ORIGINAL PAPER

In silico approach for the identification of immunologicalproperties of enolase from Trypanosoma cruzi and its possibleusefulness as vaccine in Chagas disease

Alejandro Carabarín-Lima & Olivia Rodríguez-Morales &

María Cristina González-Vázquez & Lidia Baylón-Pacheco &

Pedro A. Reyes & Minerva Arce-Fonseca & José Luis Rosales-Encina

Received: 18 June 2013 /Accepted: 4 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract Nowadays, Chagas disease is a major health prob-lem in Latin America that has been disseminated also intonon-endemic countries. Currently, a vaccine against Chagasdisease does not exist. In the present study, the gene encodingTrypanosoma cruzi enolase (TcENO) was amplified, cloned,and sequenced and the recombinant protein was purified. Weused in silico and an experimental assay to investigate theimmunological role of TcENO. The in silico assays showedthat TcENO sequence contains characteristic motifs of eno-lase; additionally, a transmembranal regionwas identified, andthis could indicate the potential membrane localization of

TcENO. Moreover, both B lymphocyte and cytotoxic T lym-phocytes (CTL) predicted epitopes were localized; these re-sults suggest the possibility that TcENO can develop bothhumoral and cellular immune responses. Furthermore, thepresence of antibodies was verified by western blot assays,showing that the purified recombinant protein was detected bysera from experimentally infected mice and sera of patientswith Chagas disease. These results indicate that TcENO isimmunogenic and could be used as a vaccine candidate.

Introduction

Chagas disease is caused by the protozoan parasiteTrypanosoma cruzi, which is a serious threat to human healthin Latin America and is one of the most important emerginghealth problems in Europe and the United States of America(USA) (Carod-Artal and Gascon 2010). In accordance withthe World Health Organization’s (WHO) report released in2012, it is estimated that 10 million people are infected and>25 million people are at risk of infection in endemic coun-tries (WHO 2010). It is assessed that more than 300,000infected patients live in the USA (Bern and Montgomery2009). Up to 40 % of chronically infected individuals developcardiac alterations and up to 10 % develop digestive, neuro-logical or mixed alterations, resulting in considerable morbid-ity and mortality (WHO 2006).

Nowadays, nifurtimox and benznidazole are the only li-censed drugs with proven efficacy against Chagas disease;both drugs have significant activity during the acute phase ofthe disease, causing a parasitological cure in up to 80 % of thepatients if they are treated during this early infection phase. Inspite of this observation, these drugs have an important

A. Carabarín-Lima :O. Rodríguez-Morales : P. A. Reyes :M. Arce-Fonseca (*)Departamento de Biología Molecular, Instituto Nacional deCardiología “Ignacio Chávez”, Juan Badiano No. 1, Col. SecciónXVI, Tlalpan, C.P. 14080 México, D. F., Méxicoe-mail: mini_arce@yahoo.com.mx

M. C. González-VázquezDepartamento de Biología Celular, Centro de Investigación y deEstudios Avanzados del I.P.N., México, D.F. 07360, México

L. Baylón-Pacheco : J. L. Rosales-EncinaDepartamento de Infectómica y Patogénesis Molecular, Centro deInvestigación y de Estudios Avanzados del I.P.N., México,D.F. 07360, México

Present Address:A. Carabarín-LimaCentro de Investigaciones en Ciencias Microbiológicas, BeneméritaUniversidad Autónoma de Puebla, Puebla 72550, México

Present Address:M. C. González-VázquezLaboratorio de Neuroendocrinología, Instituto de Fisiología,Benemérita Universidad Autónoma de Puebla, Puebla 72570,México

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limitation during the chronic phase of the disease since theyhave shown very low trypanocidal activity in whichmore than80 % of patients are not cured. The reason for this markeddifference in the antiparasitic activity between acute andchronic phases is unknown (Cancado 1999). Importantly,parasites naturally resistant to chemotherapy have been re-ported in several regions of Latin America (Camandarobaet al. 2003), and a vaccine does not exist. For this reason, itis very important to find alternatives to obtain a protectivevaccine, where reverse vaccinology represents a good strategyto this aim. In this respect, these analyses have encouraged theidentification of proteins with potential for development ofvaccines for human pathogens such as Staphylococcus aureus(Etz et al. 2002), Chlamydia pneumoniae (Montigiani et al.2002), Streptococcus suis (Liu et al. 2009) and Echinococcusgranulosus (Gan et al. 2010; Li et al. 2012).

Moreover, several studies have shown that enolase, a mul-tifunctional protein, is capable of generating a protectiveimmune response against Plasmodium falciparum (Pal-Bhowmick et al. 2007), Candida albicans (Li et al. 2011)and Ascaris suum (Chen et al. 2012).

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) isa metalloenzyme that catalyses the reversible dehydration ofD-2-phosphoglycerate (PGA) to phosphoenolpyruvate (PEP)in both glycolysis and gluconeogenesis. Enolases from avariety of sources, ranging from bacteria to higher vertebrates,show highly conserved amino acid sequences, which isparticularly apparent at the catalytic site and has led inconsequence that enzymes from diverse species share manysimilar kinetic properties. Enolase requires magnesium forboth catalysis and dimmer stabilization (Pancholi 2001).Furthermore, enolase acts as a plasminogen receptor on thecell surface of certain pathogens (Bergmann et al. 2001;Pancholi and Fischetti 1998) and has been implicated innuclear functions in protozoans (Ferguson et al. 2002; Labbeet al. 2006), plants (Lee et al. 2002) and animal cells (Feo et al.2000; Subramanian andMiller 2000). Enolase is also involvedin stress response (Wilkins et al. 2002), vacuolar fusion pro-cesses (Decker and Wickner 2006) and molecular chaperon-ing functions (Brandina et al. 2006; Entelis et al. 2006).

To evaluate whether T. cruzi enolase could be animmunogenic protein to develop a potential novel vac-cine candidate against Chagas disease, in the presentstudy, the T. cruzi enolase gene was cloned, the recom-binant protein (rTcENO) was expressed and both bioin-formatics and experimental trials were performed. Thebioinformatics assays showed that TcENO sequence hasepitopes that could be recognized by both B cells andCTL; moreover, the western blot assay showed that seraof humans with Chagas disease and T. cruzi experimentalinfected mice sera reacted against rTcENO. With theseresults, we concluded that rTcENO is a potential candi-date to develop a vaccine against Chagas disease.

Materials and methods

Parasites, RNA extraction and first strand cDNA synthesis

The T. cruzi H8 strain [MHOM/MX/1992/H8 Yucatán(T. cruzi)] used in this work was a kind gift from Dr. JorgeE. Zavala Castro from Centro de Investigaciones RegionalesDr. Hideyo Noguchi, Universidad Autónoma de Yucatán,Mérida, Yucatán, México.

T. cruzi epimastigotes forms were lysed in Trizol® (LifeTechnologies, Carlsbad, CA, USA), and total RNA was ex-tracted according to the manufacturer’s instructions. Briefly,after three rounds of freeze–thawing the T. cruzi/Trizol® prep-aration in liquid nitrogen and at 37 °C, respectively, RNAwasprecipitated with isopropyl alcohol and washed with 70 %ethanol. After centrifugation, the pelleted RNA was brieflydried at room temperature and suspended in RNase/DNase-freewater. The first strand cDNAwas synthesized from 1μg ofthis RNA using the SuperScript® II Reverse Transcriptase kit(InvitrogenTM by Life Technologies, Carlsbad, CA, USA) with1 nmol oligo dT(12-18) per reaction.

TcENO gen amplification and sequencing

The TcENO gene was amplified by polymerase chain reaction(PCR) using a forward primer (5′-GGTACCATGACAATCCAAAAGGTTCACG-3 ′) and a reverse primer (5 ′-GCCGAAGCTTACCACAAGGTCAGC-3′) that introducedboth Kpn I and Hind III restriction sites (underlined), respec-tively. These primers were used in the amplification of an1,162 bp fragment of the complete gene. The PCR reaction(25 μL) contained 1 μg of template, 2 mMMgCl, 0.2 mM ofeach dNTP, 1X PCR buffer and 2.5 U of Taq DNA polymer-ase (NEB, MA, USA). Amplification conditions were 95 °Cfor 1 min, 55 °C for 1 min, and 72 °C for 1 min for 35 cycles,and 72 °C for 10 min. The PCR product obtained was clonedin pCR®4-TOPO vector (InvitrogenTM by Life Technologies,Carlsbad, CA, USA) and transformed in Escherichia coliDH5α cells. The recombinant positive clone was picked upand sequenced. The sequence was submitted to GenBank(access number: KC862322).

In silico analysis

Homology of TcENO gene with reported enolases sequenceswas determined using Basic Local Alignment Sequence Tool(BLAST) program at the National Center for BiotechnologyInformation (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST).The most homologous sequences to TcENO and thosesequences that have already been used as vaccines in severalorganisms were selected. Amino acid sequences were alignedusing ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/)and neighbor-joining analysis was performed employing the

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Mega5 package (Tamura et al. 2011). The human sequenceswere used as an out-group. Gaps were treated as pairwisedeletions; amino acid distances were calculated using thePoisson model, and branch support was estimated using boot-strap analysis (10,000 bootstraps). In addition, the T. cruzienolase sequence was analyzed to calculate its molecularweight, isoelectric point and stability by ProtParam (http://ca.expasy.org/tools/protparam/html), post-translation modifi-cation sites by MotifScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan), functional domain and key sites by InterProScan (http://www.ebi.ac.uk/InterProScan/), transmembranalregion and topology structure by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), secondarystructure and disulfide bond by Predictprotein (http://www.predictprotein.org/), linear B cell epitopes by BepiPred 1.0Server (http://www.cbs.dtu.dk/services/BepiPred/) and CTLepitopes by NetCTL 1.2 Server (http://www.cbs.dtu.dk/services/NetCTL/).

Subcloning, expression and purification of TcENO

The TcENO DNA sequence was digested from recombinantpCR®4-TOPO plasmid by Kpn I and Hind III double enzymedigestion and subcloned into the prokaryotic expression vec-tor pRSET-B (InvitrogenTM by Life Technologies, Carlsbad,CA, USA). The correct subcloning of TcENO gene wasconfirmed by restriction enzyme analysis and sequencing(results not shown). E. coli BL21(DE3) pLysS culture harbor-ing the pRSET-B::TcENO plasmid was grown to mid-logarithmic phase at 37 °C. The expression of the recombinantfusion protein was induced by using isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 1 mM forovernight (14 h) under agitation at 30 °C in Luria–Bertani(LB) Medium containing ampicillin (100 mg mL−1). Afterinduced expression, the bacterial cells were collected by cen-trifugation at 8,000×g for 15 min at 4 °C. The His-taggedTcENO protein was purified as follows. Briefly, the finalpellet was resuspended in 20 mM sodium phosphate(pH 7.4), 500 mM NaCl, 4 M guanidine hydrochloride, and10 mM imidazole and disrupted by sonication, followed bycentrifugation at 12,000×g for 15 min at 4 °C; the supernatantwas obtained and recombinant fusion protein was purifiedthrough Ni-NTA-agarose affinity chromatography (Qiagen,Hilden, Germany) according to the manufacturer’s instruc-tions. The rTcENO protein was eluted with a 8 M urea buffer,then the purified protein was dialyzed in phosphate-bufferedsaline (PBS, pH 7.4) containing decreasing amounts of urea(4, 1, 0.2 M, and no urea). The purified rTcENO wasresolved on 12 % sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) to analyze its expression.Aliquots of the purified protein were stored at −80 °C,and the protein concentration was determined using theBradford protein assay.

Identification of rTcENO by western blot analysis

Purified recombinant rTcENO was resolved (10 μg per lane)by 12% SDS–PAGE and electrotransferred onto nitrocellulosemembrane at 70 V for 1 h. The membranes were blocked with5 % (w/v) skim milk in PBS, pH 7.4 for 1 h at 37 °C, washedthree times with PBS containing 0.05 % Tween 20 (PBS-T)10 min/per time and then incubated overnight at 4 °C withanti-histidine, anti-T. cruzi mouse sera or anti-T. cruzi humansera (1:500 dilution in 2 % skim milk-PBS). As negativecontrols either healthy human or healthy mouse sera (1:500in 2 % skim milk-PBS) were used. After the washing proce-dure, the membranes were subsequently incubated withgoat anti-mouse IgG alkaline phosphatase-conjugated sec-ondary antibody (Zymed Labs, San Francisco, CA, USA)or goat anti-human IgG alkaline phosphatase-conjugatedsecondary antibody (Zymed Labs, San Francisco, CA,USA) diluted at 1:5,000 for 1 h at 37 °C, and the blotwas revealed with nitro-blue tetrazolium chloride (NBT)(Sigma, St. Lous, MO, USA) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) (Sigma, St. Louis,MO, USA).

Results

Amplification, cloning and sequencing of T. cruzi enolasecDNA

Amplification of T. cruzi enolase cDNA by RT-PCR resultedin a PCR fragment of 1,162 nucleotides (Fig. 1a) encoding ahypothetical protein of 385 amino acids, with a theoreticalmolecular weight and pI of 41,557.23 Da and 5.23, respec-tively. This amplified PCR fragment was subcloned in thepRSET-B vector and then digested withKpn I andHind III in adouble enzyme reaction (Fig. 1b).

In silico assays with the enolase sequence obtained

A BLAST search of the Pubmed database with the deducedamino acid sequence of TcENO revealed a strong identity withthe homologous gene of T. brucei (81.03 %), as well as withthe heterologous genes of Leishmania infantum, L. donovaniand L. mexicana (73.50, 73.24 and 72.72 %, respectively).With other non-Trypanosomatid enolases, the sequence ofTcENO showed identity percentages ranging from 52.2 to54.8 % (Fig. 2). Moreover, in silico analyses of the deducedamino acid sequence did not reveal a signal peptide orglycosyl-phosphatidyl inositol anchors but they did show atransmembranal domain located in its N-terminal Gly105-Ala122 (18 residues) (Fig. 2, in shadowed gray). TcENOsequence included the four potential Mg2+ binding sites, suchas Ser40, Asp239, Glu287, Asp314 (Fig. 2, letters in bold and

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arrowhead). Furthermore, six potential sites for substrate bind-ing (His156, Glu204, Lys339, His367, Arg368 and Ser369)(Fig. 2, letters in bold), crucial active-site residues for enzymeactivity, were localized. Moreover, the amino acid sequenceLeu336-Ala349 (14 residues) (Fig. 2, underlined) was predict-ed as the signature of enolase, also a characteristic enolase“fingerprint” motif (366SHRSGETED374) (Fig. 2, boxed andshown by pentacle) and a putative plasminogen binding motif(245TYDENKKQY253) (Fig. 2, boxed) were detected, thelatter based in the motif previously reported for L. mexicana(Vanegas et al. 2007).

Furthermore, the alignment and the phylogenetic analysis(Fig. 3) of these enolases sequences indicated that theseproteins were grouped within two major clades, corre-sponding to the genus Trypanosoma (support value99 %) and Leishmania (support value 100 %). A thirdclade could be established that would include all theenolases sequences (support value 91 %).

Immunological properties of enolase sequence

In the bioinformatics assays the presence of 12 linear B cellepitopes and seven CTL epitopes (two epitopes overlapping;aa266−274 and aa273−281) in the protein sequence were ob-served, suggesting that TcENO could be immunogenic(Fig. 4). Moreover, the crucial residues His156 and Glu204

involved in substrate binding (Fig. 4, boxed sites) and twoMg2+-binding sites (Fig. 4, letters in bold and arrowhead)Ser40 and Glu287 were located in a linear B cell epitopes.Asp239, a Mg2+-binding site was located in a CTL epitope.Furthermore, two substrate binding sites Arg368 and Ser369were located in both B cell and a CTL epitopes, respectively.

Expression, purification and identification of rTcENO

The T. cruzi enolase cDNA was cloned into the expressionvector pRSET-B, and the recombinant vector was expressed inE. coliBL21(DE3) strain. After IPTG expression induction inE. coli, the hexahistidine-tagged recombinant protein waspurified under denaturing conditions (8M urea) using a nickelaffinity chromatography. Purified protein (rTcENO) showed aband between 66.2 and 45.0 kDa in the SDS-PAGE (Fig. 5),which is in accordance with the estimated molecular weight of46.01 kDa of the recombinant protein. In western blot assays,the purified protein was recognized by anti-His6x monoclonalantibodies (Fig. 6, lane 1), indicating the correct expression ofrTcENO protein. Interestingly, T. cruzi experimental infectedmice and T. cruzi-infected patient sera recognized the band of46.01 kDa (Fig. 6, lanes 2 and 4, respectively). Moreover, weobserved that either healthy mice or healthy human sera usedas negative controls did not show any recognition for thecorresponding band to rTcENO (Fig. 6, lanes 3 and 5,respectively).

Discussion

Chagas disease caused by Trypanosoma cruzi continues as amajor health problem in South, Central and North America(Bern andMontgomery 2009; Guzman-Bracho 2001; Voelker

�Fig. 2 Alignment of the amino acid sequences of the enolases. TcENO(KC862322), Trypanosoma brucei (XP_822542.1), Leishmania infantum(XP_001464303.1), L.donovani (ACE74540.1), L. mexicana(XP_003873490.1), Homo sapiens B beta-enolase isoform 1(NP_001967.3), H. sapiens G muscle-specific enolase (CAA36216.1),Ascaris suum (ADQ00605.1), Brugia malayi (XP_001896281.1),Trichinella spiralis (XP_003371233.1), Fasciola hepatica(CAK47550.1), Echinococcus granulosus (ACY30465.1), Candidaalbicans (XP_711883.1), Entamoeba dispar (XP_001735821.1) andPlasmodium falciparum (XP_001347440.1). Brackets show GenBankaccession numbers. The conserved amino acids in all sequences arelabeled with asterisks; the conservative and semi-conservativesubstitutions are labeled with two and one points, respectively. Thedashed lines demonstrate gaps introduced between the sequences. Theresidues involved inMg2+ binding are shown in boldandwith arrowhead,those forming the enolase signature are underlined, and the motifs forsubstrate binding are indicated with letters in bold. The putativeplasminogen binding site is boxed. The transmembranal domain isindicated in shadowed gray. The enolase “fingerprint” motif is boxedand shown with pentacle. The percentage of sequence identity betweenTcENO and the enolases from other organisms is indicated

Fig. 1 RT-PCR product of T. cruzi enolase cDNA sequence andsubcloning. a Lane 1: molecular size marker (λ/Hind III); lane 2: T. cruzienolase gene amplified from cDNA. bLane 1: pRSET-B::TcENO plasmiddigested with Kpn I andHind III enzymes in a double restriction reaction;lane 2: molecular size marker (λ/Hind III). For presentation purposes thecolor of images was inverted from the original image. The fragment of1,162 pb corresponding to TcENO gene is observed (arrow)

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Fig. 2 (continued)

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2009). Moreover, the urbanization and migratory populationmovements from endemic countries have led to the diseasebeing diagnosed in non-endemic areas (Schmunis 2007). Thefact that there are not effective drugs or vaccines for thetreatment or prevention of the disease is still a major problem;in spite of that, the transmission of the Chagas disease hasdiminished during the last years (Moncayo 2003; Moncayoand Silveira 2009). However, from massive sequencing ofgenomes and the development of tools for in silico trialstogether have contributed to the emergence of the reversevaccinology (He and Xiang 2013), with which it has beenpossible to appoint several vaccine candidates for humanpathogens (Etz et al. 2002; Gan et al. 2010; Liu et al. 2009;Maione et al. 2005; Montigiani et al. 2002; Wizemann et al.2001). Furthermore, several studies have shown that the gly-colytic enzyme enolase can be used as a vaccine candidatein different diseases (Li et al. 2011; Gan et al. 2010;Pal-Bhowmick et al. 2007).

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11)is a metalloenzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP)in the forward direction (catabolic reaction) of the second halfof the Emden–Mayerhoff–Parnas glycolytic pathway. In thereverse reaction (anabolic pathway) which occurs during glu-coneogenesis, the same enzyme catalyzes hydration of PEP to

PGA (Capello et al. 2011; Pancholi 2001). Enolase is foundfrom archaebacteria to mammals, and its sequence is highlyconserved (Piast et al. 2005).

In this study, we reported the cloning, expression and insilico immunological characterization of a T. cruzi cDNAenolase by molecular techniques, affinity chromatography,SDS-PAGE and immunodetection (western blot). The resultsshowed that purified His-tag recombinant protein (rTcENO)encodes a 46.1 kDa protein and can be specifically recognizedby anti-histidine monoclonal antibody (Fig. 6, lane 1),this molecular weight is corresponding with the previ-ously reported size for several recombinant purifiedenolases of P. falciparum (49 kDa), C. albicans (46 kDa),Anisakis simplex (47 kDa), Sarcocystis neurona (47 kDa),Echinococcus granulosus (49.92 kDa) and Clonorchissinensis (50 kDa) (Li et al. 2011; Pal-Bhowmick et al. 2007;Rodriguez et al. 2006; Wilson et al. 2004; Gan et al. 2010;Wang et al. 2011). Furthermore, based on multiple sequencealignment of enolases, the predicted amino acid sequence ofthe TcENO had an identity of 81.03 % to the enolase ofT. brucei and 72–73 % with the enolases of Leishmania genus(Fig. 2). With respect to the enolases of other species, theidentity was only of 52–54 %. These results indicate thatenolases of Trypanosomatidae family are conserved betweenthem and separated from the rest, including those of protozoan

Fig. 3 Neighbor-joining analysisof TcENO and enolases. T. brucei(XP_822542.1), L. infantum(XP_001464303.1), L. donovani(ACE74540.1), L. mexicana(XP_003873490.1), H. sapiensBbeta-enolase isoform 1(NP_001967.3), H. sapiensGmuscle-specific enolase(CAA36216.1), A. suum(ADQ00605.1), B. malayi(XP_001896281.1), T. spiralis(XP_003371233.1), F. hepatica(CAK47550.1), E. granulosus(ACY30465.1), C. albicans(XP_711883.1), E. dispar(XP_001735821.1) andP. falciparum (XP_001347440.1).Evolutionary distances werecomputed using the Poissoncorrection method. Branchsupport values (10,000bootstraps) for nodes areindicated. Brackets showGenBank accession numbers

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parasites P. falciparum and E. dispar as observed in theneighbor-joining tree (Fig. 3).

Meanwhile, the primary sequence of TcENO revealed sev-eral structural features of enolases. Importantly, TcENO con-tains the active site and conformation-determining metal ion-

binding site residues necessary for dimerization and enzymefunction. These sites are four potential Mg2+ binding sites(Fig. 2, letters in bold and arrowhead) and six potential sub-strate binding and catalytic sites (Fig. 2, letters boxed) in theamino acid sequence, consistent with that reported by

Fig. 4 Immunological characteristics of TcENO sequence. The codingregion of TcENO gene and its translated protein sequence are shown,linear B cell epitopes are underlined; CTL epitopes are shadowed in grey.

The residues involved in Mg2+ binding are in bold and marked with anarrowhead, and the motifs for substrate binding are boxed

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several authors (Mundodi et al. 2008; Pancholi andChhatwal 2003; Torre-Escudero et al. 2010; Gan et al.2010). In TcENO-deduced sequence, a characteristic enolasefingerprint motif (366SHRSGETED374) (Fig. 2, boxed andshown by pentacle) was located in the C-terminal region,which is highly conserved in all the enolases described to date(Babbitt et al. 1996). These results indicate that TcENO genesequence could be encoding for a protein with enolase activityas was described byNakada et al. (2005), who showed that thefull length of a TsENO protein of Trichinella spiralis had noenzymatic activity but gained enolase activity after cutting offthe signal peptide predicted after Thr41 that results in a pro-

protein, so similar phenomenon could be occurring with theTcENO since this signal sequence is also present (Fig. 2,signal peptide not indicated); however, this will be investigat-ed in further studies.

Analysis of the sequence revealed that a domain similar tothe internal plasminogen-binding site found in L. mexicanaα-enolase (AYDAERKMY) (Vanegas et al. 2007) is also foundin TcENO (TYDENKKQY) between positions 245 and 253with 44.4 % of identity. Moreover, the putative plasminogenmotif of TcENO showed high identity (66.6 %) with a plas-minogen binding motif in enolase of Trichomonas vaginalis(Mundodi et al. 2008). These data indicate that, similar toother bacterial and eukaryotic pathogens, TcENO could be ananchorless, a surface-associated glycolytic enzyme of T. cruzi,contributing as a virulence factor. However, further studiesshould be performed to test this hypothesis.

Moreover, Wilson et al. (2004) described the presence of atrue plant motif (EWGWC) in the predicted amino acid se-quence of the enolase from Sarcocystis neurona and a plant-like motif (EWGYS) in the predicted amino acid sequence ofthe enolase from Neospora hughesi and N. caninum. Thesetwo motifs may be similar to other parasitic protozoa charac-terized. This pentapeptide EWGWC or EWGYS is importantbecause it greatly contributes to enolase enzymatic activities;however, either pentapeptides are absent in the predictedamino acid sequence of the enolase from T. cruzi as well asfrom the others organisms tested in the alignment with theexception of Plasmodium falciparum (Fig. 2, pentapeptide notindicated).

With the aim to determine the probable immunogenicity ofTcENO, bioinformatics analysis was conducted; in these tri-als, we localized 12 linear B cell epitopes and seven CTLepitopes; moreover, two epitopes, aa191-199 and aa 368-365,were both facultative B cell and CTL epitopes which canstimulate humoral and cellular immune response at the sametime, indicating its strong immunogenicity. Recently, a studydemonstrated that enolase of C. albicans can stimulate bothinflammatory regulators (IL-12 and IL-10) and protectiveantibody responses (IgG1 and IgG2a) (Li et al. 2011). Somestudies have shown that enolase from many pathogens areantigen of strong immunogenicity, such as that observed inEchinococcus granulosus, where the purified protein could berecognized by E. granulosus-infected patient serum by west-ern blot assays (Gan et al. 2010). The immunogenicity ofTcENO was tested by western blot assays, after rTcENOpurification, this protein was electroblotted and T. cruzi exper-imental infected mice and Chagas disease patient serareactioned with rTcENO. Both kinds of sera were capable ofrecognizing specifically the band of 46.1 kDa correspondingto rTcENO. These results suggest that enolase of T. cruzi is animmunogenic protein and has the ability to induce the hostimmune responses. Therefore, TcENO could be a suitablevaccine candidate that may have the potential of decrease

Fig. 5 Expression and purification of rTcENO. Proteins were resolvedby SDS-PAGE on 12 % polyacrylamide gel and stained with Coomassieblue. Lane M: molecular weight marker; lane 1: E. coli Bl 21 cellcontaining the pRSET-B::TcENO without IPTG induction; lane 2: E. coliBl 21 cell containing the pRSET-B::TcENO with IPTG induction; lane 3:total protein extract unbounded to the Ni-NTA resin; lanes 4 and 5:washing material; lane 6: the purified rTcENO. The band correspondingto rTcENO (46.01 kDa) is indicated with an arrow

Fig. 6 Immunodetection of rTcENO. 10 μg per lane were immobilizedand incubated with anti-His, anti-T. cruzi mice sera and anti-T. cruzihuman sera (1:500 dilutions in 2 % skim milk–PBS). Goat anti-miceIgG and anti-human IgG alkaline phosphatase-conjugated secondaryantibody (1:5,000 dilutions in 2 % skim milk–PBS) were employed todevelop the reaction. Lane M: molecular weight marker; lane 1: anti-Histag antibody; lane 2: anti-T. cruzimice sera; lane 3: anti-healthymice sera;lane 4: anti-T. cruzi human sera; lane 5: anti-healthy human sera

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the parasite load based on protective efficacy observed inother pathogens such as Schistosoma japonicum, where afterchallenge a reduction in liver eggs, a reduction in the fecal eggcount and a high level of specific IgG antibodies were obtain-ed in mice immunized with the rSjENO protein (Yang et al.2010). In addition, the enolase has been described as a proteinthat can not only be located in membrane but can also besecreted/excreted increasing its vaccine potential (Sotillo et al.2010). It is not known whether TcENO can be secreted, so itwill be important to address this in the future studies.

Conclusions

We have demonstrated that the enolase amino acid sequenceobtained in this work contains the major characteristics ofenolases family; moreover, the in silico assays showed thatits sequence have both B cells and CTL epitopes, indicatingthat T. cruzi enolase could be immunogenic. Furthermore, serafrom Chagas disease patients and sera from experimentalinfected mice with T. cruzi were able to recognize torTcENO; together, these results indicate that rTcENO is agood candidate to be explored as a vaccine against T. cruziinfection. However, the detailed immunological property ofrTcENO will be further studied.

Acknowledgments We thank to María Guadalupe Aguilar Gonzálezfrom Departamento de Genética y Biología Molecular, CINVESTAV,Mexico for the DNA sequencing. The authors are grateful to M.Sc.Patricia Espiritu Gordillo for critically reading the manuscript. This workwas supported by a grant (236/2010 to Arce-Fonseca M.) from theInstituto de Ciencia y Tecnología del Distrito Federal (ICyTDF), Mexico.Carabarin-Lima A was recipient of a postdoctoral fellowship from theICyTDF, México.

Conflict of interest None of the authors has any potential financialconflict of interest related to this manuscript.

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