1

A

54

6.70

7 14 11+8

D k k k A D

43 43.5 43 40 29 31

5.25 5.30 5.40 6.60 6.60 4.55

3 2

D

36

5.30

2

k

30.5

4.80

15 11 8

k k k k A

28.5 28.5 26 26 8

4.85 4.90 4.90 5.60 5.20

Name

Means of analysis

1 2 3

P1408.28 P1408.28 LM24.211

MS MS-MS/MS MS-MS/MS

GNPTVEVELMTEAGVFR

4

LM24.211

MS-MS/MS

5 6 7 8 9 10

– – – LmjF36.6360 LmjF31.2150c LmjF25.1710c+ P1046.33 LmjF25.1710c+ P1046.22+ CHR27_tmp.81

Enolase Enolase 3-Hydroxy-3-methylglutarylCoA(HMG-CoA) synthase, possible 3-Hydroxy-3-methylglutarylCoA(HMG-CoA) synthase, possible Not identified Not identified Not identified Hypothetical predicted protein Prostaglandin f2-alpha synthase At5g50850/k16e14_1 Probable tubulin alpha chain At5g50850/k16e14_1 Probable tubulin alpha chain 28 kDa guide RNA-binding protein

11

Peptides

MS MS MS DITLIGFSR

MS-MS/MS

VIAPYNCEDAR LAAEGVQAEVINLR

56 126 128 (76+52) 141 60 26

20 43 28 25 9

1

SLDIERPSYTNVNR CIFLDLEPTVVDEVR QLLVSQFPQLGPR

12 13 14 15 16 17

CHR30_tmp.17

L3640.16 LmjF36.1960 P1046.33

Heat shock 70 kDa protein, mitochondrial precursor, fragment Not identified Not identified 20S proteasome alpha 5 subunit Phosphomannomutase, probable Probable tubulin alpha chain

MS-MS/MS

NNAETQLTTAER

MS-MS/MS MS-MS/MS MS* MS* MS*

SQTFSTAADNQTQVGIK

57

130 90 61

4

60 44 18

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Mowse score

Spot Accesion number BLAST database

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Table 1.

19 20 21 22 23 24 25 26 27 28 29 30 31

LmjF25.2010c LmjF36.0190

LmjF36.4910

32 33 34

L3640.20 P1046.03

35 36 37 38

CHR34_tmp.183c L2581.13 L2581.13

LM5.34

20S proteasome alpha 7 subunit, probable P28 protein, probable EF2-1. Elongation factor 2 Not identified Not identified Not identified Not identified 20S Proteasome beta 7 subunit Not identified Peroxidoxin precursor Peroxidoxin precursor Not identified Not identified Hypothetical protein, unknown function No identified Beta-tubulin Probable thermostable carboxypeptidase 1 Trypanothione reductase Not identified Not identified Not identified

MS* MS* MS MS-MS/MS MS-MS/MS MS-MS/MS MS-MS/MS MS MS-MS/MS MS MS MS-MS/MS MS-MS/MS MS MS-MS/MS MS MS MS MS-MS/MS MS-MS/MS MS-MS/MS

NNAETQLTTAER

57

29

7

¼

26

6.00

98 98

44 18

11 1

75

14

3

154 200

40 46

10 11

62

30

13

m ¼ k k ¼ ¼ m k A A D k m

23 23 19.5 20.5 20.5 20.5 30 28.5 28.5 29 29 29 43

6.25 5.30 5.20 5.25 5.20 5.05 5.10 5.20 6.20 6.45 4.30 4.20 5.80

169 59

32 18

18 11

k ¼ ¼

24 59 59

5.05 4.90 5.05

88

14

7

¼ DP DP DP

59 38 38 38

6.10 5.25 5.30 5.35

m: Upregulated; k downregulated; A: appearance; DP: disappearance; D: doubtful: ¼ : unchanged; * confirmed by MS/MS.

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CHR27_tmp.155c

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18

227

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Fig. 3. Two-DE analysis of proteins from untreated (A) and NO donor-treated (B) L. infantum extracellular amastigotes. First dimension (IEF) was performed with 400 mg of proteins using 4–7 pH range IPG strips. Second dimension (SDSPAGE) was performed on 12.5% polyacrylamide gels and stained with silver. This figure is a reliable representation of at least 4 gel runs.

Table 2.

In vitro infectivity of SIN-1-treated promastigotes to J774 macrophages Experiment 1

Experiment 2

Control

Mean SD

suitable NO generation donor among those existing. Apart from NO, the peroxinitrite (PN) is another potent reactive nitrogen species (RNS) where its microbicidal effect has been demonstrated against various pathogens such as Mycobacterium avium (Doi et al., 1993), M. tuberculosis (Rhoades and Orme, 1997; VazquezTorres and Balish, 1997), Trypanosoma cruzi (Alvarez et al., 2004), L. donovani, and L. major (Chan et al., 2005). Like NO, PN is also able to rapidly penetrate membranes and diffuse across cells thus reacting with various components eventually leading to DNA fragmentation or lipid peroxidation and protein nitration (Miller and Britigan, 1997). Chemical reagents able to generate and spontaneously sustain a release of RNS are required for components being as active and unstable as NO and PN. The sydonimines such as SIN-1 are capable of auto-disintegrating and of spontaneously releasing both NO and superoxide anion (O–2) in physiological conditions (neutral pH). The interaction of both generates PN. This system has already been extensively used in neuronal and heart pathologies (Shin et al., 2005; Choi et al., 2005) as well as in infectious diseases (Diez-Orejas et al., 2001). Based on the data previously achieved by Hernandez with Candida albicans (unpublished), we chose SIN-1 as the most suitable donor for the production of RNS (NO and PN) in conditions that mimic those occurring in infected macrophages under physiological conditions (Linares et al., 2001). We established the conditions by which the toxic effects were such as to produce highly significant but not completely lethal effects (60–70% growth inhibition) and the rationale was that these conditions are likely compatible with the maintenance of resistant populations and/or development of repairing strategies as defense mechanisms against RNS. Moreover, these conditions are highly coincidental with those described previously for C. albicans (Hernandez, unpublished), another opportunistic agent likely infecting macrophages.

SIN-1 treatment

Control

SIN-1 treatment

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

128.36 211.59 159.04 251.06 187.51 42.09

119.27 193.06 227.65 204.00 185.99 46.76

210.43 325.81 363.41 174.23 268.47* 79.71

119.27 193.06 227.65 204.00 252.18* 110.06

166.48 130.28 115.86 177.52 147.54 29.20

133.85 220.00 149.23 261.37 191.11 51.97

216.66 – 268.51 400.78 295.32* 94.94

210.46 297.81 269.01 289.55 266.71* 39.4

Results are expressed as number of amastigotes/100 macrophages. SD, standard deviation. * Significantly different from control (po0.05).

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Treatment caused dramatic changes in promastigote protein expression as shown by spot profiles in 4–7 pI 2DE maps after silver staining. However, of the 38 protein spots studied only 22 could be unambiguously matched in the L. major and Swiss-prot protein databases. Of these 22 spots, identification by peptide mass fingerprint (PMF) was only possible in 12, the remainder requiring fragmentation by tandem MS (MS/MS). Curiously, proteins that have been classically associated with oxidative stress in Leishmania and Trypanosome species, such as glyceraldehyde-3-phosphate dehydrogenase (Bourguignon et al., 1997), the cystein proteinases (Bocedi et al., 2004), and the superoxide dismutase (Ghosh et al., 2003) could not be identified in the present study. This could be an indication of protein modifications (oxidation, nitration, carbonilation) upon treatment including post-translational changes. In the case of L. infantum, S-nitrosylation of the Cys25 via NO is the way of inactivation of cystein proteins in promastigotes. In a similar way, transnitrosation of proteins has been suggested as the mechanism of action for S-nitrosoglutathione (GSNO), another NO donor, on L. major and L. amazonensis (de Souza et al., 2006). Unlike other studies (Augustyns et al., 2001; Thomson et al., 2003), the expression of trypanothyone-reductase was not affected by our oxidative treatment. Logically, proteins that have been classically associated to oxidative stress were also clearly up-regulated here. Among them are enolase, already reported in L. mexicana (Nugent et al., 2004), peroxidoxin precursor, also described as detoxifying enzymes in L. chagasi, L. major, and L. aethiopica (Levick et al., 1998; Jirata et al., 2006), and the mitochondrial precursor of the Hsp70 protein, reported as antioxidant in L. chagasi (Miller et al., 2000). Hypothetical fragments of Hsp70 were apparent in the map of treated promastigotes with slightly varied position to that exhibited in the map of untreated controls. Again, as pointed out above this variation could be indicative of a possible post-translational modification induced by NO. Three protein spots (the possible HMG-CoA synthase, the hypothetical predicted protein 8LmjF36.6360, and the PGD2 synthase) were present only in maps from treated promastigotes. Although association of HMGCoA synthase with oxidative stress has been reported in other systems, to our knowledge no linkage of this enzyme to ROS or to RNS has been established in trypanosomatids. The other protein of known function is the PGD2 synthase. This enzyme has been previously described in several species of Leishmania of the Old World while being absent in the New World species. It is localized in the promastigotes cytosol (Kabututu et al., 2003). The species-specific restriction of this enzyme is supposedly related to midgut environmental conditions of the sandfly vectors from both parts of the world.

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Recently, its participation in programmed cell death (PCD) of bloodstream forms of Trypanosma brucei by means of the oxidative stress has been demonstrated (Figarella et al., 2006). Among the various proteins that were clearly under-expressed in NO-treated promastigotes, only the phosphomannomutase and the 20S proteasome alpha 5 could be identified following fragmentation by tandem MS/MS. In contrast, the 20S proteasome alpha 7 was not affected. However, the involvement of proteasome in PCD of Leishmania has been reported as well (Nguewa et al., 2004). Treatment did not seem to affect the expression of EF2. In other studies carried out in liver rats, it has been shown that oxidative stress decreased the production of EF2, the main protein involved in the peptide chain elongation step along polyribosomes (Ayala et al., 1996). However, so far no information is available concerning trypanosomatids and oxidative stress with regard to this protein. Treatment with SIN-1 increased the infectivity of the surviving promastigotes to macrophages in the in vitro assay with regard to untreated controls. This is an interesting result and deserves some comment. As described above, we adopted assay treatment conditions that allowed survival of about 30% of the promastigotes and, although treatment killed the majority of the parasite population, the surviving resistant forms had their infectivity strengthened. So the first conclusion that can be drawn from these results is that under the oxidative treatment the selection of viable parasites is promoted. The mechanism that underlies this population selection deserves further investigation but information is already available that can suggest a speculative hypothesis. PCD, a physiological regulatory system of mammalian cells, has already been described in various protozoa including trypanosomatids (Nguewa et al., 2004). In Leishmania species, when providing different stimulus such as serum deprivation, heat shock, and treatment with oxidant agents (NO, H2O2), PCD caspase-independent apoptosis-like death is induced (Moreira et al., 1996; Das et al., 2001; Zangger et al., 2002, Lee et al., 2007). In the revision by Nguewa et al. (2004), it is stated that PCD could have a functional role for trypanosomatids as a way to maximize their biological fitness, thus serving as a mechanism of adaptation and defense against the host. In clonal organisms such as trypanosomatids, PCD could serve as cell-sorting to select infectious forms (e.g. select metacyclic from procyclic stages) operating in the digenic cycle (both the insect and the vertebrate host). In this regard, inducible NO synthesis has been reported in the response of Anopheles stephensi to limit the infection with Plasmodium berghei and in the response of Rhodnius prolixus against infection with Trypanosoma rangeli (Rivero, 2006). Moreover, it was found that promastigote preparations with high

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proportion of metacyclic forms produce NO, thus suggesting an association of NO with differentiation and infectivity of the parasite (Genestra et al., 2006). We also found that amastigote-like cells were resistant to treatment given in similar conditions to promastigotes showing no changes at the proteomic level with regard to controls. A similar observation on differential developmental resistance to NO was reported by Lemesre et al. (1997) with L. mexicana, L. amazonensis, and L. chagasi and using authentic NO gas flushed into cultures. In their work, incubation times longer than 4 h were needed to achieve in amastigotes a killing effect comparable to that reached in promastigotes. To explain this different behavior, they suggested that amastigote stages which replicate inside the phagolysosome of macrophages may – under exposure to oxidant agents – evolve compensatory mechanisms that evade the antimicrobial effects of these agents. Accordingly, Holzmuller et al. (2006) selected NO-resistant clones of L. infantum that overexpressed cis-aconitase, glyceraldehyde-3-phosphate dehydrogenase with no evidence of apoptotic signs. Although clear evidence for PCD was not provided in our study, the existence of apoptosis-like features operating in our systems should not be excluded as the presence of PGD2, a protein already associated to PCD in T. brucei via oxidative stress, was indeed evident in treated promastigotes. In summary, by adapting an in vitro model system used in combination with proteomics to measure oxidative stress in L. infantum, we were able to provide some new potential molecular targets of RNS. Moreover, we identified some proteasome units, PGD2 and likely some HSP isoforms, which have been previously reported to be involved in PCD events in Leishmania species. The enhanced infectivity of promastigotes that survive RNS lethal effects could suggest the existence of an oxidative stress-mediated PCD as a mechanism of population regulation and selection of good infecting forms of trypanosomatids that may operate preferentially inside the digestive tract of the invertebrate vector. Confirmation of this suggestion will require optimized experimental approaches including the characterization and purification of metacyclic forms as well as to perform specific PCD assay to measure DNA fragmentation and caspase-like activities. Furthermore, new proteomic tools combined with the availability of the annotated L. infantum data base will help for a better assessment and understanding of NO-induced changes in Leishmania proteins.

Acknowledgments The authors thank to M. Colmenares from the Centro de Investigaciones Biolo´gicas, CSIC (Madrid), for

kindly providing us with the axenic extracellular amastigotes and to M.D. Gutierrez-Blazquez of the Complutense University Proteomics Facility for providing technical help. The authors also thank B. Simmons Dı´ ez for her help in the English revision. This work was funded by Grant no. GR/SAL/0546/2004 from Direccio´n General de Universidades e Investigacio´n de la Comunidad de Madrid (Spain). M.D.A. was a Postdoctoral research fellow of the Comunidad de Madrid.

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