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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.43 no.2 Santiago  2010

http://dx.doi.org/10.4067/S0716-97602010000200011 

Biol Res 43: 233-241, 2010

Article

Sera of Chagasic patients react with antigens from the tomato parasite Phytomonas serpens

 

Viviane K. Graça-de Souza1, Viviane Monteiro-Góes2, Patrício Manque3, Tatiana A.C.B Souza1, Paulo R.C. Corrêa1, Gregory A. Buck3, Andréa R. Ávila2, Lucy M. Yamauchi1, Phileno Pinge-Filho4, Samuel Goldenberg2, Marco A. Krieger2 and Sueli F. Yamada-Ogatta1,*

1Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid s/n, Campus Universitário, 86055-900, Londrina, Paraná, Brazil.
2Instituto Carlos Chagas, FIOCRUZ, Avenida Algacyr Munhoz Mader 3775, Cidade Industrial, 81350-010, Curitiba, Paraná, Brazil.
3Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284-2030 Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.
4Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid s/n, Campus Universitário, 86055-900, Londrina, Paraná, Brazil.

Dirección para Correspondencia


ABSTRACT

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These fagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identifed, resulting in 22 different putative proteins. The identifed proteins were classifed into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

Key terms: Phytomonas serpens, Trypanosoma cruzi, antigenic cross-reactivity, trypanosomatids.


 

INTRODUCTION

Some species of the family Trypanosomatidae are responsible for diseases that affect humans, animals (Leishmania and Trypanosoma) and plants (Phytomonas). Trypanosoma cruzi is the etiologic agent of Chagas disease (Chagas, 1909), an illness that affects millions of people, particularly in Latin America (Guhl and Lazdins-Helds, 2007). There is no vaccine against infection by T. cruzi and chemotherapy remains the only means of treatment for Chagas disease. Meanwhile, the drugs available for treatment are few and their effcacy is limited, mainly due to the development of resistance and the lack of host specificity (Filardi and Brener, 1987). Despite this scenario, efforts directed at the discovery of new antitrypanosomal agents and/or vaccines are insuffcient (Tarleton et al., 2007).

Species of the genus Phytomonas alternate their biological cycle between phytophagous insects of the order Hemiptera and many species of plants (Jankevicius et al., 1989; Batistoti et al., 2001).

Promastigote forms of the parasite colonize the digestive tract of these insects, cross the intestinal barrier and reach the salivary glands through the hemolymph. These microorganisms are transmitted by the host through the saliva when feeding. In the plant, promastigote forms, and rarely amastigote forms, are found in the phloem, lactiferous tubes, fruits or seeds (Jankevicius et al., 1989). However, only the trypanosomatids found in the phloem of some economically important plants cause fatal phytopathological conditions (Camargo, 1999).

The lack of specificity and extensive cross-reactivity among flagellates of the Trypanosomatidae family was previously observed.

One of the first lines of evidence of antigenic cross-reactivity among these trypanosomatids was reported by Noguchi (1926), when utilizing serologic tests to differentiate trypanosomatids of insects of the genus Herpetomonas from various species of Leishmania. Since then, several studies have demonstrated immunologic cross-reactivity between T. cruzi and monoxenous species of the family Trypanosomatidae. One approach broadly used in those studies was the utilization of sera from chagasic patients to detect common antigens among these trypanosomatids (Lopes et al., 1981; Monteón et al., 1997). It has also been demonstrated that polyclonal antibodies against cruzipain, the main cysteine peptidase from T. cruzi, recognize two peptides of 38 and 40 kDa in P. serpens. The 40 kDa protein is located on the cell surface, and both have cysteine peptidase proteolytic activity and other features similar to cruzipain (Santos et al., 2007).

Previous works of our research group have also demonstrated that P. serpens 15T shares antigens with T. cruzi. We have demonstrated that sera from patients with Chagas disease display a strong reactivity with P. serpens antigens by indirect immunofluorescence (IIF) assay. When the sera of Chagas disease patients were adsorbed with living P. serpens 15T promastigotes, a significant reduction in IIF titers to T. cruzi antigens was observed. Moreover, rabbit hyperimmune serum raised against living forms of P. serpens 15T or T. cruzi was able to recognize both trypanosomatids antigens by IIF assay. It was also observed that there was partial protection against infection of BALB/c mice immunized with living P. serpens 15T by the intraperitoneal or oral route and later challenged with a lethal inoculum of blood trypomastigotes of T. cruzi. Infected and previously immunized mice showed a reduction in blood trypomastigote counts and in mortality compared to non-immunized animals (Breganó et al., 2003). The protection afforded by P. serpens immunization is due to nitric oxide production, as observed from the higher parasitemia and mortality of inducible nitric oxide synthase (iNOS) deficient mice when compared to wild-type C57BL/6 mice. Moreover, immunized and infected wild-type mice showed fewer amastigote nests in their hearts, although immunization with P. serpens did not induce inflammation in the myocardium (Pinge-Filho et al., 2005). In view of these results, a proteomic-based study was carried out to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. The identified proteins may be further explored as antigens for vaccine or immunologic diagnosis studies.

METHODS

Microorganism

P. serpens 15T was isolated from Lycopersicum esculentum in Londrina, Paraná, Brazil. Promastigote forms of the flagellate were maintained at 28 oC in GYPMI medium [10.0 g/l Glucose, 2.5 g/l Yeast extract, 2.5 g/l Peptone, 20% Meat Infusion, 0.001% Hemin, 10.0 g/l KCl, 8.5 g/l NaCl pH 7.0 (Jankevicius et al., 1989)]. The genus identifcation was confrmed by PCR amplifcation of a genus-specific sequence of the spliced leader gene as described by Serrano et al., (1999).

Human sera

Sera from normal individuals and chronic chagasic patients were supplied by the Blood Bank of the University Hospital, Universidade Estadual de Londrina, Londrina, Paraná, Brazil. The presence of antibodies to T. cruzi was determined as described in Breganó et al., (2003). A pool of 14 sera from chagasic patients was used in a Western blot assay.

Protein solubilization

Aliquots of 2 x 108 log-phase promastigotes, cultivated in GYPMI medium, of P. serpens were harvested by centrifugation at 800 g for 10 min at 4 oC and washed three times in phosphate-buffered saline (PBS), pH 7.2. Total protein extracts were obtained by lysing the fagellates in a buffer containing 40 mM Tris base, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]1-propane sulfonate - CHAPS (General Electrics Life Sciences, São Paulo, Brazil) and 1 mM Na-tosyl-L-lysine chloromethyl ketone hydrochloride - TLCK (Sigma-Aldrich Co, São Paulo, Brazil). After 30 min incubation at room temperature with gentle agitation, lysed fagellates were centrifuged for 30 min at 13,000 g and the supernatant was kept at - 80 oC. The protein concentration was determined using the Bradford reagent, and the proteins were loaded for isoelectric focusing (IEF).

2D electrophoresis

For the first dimension, aliquots of solubilized proteins (500 µg) were diluted to a fnal volume of 250 µl in rehydration solution (8 M urea, 2% CHAPS, 40 mM dithiothreitol - DTT, 0.5% ampholytes 3-10, 0.002% bromophenol blue). This solution was applied to 13-cm IPG-strips (Amersham Biosciences, Uppsala, Sweden) with a non-linear separation range of gradient pH 3-10 by in-gel rehydration. After 10 h of rehydration at 20 oC, IEF was performed on an Ethan™ IPGphor™ unit (Amersham Biosciences), at the same temperature, with the following conditions: 500 V for 1 h, 1000 V for 1 h, 4000 V for 1 h, 6000 V for 2 h, 8000 V for 9 h and 100 V for 1h. Before second dimension electrophoresis, proteins were reduced and alkylated by incubation of the strips as follow: 15 min in equilibration buffer (6 M urea, 50 mM Tris-HCl pH 8.8, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 5.0 mg/ml DTT, and for an additional 15 min in the same buffer containing 12.5 mg/ml iodoacetamide instead of DTT. Equilibrated IPGphor strips were separated across 12.5% SDS-PAGE gels, using a vertical system (Hoefer™ SE 600 Ruby™, Amersham Biosciences), at 30 mA/gel constant current at 10 oC until the dye front reached the lower end of the gel. Proteins were visualized by silver staining according to the following procedures: gels were fxed in 12% v/v acetic acid, 50% v/v ethanol and 0.5 ml/l formaldehyde (37% v/v) for 30 min. After 3 x washes (5, 10 and 15 min) in 50% v/v ethanol, the gels were incubated in 0.02% w/v sodium thiosulfate for 30 s followed by three 5-min washes in water. Another 30-min incubation was carried out in a solution containing 0.2% w/v silver nitrate and 0.75 ml/l formaldehyde. After 3 x washes in water, development was performed by incubation in a solution containing 3% w/v sodium carbonate, 2% w/v sodium thiosulfate and 0.5 ml/l formaldehyde. The reaction was stopped with 50% v/v ethanol and 12% v/v acetic acid.

Western blotting

Parasit e pro te in ex tr a c ts sep a rat ed by electrophoresis, as above, were electrotransferred onto Hybond-C membranes (GE Healthcare Life Sciences, São Paulo, Brazil) according to standard procedures (Towbin et al., 1979). The membranes were blocked by incubation in 5% skim milk powder in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 0.05% Tween-20. The blots were probed using 1: 80 dilution of chagasic sera. Bound antibodies were detected with 1: 7,500 dilution of phosphatase alkaline-conjugated anti-human IgG (Promega, Wisconsin, USA) and developed with 5-bromo-4-chloro-3-indoxyl phosphate and nitroblue tetrazolium.

Protein digestion and mass spectrometric analysis

Some of the P. serpens protein spots that reacted with sera from Chagas' disease patients were manually excised from the gel and destained for 15 min with a freshly prepared solution of 30 mM potassium ferricyanide, 100 mM sodium thiosulfate. After several washings in water, the gel pieces were further washed 2 x 30 min in 0.1 M ammonium bicarbonate, pH 8.3, 50% v/v acetonitrile solution. The gels were dried using a Savant Speed Vac" evaporator (TeleChem International, Inc. Sunnyvale, USA) and were rehydrated in 15 µl of 50 mM ammonium bicarbonate. The proteins were enzymatically digested overnight at 37 oC with sequencing grade porcine trypsin (0.5 µg/ml, Promega). The tryptic peptides were extracted twice with 40 µ l of 60% v/v acetonitrile, 0.1% v/v TFA (trifuoroacetic acid) solution in a sonicator for 10 min. The extracts were concentrated under vacuum to an approximate volume of 10 µl. The resulting tryptic peptides were desalted on C8 cartridges (Michrom BioResources Inc., California, USA) and subjected to 2D Nano LC/MS/MS analyses on a Michrom BioResources Paradigm MS4 Multi-Dimensional Separations Module, a Michrom Nano Trap Platform and a LCQ Deca XP plus ion trap mass spectrometer. The mass spectrometer was operated in data-dependent mode and the four most abundant ions in each MS spectrum were selected and fragmented to produce tandem mass spectra. The MS/MS spectra were recorded in the profle mode.

Database search

Proteins were identified by searching MS/MS spectra against the National Center for Biotechnology non-redundant and GeneDB databases, and its reverse complement using Bioworks v3.2. Peptide and protein hits were scored and ranked using the probability-based scoring algorithm incorporated in Bioworks v3.2 and adjusted to a false positive rate of < 1%. Only peptides identified as possessing fully tryptic termini with cross-correlation scores (Xcorr) greater than 1.9 for singly charged peptides, 2.3 for doubly charged peptides and 3.75 for triply charged peptides were used for peptide identifcation. In addition, the delta correlation scores (D Cn) were required to be greater than 0.1, and for increased stringency, proteins were accepted only if their probability score was <0.0005 and the result was repeated with the same spot picked from a parallel gel.

RESULTS

2D electrophoresis and Western blotting analyses

Proteins from whole-cell lysates of log-phase promastigotes of P. serpens 15T were separated by 2DE in pH range 3-10. Approximately 150 spots were detected in the silver-stained gels. There was homogeneous distribution of the peptides in relation to isoelectric focusing, but with the greatest concentration of peptides with an estimated molecular mass above 30 kDa (Fig. 1A). The 2DE protein spot profles obtained from 3 independent experiments were highly reproducible in terms of the total number of protein spots and their positions, and the reactivity with sera from chagasic patients.


Despite the intrinsic limitation of the Western analysis used here, more than 50 polypeptide spots reacted strongly with the sera from patients with Chagas disease (Fig. 1B), and most of them showed a molecular mass range of 25-100 kDa. Although some reactive polypeptides were detected in Western assay, they could not be visualized with the 2DE gels. One possible limitation for spot detection resides in the sensitivity of the silver staining method used here, which is compatible with MS analysis. Healthy human sera were also tested, and the result is shown in Fig. 1C. A total of 31 tryptic peptide samples that strongly reacted with sera of chagasic patient and clearly resolved on 2DE gels (shown in the Figs. 1A and 1B) were selected and analyzed by MS/MS.

Protein identifcation

Automatic and blast searching allowed the identifcation of 28 spots, resulting in 22 different putative proteins (Table 1). One limitation of this approach is that few genes and proteins of Phytomonas spp. have been sequenced and characterized to date, thus we assigned the protein identity by homology with other trypanosomatids. Pappas et al. (2005) generated expressed sequence tags (EST) from a cDNA library of P. serpens 10T. Most of the protein identifed in our study matched with the deduced amino acid sequence from these ESTs (Table 1), validating previous gene predictions. The results of this analysis showed that two proteins, from spots 6 and 18, identifed in this analysis are ortholog genes annotated as hypothetical in L. braziliensis and T. cruzi databases, respectively. Some proteins were detected in more than one spot: cystathionine beta synthase (4 and 27), fructose-bisphosphate aldolase (9 and 11) glyceraldehyde-3-phosphate dehydrogenase (10 and 12), enolase (16 and 24), and malic enzyme (29, 30 and 31).



Biological functions of the identifed peptides

According to the gene ontology annotation of biological processes, the identifed proteins of P. serpens 15T could be classified into 8 categories (Table 2). Of the 22 proteins identifed, 7 (31.8%) were associated with carbohydrate metabolism. Protein synthesis represented the second most Alcohol dehydrogenase; Enolase; Fructose-biphosphate aldolase, glycosomal; Glyceraldehyde-3-phosphate dehydrogenase, glycosomal; Isocitrate dehydrogenase, mitochondrial; Malic enzyme; 2-Oxoglutarate dehydrogenase 60S ribosomal protein L7; 60S ribosomal protein L13; 60S ribosomal protein P0; 60S acidic ribosomal protein P2; 40S ribosomal protein SA; 40S ribosomal protein S4; 25 kDa translation elongation factor 1-beta abundant category, where 6 (27.3%) different proteins were identifed. The other proteins assigned in this study are associated with various activities, including: amino acid and lipid metabolism, nucleosome assembly, proteolysis, endomembrane system, and intracellular protein/RNA transport.


DISCUSSION

In this study, 22 putative proteins from P. serpens 15T were identifed and most of them were clustered into a cellular metabolic process category. As there is little Phytomonas genomic information, the mass spectra derived from reactive polypeptides were searched against all available trypanosomatid sequences at the time to maximize the possibility of identifying the proteins. But we cannot exclude the possibility of some protein misidentifcation. Because of the hydrophobic nature of membrane proteins, they are usually under-represented in proteomic analyses (Santoni et al., 2000). Indeed, in previous 2-D electrophoresis mapping of whole-cell lysates of T. cruzi, membrane proteins were not identifed (Paba et al., 2004, Andrade et al., 2008). This could explain why membrane proteins of P. serpens were not identifed in this study.

Three enzymes of the glycolytic pathway were identified in this study, fructose-bisphosphate aldolase, glycosomal glyceraldehyde-3-phosphate dehydrogenase and cytosolic enolase. We also identifed one peptide as isocitrate dehydrogenase and three as mitochondrial malic enzyme. Glyceraldehyde-3-phosphate dehydrogenase is a glycosomal enzyme that has also been detected in the cytosol of trypanosomatids (Hannaert et al., 1998), which could explain the isoenzymes found in this study. The presence has been reported of two isoforms of malic enzymes in Phytomonas sp., as well in T. cruzi, a mitochondrial and a glycosomal isoenzymes (Cannata et al., 1979; Uttaro and Opperdoes, 1997). It is probable that at least three isoforms of malic enzymes are expressed in P. serpens 15T, as indicated by their different isoelectric points. We also cannot exclude the possibility that some protein spots may have resulted from protein degradation.

Like other trypanosomatids, species of Phytomonas degrade carbohydrates via glycolysis, and the first reactions of the classical Embden-Meyerhof pathway occur inside glycosomes (Sanchez-Moreno et al., 1992). Since Phytomonas spp. lacks a functional citric acid cycle (Sanchez-Moreno et al., 1992; Chaumont et al., 1994) and the genes for cytochrome mediated respiration are missing in the maxicircle kinetoplast DNA (Maslov et al., 1999; Nawathean and Maslov, 2000; Opperdoes and Michels, 2008), they depend on glycolysis to obtain energy. One important aspect of this metabolism is that enzymes from the glycolytic pathway have been selected as targets for drugs against members of Trypanosomatidae family, including fructose-bisphosphate aldolase (Dax et al., 2006) and glyceraldehyde-3-phosphate dehydrogenase (de Marchi et al., 2004). The same approach has been used for sterol biosynthesis since trypanosomatids synthesize ergosterol and related 24-alkylated sterols, whose structure and biosynthetic pathway show differences compared to that for cholesterol found in mammalian cells. One principal difference is the reaction catalyzed by S adenosyl-L-methionine C24-delta sterol methyltransferase, which introduces a C24-methyl group to the ergosterol and stigmasterol side chains (Roberts et al., 2003). Inhibitors of this enzyme have been shown to have antiproliferative effects in several trypanosomatids (Lorente et al., 2004). Another potential target for the development of new drugs in the treatment of diseases caused by trypanosomatids is the metabolic pathway of sulfur-containing amino acids. The cystathionine beta synthase catalyzes the trans-sulfuration reaction of homocysteine to cysteine, a sulfur-containing amino acid that plays an important role in the structure, stability and catalytic functions of many proteins. T. cruzi cystathionine beta synthase lacks the 90-120 amino acids in the carboxyl terminal, is not activated by the presence of S-adenosylmethionine, and does not contain heme, which differs from the corresponding mammalian enzymes (Nozaki et al., 2001).

Of particular interest is a small phosphorylated protein located at the ribosome "stalk," the 60S acidic ribosomal protein P. The biological function of eukaryotic P proteins is still unclear. Their participation in protein synthesis has been shown, and these proteins may also be involved in transcription and DNA repair processes. In addition, P proteins have been implicated in several diseases associated with the immune response, including systemic lupus erythematosus, allergies caused by some flamentous fungi and protozoan infections (Tchórzewski, 2002). Antibodies to ribosomal P proteins are prevalent in patients with chronic Chagas heart disease and they are directed against the carboxy-terminal region of the T. cruzi proteins (Levin et al., 1989). These antibodies were able to cross-react with the acidic motif present on the second extracellular loop of human cardiac b1 adrenergic receptor (Smulski et al., 2006). Anti-P antibodies are believed to take part in the induction of heart dysfunctions, such as arrhythmias and/or other electrical disorders (Lopez Bergami et al., 2001). Corroborating this, mice immunized with the recombinant T. cruzi P protein (TcP2b) demonstrated a strong response against the C-terminal region of this protein and developed lethal supraventricular tachycardia (Lopez Bergami et al., 1997). As mentioned, mice previously immunized with live P. serpens 15T and infected with T. cruzi did not display inflammation in the myocardium (Pinge-Filho et al., 2005). Altogether, these data open perspectives for exploring the role of the 60S acidic ribosomal protein P from P. serpens in the protection against T. cruzi infection.

The other proteins assigned in this study are associated with various important activities in trypanosomatids. A protein involved in proteolytic activity, the proteasome alpha 2 subunit, was identifed in this study. De Diego et al., (2001) have reported that the ubiquitin-proteasome pathway has an essential role in protein turnover during T. cruzi differentiation. Calmodulin, a universal Ca2+-binding protein that can modulate the activity of other proteins, has been shown to have a role in T. cruzi differentiation (Lammel et al., 1996) and motility (Ridgley et al., 2000). Finally, one peptide was identifed as a GTP-binding rtb2 protein by homology with the ortholog protein in T. brucei. In this parasite, the protein has homology to Ran, a member of G protein superfamily, which is an essential element in the transport of proteins and RNA across the nuclear membrane (Field et al., 1995).

Promastigo te forms of P. serp ens, a trypanosomatid isolated from edible tomatoes, are easily cultivated in in vitro conditions (Batistoti et al., 2001). This non-human pathogenic flagellate is highly immunogenic and expresses important protein homologs of trypanosomatids that cause human infections. The antigenic and metabolic pathway similarities between P. serpens and T. cruzi raise important questions about obtaining and utilizing antigens from microorganisms that are innocuous to humans for immunologic diagnosis of Chagas disease, as well as for the development of new strategies of immunization against infection by T. cruzi, and studying target molecules for the development of new chemotherapeutic agents against trypanosomatids. Therefore, as pointed out by Santos et al., (2007) this fagellate is useful as a model for the immunological and biochemical studies among the Trypanosomatidae family. Now, we are faced with the task of trying to understand the signifcance of these proteins in T. cruzi cross-reactivity and P. serpens biology.

ACKNOWLEDGMENTS

This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Araucária -Paraná and Pro-Reitoria de Pesquisa e Pós Graduação (PROPPG) of Universidade Estadual de Londrina (UEL). This work was part of the Ph.D. dissertation of V.K. Graça-de Souza. We thank Dr. A. Leyva for English editing of the manuscript and Ediel Clementino da Costa for technical support.

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* Corresponding author: Sueli Fumie Yamada-Ogatta, Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de Microbiologia. Rodovia Celso Garcia Cid, PR 445, km 380. CEP 86055-900. Tel: +55-43-3371-4297; fax: +55-43-3371-4788. e-mail: ogatta@uel.br

Received: November 18, 2009. Accepted: December 12, 2009.

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