Proteomic Analysis of Trypanosoma cruzi Resistance to Benznidazole

Proteomic Analysis of Trypanosoma cruzi Resistance to Benznidazole He´lida M. Andrade,*,†,‡ Silvane M. F. Murta,† Alex Chapeaurouge,§ Jonas Perales,§ Phillipe Nirdé,| and Alvaro J. Romanha† Laborato´rio de Parasitologia Celular e Molecular, Centro de Pesquisa Rene´ Rachou/FIOCRUZ, Brazil, Universidade Federal do Paiuı´, Lab Imunogene´tica e Biologia Molecular, Teresina, Piauı´, Brazil, Laborato´rio de Toxinologia, Departamento de Fisiologia e Farmacodinaˆmica, Instituto Oswaldo Cruz/FIOCRUZ, Brazil, and INSERM U540, 60 rue de Navacelles, 34090 Montpellier, France

J. Proteome Res. 2008.7:2357-2367. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 09/29/18. For personal use only.

Received October 12, 2007

The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole-in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole. Keywords: Trypanosoma cruzi • Benznidazole • resistance • proteome • 2-DE

Introduction Chagas disease or American Trypanosomiasis is a chronic parasitary infectious disease that appears as the main cause of heart disease in Latin America. It affects from 16 to 18 million people in the Americas, causing 21 000 deaths yearly.1 The only medicines prescribed in medical practice to treat the infection are Nifurtimox (NFX), a 5-nitrofuran, and Benznidazole (BZ), a 2-nitroimidazole. Their action is very limited, and the cure depends on the phase of the disease, on the susceptibility of the Trypanosoma cruzi strain, and on the host’s physiological conditions. The medicines have been effective in treating the disease in the acute phase and in preventing the progress and worsening of infection in the chronic phase, although they present low efficacy in this stage.2–4 In addition, the therapeutic is ineffective in immunosupressed and AIDS patients.5,6 Among the obstacles to the accomplishment of a parasitological cure, * To whom correspondence should be addressed. Dr. He´lida Monteiro Andrade, Laborato´rio Imunoparasitologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerias, Brasil. Phone, (55) 0XX31-3499-2634; e-mail, [email protected]. † Centro de Pesquisa Rene´ Rachou. ‡ Universidade Federal do Paiuı´. § Instituto Oswaldo Cruz. | INSERM U540. 10.1021/pr700659m CCC: $40.75

 2008 American Chemical Society

one can mention the wide genetic variability of the T. cruzi population and the existence of strains which are naturally resistant to the chemicals used against them.7 Therapeutic options for the treatment of Chagas disease are limited to Benznidazole and Nifurtimox, which were discovered about 30 years ago, and the latter is no longer produced in Brazil.8 Currently, vectorial transmission is controlled in nearly all the areas of occurrence, and in most cases, the disease is diagnosed in the chronic phase, when the medicines are ineffective and the cure rates are low. In addition, the chemicals are very toxic and cause considerable side effects. The need for new options to treat Chagas disease has led to the performance of several tests utilizing compounds chosen through studies that identify metabolic targets in the parasite or that are chosen empirically. One of the possibilities for the identification of new therapeutic targets is proteomic analysis. The use of proteomic techniques in trypanosomatids is particularly important because these organisms do not use transcription initiation as a regulatory step to control gene expression. All protein-encoding genes in trypanosomatids are organized in large polycistronic transcription units that generate polycistronic precursor RNAs which are then processed to monocistronic mRNAs by the mechanism of trans-splicing. Gene regulation occurs by controlling stability and/or translaJournal of Proteome Research 2008, 7, 2357–2367 2357 Published on Web 04/25/2008

research articles tion of specific mRNAs. In addition, post-translational modifications play an important role in modulation of protein function in these parasites.9 Taken together, these considerations support proteomic analysis as an ideal approach to evaluate protein expression levels in specific stages or upon specific treatments of trypanosomatids. Moreover, a proteomic approach for T. cruzi becomes even more significant given that the T. cruzi genome is already available on database.10 Taking into account the fact that the molecular mechanisms by which T. cruzi becomes resistant or susceptible to drugs are not known and that the proteins are ultimately responsible for the cell phenotypes, a comparative study of the proteome of T. cruzi clones and strains susceptible and resistant to Benznidazole was performed to identify the differential expression of proteins in each phenotype. The Benznidazole-resistant T. cruzi population (17LER) used in this study was derived from the Tehuantepec cl2 strain (17WTS)11 by exposing in vitro to increasing concentrations of Benznidazole. The 17LER parasites are resistant to a dose of Benznidazole 23 times higher than that required to kill 50% of the 17WTS parasites. The Benznidazole-resistant T. cruzi population (BZR) was derived from the Y strain, selected in vivo in a previous study after 25 successive passages in mice treated with a single high dose of Benznidazol.12 A Benznidazole-susceptible population (BZS), a Benznidazole-susceptible clone (Cl 9S), and a Benznidazole-resistant clone (Cl 27R) were also included in the study.

Experimental Section T. cruzi Samples. Epimastigote forms of a population and of T. cruzi clones showing selected in vivo resistance to Benznidazole (N-benzyl-2-nitro-1-imidazolacetamide, Rochagan/Roche, SP, Brazil)sBZR and Clone27Rsand their respective susceptible pairs (BZS and Clone9S),12 as well as clones showing susceptibility and induced in vitro resistance to Benznidazoles17LER (resistant) and 17WTS (susceptible) produced by Nirde´11swere utilized. The pairs of samples Susceptible/Resistant were cultivated under identical conditions (exponential growth phase, temperature, parasite concentration and medium). For each pair of samples, masses of parasites from three independent cultivates were obtained and maintained at -70 °C for the protein extract preparation. Protein Extract. The protein extracts were obtained simultaneously for each pair susceptible/resistant sample. Epimastigote forms were obtained in the exponential growth phase in Liver Infusion Tryptose (LIT) medium according to Camargo.13 The epimastigote were washed three times in (RPMI) medium by centrifugation at 200g for 5 min at 4 °C. Lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithiothreitol (DTT), 40 mM Tris base and a protease inhibitor mix) was added to the cell sediment, in a proportion of 500 µL of lysis buffer for 109 parasite forms. After centrifugation at 20 000g for 1 h at room temperature, the supernatant (protein extract) was kept at -70 °C until use. Protein concentration was determined by the modified Bradford method.14 Two-Dimensional Gel Electrophoresis (2-DE). 1. Isoelectric Focusing (IEF). IEF for the 2-DE was performed using the IEFCell system (Bio-Rad Hercules, CA). Aliquots of 500 µg of protein were diluted to a final volume of 350 µL in a rehydration solution (8 M urea, 2 M thiourea, 2% CHAPS, 40 mM DTT, 0.5% IPG buffer, pH 3-10, trace bromophenol blue). Samples were then applied to the immobilized pH gradient (IPG) strips (17 cm, pH 3-10 nonlinear; Bio-Rad) by in-gel rehydration. Passive 2358

Journal of Proteome Research • Vol. 7, No. 6, 2008

Andrade et al. rehydration was performed for 4 h at 20 °C, followed by an active rehydration at 50 V for 12 h at 20 °C. Isoelectric focusing was increased gradually to 8000 V and run for 60 000 Vh at 20 °C and a maximum current of 50 µA/strip. After IEF, each strip was incubated for 15 min in 10 mL of 50 mM Tris-HCl buffer, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% BPB, and 125 mM DTT, followed by a second incubation step in the same buffer solution, except for DTT, which was replaced by 125 mM iodacetamide. 2. SDS-PAGE. IPG strips were transferred to a 12% polyacrylamide gel, and run on a Protean II system (Bio-Rad) connected to a Multitemp II cooling bath (Amersham Biosciences). Electrophoresis was performed in Tris/Glycine/SDS buffer according to the conditions previously reported. 15 Proteins were separated for 1 h at 50 V and then at 200 V until the dye front reached the bottom of the gel. After electrophoresis, gels were stained with colloidal CBB G-250 following procedures described elsewhere.16 3. 2-DE Image Analysis. Two-dimensional gel images were digitized from stained gels using a GS-800 calibrated densitometer (Bio-Rad). For each pair of samples, proteins from three independent cultivates were obtained, and gels in duplicate were made for each cultivate. Thus, six gels were obtained for each sample. Data from 2-DE gels were analyzed using the Discovery Series PDQuest software (Version 7.3.1; Bio-Rad). The authenticity and outline of each spot were validated by visual inspection and edited when necessary. The intensity of each protein spot was normalized relative to the total abundance of all valid spots. After normalization and background subtraction, a matched set was created for each pair of samples: BZR and BZS, 17LER and 17WTS, Cl27R and Cl9S (six replicates/ sample). The differential expression analysis was performed comparing the quantity of matched spots in each pair of sample, Benznidazole-resistant versus Benznidazole-susceptible gels. The program created a quantitative table with all normalized optical spot densities that permitted an analysis of variance (ANOVA) to detect statistical differences between the measurements of the same spot in all replicates in each sample pair. A protein was considered differentially expressed when the ratio between the intensities of S (susceptible) and R (resistant) spots were g2.5-fold. An ANOVA was accomplished using StatView 4.5 software (Abacus Concepts, Berkeley, CA), and p < 0.05 was adopted as the level of significance. 4. Protein Identification. Spots with differential expression were excised, and gel fragments were washed in 25 mM ammonium bicarbonate/50% acetonitrile until completely destained. After drying, gel fragments were placed on ice in 50 µL of protease solution (sequence grade modified trypsin, Promega, at 20 ng/µL in 25 mM ammonium bicarbonate) for 30 min. Excess protease solution was then removed and replaced by 20 µL of 25 mM ammonium bicarbonate. Digestion was performed at 37 °C for 16 h. Peptide extraction was performed twice for 15 min with 30 µL of 50% acetonitrile/5% formic acid. Trypsin digests were then concentrated in a SpeedVac (Savant) to about 10 µL and desalted using Zip-Tip (C18 resin; P10, Millipore Corporation, Bedford, MA). Peptide elution from the column was performed in 50% acetonitrile/ 0.1% trifluoroacetic acid.17 Controls for each group of spots/ samples identified by MS were included: (a) a gel fragment with no protein (negative control), (b) a gel fragment from the molecular weight standard bovine albumin (positive control)

research articles

Proteomic Analysis of T. cruzi Resistance to Benznidazole

Figure 1. 2-DE gels of proteins from T. cruzi Benznidazole-resistant samples. IEF was performed with 500 µg of protein using 17 cm, 3-10NL pH range strips. SDS-PAGE was performed on 12% polyacrylamide gels and stained with Coomassie blue G250. Inside circles are the proteins overexpressed in Benznidazole-resistant compared to Benznidazole-susceptible samples. The numbers refer to the spot identification used in the Tables. The rectangle shows a gel region to be amplified (Figure 3). This gel is representative of six gel runs.

and (c) gel fragment of proteins from T. cruzi (chaperones, actin and tubulin) previously identified by 2-DE. MALDI-TOF-TOF mass spectrometry (MS) analysis was performed on the 4700 Proteomics Analyzer with version 3.0 software (Applied Biosystems, Foster City, CA). Briefly, the tryptic peptide samples (0.3 µL) were mixed with the same volume of a saturated solution of R-cyano-4-hydroxycinnamic acid matrix (Aldrich, Milwaukee, WI), in 50% acetonitrile/0.1% trifluoroacetic acid on the MALDI plate and allowed to cocrystallize at room temperature. MS and MS/MS mass spectra were acquired in reflector mode and internally calibrated with trypsin autolysis peptides. Up to eight of the most intense ion signals were selected as precursors for MS/MS acquisition. External calibration in MS mode was performed using a mixture of four peptides: des-Arg1-Bradykinin (m/z ) 904.468), angiotensin I (m/z ) 1296.685), Glu1-fibrinopeptide B (m/z ) 1570.677) and ACTH (18-39) (m/z ) 2465.199). MS/MS spectra were externally calibrated using known fragment ion masses observed in the MS/MS spectrum of angiotensin I. Tandem mass spectra were searched against the NBCI nonredundant database using the MASCOT software (http://www.matrixscience.com). The search parameters were as follows: no restrictions on protein molecular weight, the Trypanosomaspecies of origin, two tryptic miss-cleavages allowed, variable modifications of methionine (oxidation), cysteine (carbamidomethylation) and pyroglutamate formation at N-terminal glutamine of peptides. All automatically interpreted MS/MS spectra were manually validated for correct ion series. Only proteins identified by MOWSE (molecular weight search) scores >50 are reported. In cases were the identification of the protein was based on only one or two peptides, an additional peptide mass fingerprint search was performed to confirm protein identity. The false discovery rate was estimated using the Decoy function as a search parameter in the Mascot search engine. No false-positive hits were observed. Furthermore, good correlation between experimental and theoretical molecular mass and pIvalues was also taken into account.

Results and Discussion Drug resistance is one of the major clinical problems that affect not only bacterial, but also parasitic diseases. In Chagas disease, drug resistance has a strong impact on the increase in the number of therapeutic failures, significantly limiting the treatment options. In addition, treatment failures may also be a result of the genetic diversities of T. cruzi and/or the hosts.18 Among the methods used to study differential expression of the genes involved in cell resistance to drugs, “Differential Display” has been the most widely used. Nevertheless, the control of gene expression by means of post-transcriptional events in T. cruzi results in a weak correlation between mRNA and protein levels.19 As a consequence, studies on gene differential expression by proteomics are very suitable. Previous studies on T. cruzi proteome have been performed for 2-DE standardization and the identification of proteins showing differential expression in the various evolutive stages of the parasite.9,19–21 In the present study, the proteomic analysis was performed on three pairs of T. cruzi samples which were different with respect to one another only for the Benznidazole resistance (R)/susceptibility (S) phenotypes. Small differences among the protein profiles of the replicates were observed. The 2-DE gels of proteins from T. cruzi Benznidazoleresistant and -susceptible samples are shown in Figures 1 and 2, respectively. The analysis of the gel images showed that the number of spots per gel ranged from 176 to 398. The average number of spots per pair of T. cruzi samples Susceptible/ Resistant was 389 spots for 17WTS/17LER, 248 for CL9S/CL27R, and 194 for BZS/BZR (Table 1). The samples showing the Benznidazole-resistant phenotype presented a larger number of total and exclusive (orphan) spots in comparison to the susceptible phenotype. These results suggest that the selection of Benznidazole-resistant parasites induces proteins’ overexpression for adapting to the unfavorable drug stress conditions. Besides, it is possible that T. cruzi genomic plasticity, susceptible/ Journal of Proteome Research • Vol. 7, No. 6, 2008 2359

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Andrade et al.

Figure 2. 2-DE gels of proteins from T. cruzi Benznidazole-susceptible samples. IEF was performed with 500 µg of protein using 17 cm, 3-10NL pH range strips. SDS-PAGE was performed on 12% polyacrylamide gels and stained with Coomassie blue G250. Inside circles are the proteins PGF-2∝ overexpressed in Benznidazole-susceptible compared to Benznidazole-resistant samples. The rectangle shows the same gel region of the Figure 1 to be amplified (Figure 3). This gel is representative from six gel runs. Table 1. Analysis of 2-DE Protein Profiles from T. cruzi Samples Susceptible (S) and Resistant (R) to Benznidazole spots T. cruzi sample

17 LER 17 WTS Clone 27R Clone 9S BZR BZS Total

spots/gel

398 380 279 217 212 176

exclusive (orphans)a

differentially expressedb

MSc

IDd

30 12 75 7 54 10 188

26 33 52 20 9 20 160

26 16 44 16 22 13 137

18 9 36 13 21 13 110

a Present in one of the samples of the pair BZ susceptible/resistant. Present in both samples of the pair but overexpressed in one of them (ANOVA p < 0.05). c MS, mass spectrometry. d ID, identified proteins.

b

Figure 3. Viewing detail by magnification of the same region inside rectangles of the 2DE gels from T. cruzi Benznidazoleresistant (17LER) and -susceptible (17WTS) populations. The protein PGF-2a (in circle) is overexpressed in 17WTS as compared to 17LER.

resistant samples’ origin from Tehuantepec or Y strain, and the way, in vitro or in vivo, the Benznidazole resistence was obtained had influence. The success in protein identification utilizing databases for peptide sequences obtained by MS is directly related to the genomic databases of the same organism. Fortunately, the T. cruzi genome was recently published.10 Table 1 presents the number of total and differentially (over) expressed spots in each sample and the number of spots that were submitted to MS and identified in each group. Of the 348 spots exclusive 2360


(orphans) and differentially expressed in all samples analyzed, 137 spots were submitted to MS for protein identification because they were well-defined and reproducible. Among these, 117 (85.4%) presented good spectra, and 110 (80.3%) spots were identified as 56 distinct proteins. Out of the 56 proteins identified, 36 (64.3%) were only expressed or overexpressed in resistant samples, 9 (16.1%) in sensitive samples and 11 (19.6%) in both phenotypes. Of the 137 spots, 14 (10.2%) contained a mixture of 2 proteins and 2 spots contained a mixture of 3 proteins. Among the overexpressed proteins identified in samples of the resistant phenotype, 5 were common to Cl27R and BZR (calpain like cysteine peptidase, 26 kDa hypothetical protein conserved, putative peptidase, peroxiredoxin and tyrosine amino transferase), 4 were common to Cl27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase, and putative nucleoside diphosphate kinase), 13 were exclusive for Cl27R, 9 exclusive for 17LER and 5 exclusive for BZR. Among the overexpressed proteins identified in samples of the susceptible phenotype, only prostaglandin F 2R synthase (PGF-2a) was common to BZS and 17WTS. Three proteins were exclusive for Cl9S, 3 exclusive for 17WTS and 2 exclusive for BZS. No common overexpressed protein was present in the three samples that were either Benznidazoleresistant or -susceptible. The 56 proteins identified were classified according to the “Functional Catalogue 2004” (FunCat).22 The proteins were grouped in 10 categories: (1) Metabolism (30.4%), (2) Hypothetical proteins (12.5%), (3) Cellular defense (12.5%), (4) Biogenesis of cell constituents (8.9%), (5) Interaction with cell environment (7.1%), (6) Synthesis of proteins (7.1%), (7) Transcription (5.4%), (8) Cellular communication (5.4%), (9) Destination proteins (5.4%), and (10) Unknown (5.4%). The proteins with the roles of transcription and destination were identified only in the resistant phenotype samples. On the other hand, no exclusive functional categories were identified for the proteins of the susceptible phenotype. A list of the proteins identified showing increased expression in the T. cruzi samples that were Benznidazole-resistant and Benznidazole-susceptible is presented in Tables 2 and 3, respectively. The Tables also show in which T. cruzi samples

Cl-27-5520

L-9a

Cl-27-13

2

3

4

BZR Cl27R

BR-6112 Cl-27-4328

6

Cl27R

BZR

Cl-27-6207 L-11

Cl-27-6107

Cl-27-5726

Cl-27-18 BR-16

8

9

10

11

Cl27R

Cl27R 17LER

Cl-27-3112

7

Cl27R

Cl27R

Cl-27-4118

Cl27R

17LER

Cl27R

17LER

T. cruzi sample

5

Cl-27-14 Cl-27-15 Cl-27-16

L-1205

1

spot

Orphan Orphan

5.58

17.23

10.89 orphan

10.14

3.41 2.75

21.01

Orphan Orphan Orphan

Orphan

orphan

2.83

3.21

ratio

specific 2-hydroxyacid dehydrogenase protein-putative

D-isomer

Dihydrolipoyl dehydrogenase

Cytochrome C oxidase subunit VI-putative

Cyclophilin A

Cofilin/Actin depolymerizing factor

Co-chaperone GrpE

Calpain-like cystein peptidase

Aspartate aminotransferase mitochondrial

Aromatic L-R hydroacid dehydrogenase

Arginine kinase

Actin

protein

GITQNEDDLACALR (1575.8)+carbamidomethyl

Metabolism

Metabolism

GAYEFWLDR (1156.5) LGAEVTVVEFAPR (1387.7) ETLTCEALLVSVGR (1547.8) RLDTYLVDPVR (1346.7)

Metabolism

Protein fate

Biogenesis of cellular components

Protein fate

DATFQQFVDSIDK (1513.7) FADESFAGK (971.5) NFGYAGSGFHR (1212.6) VVFELFADAVPK (1334.8) VFFDVSIGGQSAGR (1439.8) HVVFGQVLEGIEVVK (1652.9) DFEPLYDADAADFR (1644.7)

VTFGEDCDIK (1183.5) SYGISSFGK (945.4) TPASSEFPSGHISIVLK (1769.9) LILVSWNPDSGLPR (1566.9)

Metabolism

Protein synthesis

AELMDCAFPLLDR (1550.9) AELMDCAFPLLDR (1566.9) VVVSGAAGQVGYALLPLIAGGR (2068.4) EAIKDDAYLDGEFMTTVQQR (2330.3) NCIIWGNHSGTQVPDVNSATVR (2425.4) VVITHKPAVAFENVDIAILCGSFPAKPGTLR (3321.1) NFGLYGLR (939.5) VNLAVGVYR (990.6) VASCHTLGGTGALR (1399.7)+carbamidomethyl DDANRPFVLESVK (1489.8) AAGITLPPYTYYSPATK (1813.9) ELGECGSVLDWSHIER (1886.8)+ carbamidomethyl GFDEGNGLLFR (1224.8)

Metabolism

Cellular communication/ signal transduction mechanism


functional category

TFLVWVNEEDHLR (1657.8) FLQAAHACEFWPTGR (1790.8) + carbamidomethyl LGFLTFCPTNLGTTIR (1810.9)+carbamidomethyl NAAIFSEHGR (1101.6)

SEYDEAGPSIVHNK (1545.7) SLEGYPFNPCLK (1424.7)+ carbamidomethyl

AGFSGDDAPR (992.4)

sequence of identified peptides (m/z)

Table 2. Proteins with Increased Expression in T. cruzi Samples Resistant to Benznidazole Compared to Their Susceptible Pairsa

gi|71420052

gi|71422952 XP_811353

gi|71412035 XP_812294

XP_808220

gi|71407235 XP_821578 gi|71659715

gi|71407848 XP_803261 gi|71401098 XP_806100

XP_806365

gi|71412236

XP_808313

gi|998496

AAB33762

gi|71407949

gi|71409880 XP_806409

XP_807262

accession number


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BR-16

L-5

Cl-27--1330

L-3

Cl -27-9a

Cl-27-12

BR-19

L-7

18

19

20

21

22

23

24

Cl-27-8422

Cl-27-7403

BZR

Cl-27-1410

16

17

Cl27R

Cl-27-10

15

17LER

BZR

Cl27R

Cl27R

17LER

Cl27R

17LER

Cl27R

Cl27R

Cl27R

Cl-27-9b

14

BZR

Cl27R

Cl-27-17b

BR-7

17LER

L-7416

T. cruzi sample

13

12

spot

Table 2. Continued

Orphan

Orphan

Orphan

Orphan

Orphan

3.46

Orphan

3.02

3.22

orphan

5.6

Orphan

Orphan

Orphan

Orphan

2.53

ratio

Mitochondrial RNA biding-putative

Metallocarboxypeptidase

3 dehydrogenase-putative

L-threonine

Iron superoxide dismutase B

Iron superoxide dismutase A

IgE-dependent histamine releasing factor

Hypothetical protein Tc00.1047053504001.10

Hypothetical protein conserved

Hypothetical protein conserved (26 kDa)



HSP70 mitochondrial precursor

Glutamate dehydrogenase-putative

protein

Hypothetical protein

LMHLDCTVTPLPLAR (1736.9)+carbamidomethyl LYWQEPAK (1034.5)

Metabolism

LADEINASFGSFAK (1469.8) VFTEMIGTYYR (1379.8)

QHFVHFSALQTETGGFR (1962.0)

Transcription

Metabolism

Cell defense

LGFNWKDGCAPVFSPR (1850.9) GLFNQAAQHFNHTFYFR (2098.0) HHQGYVTK (969.5)

AVYNISGFSFTPEQLR (1829.1) SYLGIETLGNDR (1337.7) SLGVHEGQSLFAER (1529.8) SLTVGQEVEFEVASQDGR (1951.0)

Cell defense

Unknown

DQQMVLVK (976.5) AINEAVSVVEMLK (1402.8) APGFNEKYEEQQK (1567.7) HLLHDGEKEVSISALGK (1832.9) GSYIEVGGEDYGIAANVDEDAGEGAK (2586.2)

DGCAPVFSPR (1105.6)


LVDNWTAAVTLDK (1445.7) GQGWTGTLGFETACVLFK (1971.9) HLLHDGEK (948.5)



SVYLGNSDYAYR (1407.6) VIGFPTSTHVR (1213.7)

DFHEFAVYLNAE (1454.7) LMDQSLPVYDDVVTGSGR (1967.9) FSVDVEYAPR (1182.5)


Protein fate

Metabolism

functional category

SAVFQPIPR (1014.6)

FCQALVTELYR (1399.9) VSWVDDKGEVQVNR (1630.9) HIGPDTDVPAGDIGVGGR (1733.2) SQTFSTAADNQTQVGIK (1795.9)

VLWIYSLTAGVDVYR (1754.9) ESDFVVNILPGTEETKR (1934.0) FLGFEQTFK (1116.7)


gi|70876741

gi|70877737 CAG28318 gi|47076076 EAN90161

gi|2149612 EAN91053

AAC47549

gi|62529260

gi|71408210 AAX84936

XP_806523

gi|71407756

XP_806325

gi|71398774

XP_802643

gi|71404579

gi|71405542 XP_804983

gi|70878132 XP_805380

gi|71407386 EAN91412

XP_806165en

gi|2981039

AAC06213

accession number

research articles Andrade et al.

34

a

17LER

17LER

Cl27R BZR

BZR

17LER

17LER

ratio

Orphan

14.68

3.16 Orphan

Orphan

Orphan

Orphan

Orphan

Orphan Orphan 3.46 Orphan Orphan

Orphan

Orphan

2.54

3.91

Orphan

protein

Variant superface glycoprotein

Tryparedoxin peroxidase

Tyrosine amino transferase

TcC 31.14

TcC 1a22.6

Reductase-thymidylate synthase

Proteasome beta 5 subunit

Procyclin PARP-A

Peptidase M20/M25/M40

Peroxiredoxin (triparedoxin peroxidase)

P 22 protein precursor

Nucleoside diphosphate kinase-putative

Mitochondrial processing peptidase subunit beta

AGSITEEQAFK (1180.6) AGSITEEQAFKK (1308.7) SEYTDDSGSLTAKIWEPLGR (2225.2) YILALLVTVQASPAADLHDTK (2239.3) KAVALAYIPGADLSKLHELNDK (2266.3)

NLVVPGWR (940.6) EAVATWWR (1018.6) LTTTRPVEVYR (1334.8) LLVTNPSNPCGSNFSR (1875.9) GKDPNATFTSVADFETTVPR (2153.0) LLEEENVQVLPGTIFNSPGFTR (2460.5) QITVNDLPVGR (1194.6)+pyro-glu QITVNDLPVGR (1211.8) GFTPTLVEFYEK (1430.7) SYGVLKEEDGVAYR (1585.9) HGEVCPANWKPGDK (1594.8) ADSADKTGESR (1136.6)

GVHIWDDNGSRAFLDSR (1944.9) MSQLLLLLR (1086.7) THTYEVILALHR (1452.9) SIAQVLWGHVEGALYHMK (2055.1) YHIVYFDDFQTVAANDPSAR (2329.2)

ILANITYSYR (1213.6) NLTVEEACELAR (1404.6) FRPLPGR (842.5)

KASGTVLPDDLCPVR (1641.9) VFSVHYVIYGLAFR (1670.9) SIETRPRR (1014.5)

QPPLPPWDEGLHPQK (1721.8) GAADDGYAVFSSLTALAAVQK (2055.0) AENVYVLAWMSRK (1566.9)

KKHEDEEIVIR (1395.7) SEKPAKPELPAGWTLER (1908.9) DYGVLIEEQGISLR (1591.8)

VLLGATNPADSLPGTIR (1695.0)

Unknown

Cell defense

Metabolism

Unknown

Transcription

Metabolism


Cellular com/signal transduction mechanism

Metabolism

Cell defense

Transcription

Metabolism

ALDEIGGQLTVQVGR (1555.8) TFIAVKPDGVQR (1330.7)

functional category

Metabolism


AVGLLADVVR (1012.6)

Note: Proteins no. 36 is the same as no. 28. In bold are the proteins identified in more than one sample.

L-14

33

37

BR-18

32

L-2105

L-1

31

36*

L-6

30

Cl-27-4530 BR- 15

Cl- 27-9a

29

35

Cl-27R Cl27R BZR 17LER

BR-14 Cl-27-3 Cl-27-1330 BR- 10 L-7

Cl27R

BZR

BR-12

28*

BZR

27R

BR-10

Cl

Cl-27-7124

27

17LER

L-7118

26

Cl27R

T. cruzi sample

Cl-27-19

25

spot

Table 2. Continued

gi|13432319

AF335471

AAL37182 gi|17224953

AAN78337 gi|26105959 P33447 gi|1168606

gi|2833338 AAL82706 gi|18958725

Q27793

gi|51340809

AAC97957

gi|535936

XP_811760 gi|71421293 CAA85390

gi|4388655

gi|70886070 XP_820714 gi|71667532 XP_820245 gi|71666575 CAA06923

gi|71408412 EAN98863

XP_806612

accession number


research articles


2364


W-3

Cl-9-3539

Cl-9-3330 Cl-9-3326

BS-5304

W-9

W-1 W-2 W-6 BS- 3403

BS-5204

Cl-9-3539

2

3

4

5

6

7

8

9

a

17WTS

1

ratio

protein

60S ribosomal L-40

40S ribosomal protein S12-putative


ALASQANIDFVEVESR (1748.9)

YNWEKKVCR (1296.6) ACGHCSNLRMKK (1475.7) KACGHCSNLRMK (1475.7) 2.8 Cytocrome C oxidase subunit IV YHIVYFDDFQTVAANDPSAR (2329.1) SVLPHVDFASSYECLLFDADR (2441.2) 4.6 Cytocrome C oxidase subunit V VFLPPHLGDPHR (1384.7) 3.89 VVIPHIELVEYLAK (1622.9) GADIPDHVFQTPAVIER (1864.9) 2.57 HSP 70 FELSGIPPARP (1187.7) GDDKPVIQVQFR (1401.9) SDIHEIVLVGGSTR (1482.8) TTPSYVAFTDTER (1487.8) AVVTVPAYFNDSQR (1566.9) QSTKDAGTIAGLNVVR (1630.0) IINEPTAAAIAYGLDK (1659.9) NAVVTVPAYFNDAQR (1664.9) Orphan Hypothetical protein conserved CDVVNSDNVSKK (1378.7) MATAGGHHTTASTVR (1497.7) IIYVQHADSTPMNDWQR (2073.9) ENREIAAFLEEEVHQYVQQYRR(2807.3) Orphan Prostaglandin F2a synthase (PGF 2-r) WAIEAGYR (965.5) Orphan FIANPDLVER (1173.7) Orphan GGLIFLQLIHAGR (1394.9) 3.57 YDFEEADQQIR (1413.7) IQENFNVWDFK (1439.7) AIGWSNFEPHHLTELFK (1939.1) 4.14 Pyruvate dehydrogenase E1 beta subunit EGIEAEVINLR (1242.7) VFAPYNSEDAR (1268.6) DLEVASQPQVSDVLAVAR (1897.1) 2.83 TcC 31.34 EFGNEVTFRLEK (1468.9) AEIGARLTAWLPEK (1554.9) IFDEEHVQAEMKYVKCIR (2309.2) HITPEAIKACTDLNQLEEWSR (2511.4)

Orphan

2.68

In bold are the proteins identified in more than one sample.

Cl9S

BZS

BZS

17WTS

17WTS

BZS

Cl-9S

Cl-9S

17WTS

T. cruzi sample

W-2108

spot

functional category


Metabolism

Cell defense


Interaction with the cellular environment

Metabolism

Metabolism

Protein synthesis

Protein synthesis

Table 3. Proteins with Increased Expression in T. cruzi Samples Susceptible to Benznidazole Compared to Their Resistant Pairsa

AAC14088 gi|3063552

XP_811646 gi|71420903

BAC24024 gi|25006239|

AAC05480 gi|2952238

AAA30205 gi|162117

XP_821926 gi|71660419 XP_816256 gi|71655308

EAN96753 gi|70883865 P14795 gi|133106

accession number

research articles Andrade et al.

Proteomic Analysis of T. cruzi Resistance to Benznidazole these proteins were higher, how many times they are overexpressed, sequence of all identified peptides, their functional categories and accession number. The proteins calpain-like cysteine peptidase, cyclophilin A, hypothetical protein conserved (26 kDa), glutamate dehydrogenase, iron superoxide dismutase, nucleoside diphosphate kinase, putative peptidase, peroxiredoxin and tyrosine amino transferase were simultaneously identified in two of the three samples of the resistant phenotype (Table 2 and Figure 1). PGF-2a was identified in two of the three samples of the susceptible phenotype (Table 3, Figure 2 and Figure 3). The tubulins were identified in 16 spots from both Benznidazole-resistant and -susceptible T. cruzi samples, six spots being of alpha-tubulin and 10 spots of betatubulin (data not shown). Calpain-like cysteine peptidases ostensibly participate in a variety of cellular processes, including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways and apoptosis. T. cruzi contains cysteine, serine, threonine and metallo-proteases, the most abundant being Cruzipain, another cysteine protease expressed as a complex mixture of isoforms by the developmental stages of the parasite. It seems to be important in the host/parasite relationship. Inhibitors of Cruzipain kill the parasite and cure infected mice, thus, making the enzyme a promising target for the development of new drugs against Chagas disease.23 A cysteineprotease inhibitor protected dogs from cardiac damage during infection by T. cruzi.24 Tyrosine aminotransferase (TAT) catalyzes the transamination of tryptophan, phenylalanine and tyrosine. This particular enzyme may also be an alanine aminotransferase. This family includes the tyrosine aminotransferase found in animals and T. cruzi. TAT (EC: 2.6.1.5) is the first enzyme involved in tyrosine catabolism; however, it differs from other transaminases in some aspects.25 T. cruzi TAT gene has been characterized in 14 BZ-resistant and -susceptible strains and clones. A unique transcript of 2.0 kb and similar levels of TAT mRNA were observed in all parasite populations. The results suggest that TAT is not directly associated with the T. cruzi drug resistance phenotype.26 Peroxiredoxins (Prxs) represent a ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels, which mediate signal transduction in mammalian cells.27 T. cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses, in addition to those produced by endogenous processes. It has been shown that two distinct trypanothione-dependent enzymes located in the cytosol and mitochondrion catalyze the reduction of peroxides in T. cruzi.28 Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox-active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasitespecific features of cytosolic and mitochondrial peroxiredoxins of T. cruzi may be exploitable for drug development.29 Superoxide dismutases (SODs) catalyze the conversion of superoxide radicals to molecular oxygen. Their function is to neutralize toxic radicals produced inside the cells. Prx was enhanced in two out of three Benznidazole-resistant samples in vivo, while SOD was enhanced in Benznidazole-resistant samples in vivo (Cl27R) and in vitro (17LER). Prxs and SOD are components of the parasite oxidative defense system that may have a potential as chemotherapeutic targets. In addition

research articles to SOD, the Benznidazole-resistant parasites may also overexpress other antioxidant enzymes involved in the hydrogen peroxide metabolism, thus, detoxifying the parasite and making it Benznidazole-resistant. Consistent with this mechanism, SOD and Prx enzyme expressions were increased in metronidazoleresistant Entamoeba histolytica.30 The increased expression of the SOD and/or Prx in the Benznidazole-resistant parasites would provide efficient protection against the accumulation of superoxide anion and hydrogen peroxide. SOD-A gene was characterized from 25 samples of T. cruzi susceptible/resistant to Benznidazole, and the author showed an increased expression of the SOD-A enzyme in the T. cruzi population with in vitro-induced resistance to Benznidazole.31 Cyclophilin is the major high-affinity binding protein in vertebrates for the immunosuppressive drug cyclosporin A (CSA).32 It has also been encountered in other organisms. The anti-T. cruzi activity of cyclosporin A analogues was tested in vitro. The results showed that the enzymatic activity of cyclophilin was inhibited by all CSA derivatives, suggesting the involvement of cyclophilin in the observed trypanocidal effects.33 Glutamate dehydrogenases (EC: 1.4.1.2-4) (GluDHs) are enzymes that catalyze the NAD- and/or NADP-dependent reversible deamination of L-glutamate to form alpha-ketoglutarate.34 GluDHs are essential enzymes for the metabolism of amino nitrogen in organisms ranging from bacteria to mammals. In T. cruzi, the GluDH properties were studied in some detail.35 T. cruzi has a metabolism largely based on the consumption of amino acids, mainly, proline, aspartate and glutamate, which constitute the main carbon and energy sources of the T. cruzi epimastigotes forms. It has been suggested that amino acid metabolism may provide multiple as yet unexplored targets for therapeutic drugs in Chagas disease.8 Nucleoside diphosphate kinases (EC: 2.7.1.143-NDK) are enzymes required for the synthesis of nucleoside triphosphates (NTP) other than ATP. The purification and characterization of a soluble NDK in T. cruzi has already been reported.36 NDK are 17 kDa proteins that act via a ping-pong mechanism in which a histidine residue is phosphorylated by transfer of the terminal phosphate group from ATP. In the presence of magnesium, the phosphoenzyme can transfer its phosphate group to any NDP to produce NTP. The only protein that was overexpressed in two out of the three susceptible samples was PGF-2a. This protein, also named Old Yellow enzyme (OYE), is a NAD(P)H flavin oxidoreductase that in T. cruzi catalyzes prostaglandin PGF2a synthesis and reduction of some trypanocidal drugs.37 The DNA microarray analysis revealed that the levels of transcription of the PGF2a gene were 6-fold lower in a T. cruzi population with in vitroinduced resitance to Benznidazole (17LER) than in wild-type (17WTS). Furthermore, the PGF2a mRNA levels and the protein expression were lower in 17LER than in 17WTS. Thus, PGF-2a may be involved in Benznidazole metabolism by generating toxic drug anion radicals that cause the death of susceptible parasites.38 This escape mechanism has been observed previously in metronidazole-resistant (a nitroimidazole derivative similar to Benznidazole) E. histolytica in which reduced levels of flavin reductase activity have also been described.30 In the present discussion, nine proteins identified simultaneously in two of the three resistant samples were emphasized. Among these, five have been suggested by other authors as potential drug targets. However, another 27 proteins were Journal of Proteome Research • Vol. 7, No. 6, 2008 2365

research articles identified as overexpressed in the resistant phenotype, 13 being exclusive for Cl27R, nine exclusive for 17LER and five exclusive for BZR. Among the proteins identified in samples with the susceptible phenotype, only PGF-2a was common to BZS and 17WTS. Recently, a study was published analyzing the differential gene expression in T. cruzi strains selected as representative of the genetic variability of the parasite with regard to transient Benznidazole exposure or Benznidazole-induced resistance.39 Interestingly, the authors observed that each strain acts independently of its own genetic cluster when submitted to a drug stress. They hypothesized that the mechanisms involved in natural drug susceptibility are different from those involved in induced chemoresistance.38 Several mechanisms that act together to produce the chemoresistance status could be involved in Benznidazole resistance in T. cruzi. In addition, comparative studies of drug susceptibility between T. cruzi strains have found no correlation between parasite drug susceptibility in vitro and in vivo.40–42 The proteins identified in this study came from T. cruzi showing resistance to Benznidazole in vitro and in vivo. They were classified in various functional categories, while being grouped in the same phenotype, although they were only partly shared. Most of the proteins were exclusive for each particular sample. This diversity suggests that the mechanisms involved in induced, selected or natural resistance of T. cruzi to drugs may be different from one another. Altogether, the results presented herein have provided large-scale, protein-related information for the investigation of the mechanisms of T. cruzi resistance to Benznidazole This information may provide the basis for revealing new drug targets and leading to the rational development of new chemotherapeutic agents against Chagas disease.

Andrade et al.

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Acknowledgment. Financial support: Programa para Desenvolvimento de Insumos para a Sau ´ de da Fundaçaõ Oswaldo Cruz (PDTIS/FIOCRUZ), Conselho Nacional de Pesquisa (CNPq), Fundaçaõ de Apoio a Pesquisa de Minas Gerais (FAPEMIG) and Fundaçaõ de Amparo a Pesquisa do Estado de Rio do janeiro (FAPERJ).

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PR700659M


Proteomic Analysis of Trypanosoma cruzi Resistance to Benznidazole

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