Expression and characterization of the Babesia bigemina cysteine protease BbiCPL1

Acta Tropica 121 (2012) 1–5

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Expression and characterization of the Babesia bigemina cysteine protease BbiCPL1 Tiago M. Martins ∗ , Virgílio E. do Rosário, Ana Domingos Centro de Malária e Doenc¸as Tropicais, Instituto de Higiene e Medicina Tropical, Rua da Junqueira 96, 1349-008 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 29 November 2010 Received in revised form 25 August 2011 Accepted 12 September 2011 Available online 1 October 2011 Keywords: Babesia bigemina Cysteine protease BbiCPL1 Recombinant expression

a b s t r a c t BbiCPL1 was the first papain-like cysteine protease from a piroplasm to be identified with proteolytic activity. Here we report the improved production of the active recombinant enzyme, and the biochemical characterization of this potential drug target. BbiCPL1 showed characteristic properties of its class, including hydrolysis of papain-family peptide substrates, an acidic pH optimum, requirement of a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors such as E-64, leupeptin, ALLN and cystatin. The optimum pH for the protease activity against peptide substrates was 5.5, but enzymatic activity was observed between pH 4.0 and pH 9.0. At slightly basic pH 7.5, BbiCPL1 maintained 83% of maximum activity, suggesting a role in cytosol environment. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Babesia bigemina is an intraerythrocytic protozoan parasite of the phylum Apicomplexa, and a causative agent of bovine babesiosis, a tick-borne disease of worldwide distribution (Uilenberg, 2006). Babesiosis has long been recognized as an economically significant disease of cattle (Bock et al., 2004). Live vaccines are the main measure to control bovine babesiosis in many regions of the world, but breakdowns of protective immunity are common as the vaccines might not offer cross protection against the several local existing variants (de Waal and Combrink, 2006; Fish et al., 2008). Chemoprophylaxis is also used as a method of short-term protection, during outbreaks, and other temporary circumstances, as when pregnant cows are at increased risk of transmission and consequent abortion or when animals are momentarily relocated (Bock et al., 2004; de Waal and Combrink, 2006). Extensive outbreaks of the disease also occur in herds of susceptible unvaccinated cattle transported to natural and stable endemic areas (Alfredo et al., 2005; Bock et al., 2004). In addition, control of babesiosis currently requires more specific, fast acting, reliable and safer chemotherapeutic treatments (Vial and Gorenflot, 2006). Thus, the identification and characterization of new drug targets

∗ Corresponding author. Present address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Tel.: +351 214469848; fax: +351 214411277. E-mail address: [email protected] (T.M. Martins). 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.09.008

for chemotherapy of bovine babesiosis is considered a pressing priority. The members of the Clan CA, or papain-family of cysteine proteases are the most common proteases in protozoan parasites and are essential to the life cycle and pathogenicity of these organisms (Sajid and McKerrow, 2002). Hence, cysteine proteases of protozoan parasites are recognized drug targets and specific inhibitors are in validation for chemotherapy of leishmaniasis, malaria, and trypanosomiasis (McKerrow et al., 2008). In Babesia bovis, another agent of bovine babesiosis, essential roles for cysteine proteases were suggested when it was demonstrated that cultured parasites showed reduced growth when incubated with the cell permeable cysteine protease inhibitors ALLN and E-64d (Okubo et al., 2007). Cysteine proteases of Babesia spp. may therefore be potential new chemotherapeutic targets. Three cathepsin L-like cysteine proteases, BbiCPL1–3, were previously identified in the B. bigemina genome (Martins et al., 2011). BbiCPL1 was cloned, expressed as a fusion protein with gluthatione-S-transferase (GST) and the soluble recombinant protein purified by affinity chromatography. The recombinant BbiCPL1 had activity against typical peptide substrates of cysteine proteases, and was inhibited by E-64 (Martins et al., 2011). However, a low yield was obtained with the purification of the recombinant protein from the soluble fraction which prevented additional characterization of BbiCPL1. Here, we report the improved purification of recombinant BbiCPL1 from the insoluble fraction, refolding of the denatured protein and the additional biochemical characterization of this potential drug target.

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T.M. Martins et al. / Acta Tropica 121 (2012) 1–5

2. Materials and methods 2.1. Materials The inhibitors 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 3,4-dichloroisocoumarin (DCI), ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), egg cystatin, 1,10-phenanthroline, N-acetyl-l-leucyl-l-leucyl-largininal (leupeptin), pepstatin-A, N-(trans-epoxysuccinyl)l-leucine 4 guanidinobutylamide (E-64), and N-acetylleucineleucine-norleucinal (ALLN); and the chemical 7-amino-4methyl coumarin (AMC) were obtained from Sigma–Aldrich. 2.2. Expression and purification of recombinant BbiCPL1 The 1377 bp full-length BbicpL1 gene (GenBank accession no. FJ859910) was previously amplified and cloned into the pGEX6P-1 (GE Healthcare, Buckinghamshire, UK) expression vector (Martins et al., 2011). The pGEX-6P-BbiCPL1 construct was transformed using the TransformAidTM Bacterial Transformation System (Fermentas, EU) into E. coli BL21 cells (GE Healthcare, Buckinghamshire, UK), and cultures were induced at an Abs 600 nm of 1.0 for 3 h with 1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG). The molecular weight of the recombinant GST-BbiCPL1 protein was estimated to be 78.7 kDa based on the recombinant protein sequence and calculations using ExPASy tools (Gasteiger et al., 2005). The induced cell pellet from 750 ml of culture was collected, resuspended with 30 ml of 100 mM Tris pH 8.0, 0.2% (v/v) Triton X100, and lysis was performed by sonication. Lysed cells were diluted to 250 ml in Urea Wash Buffer (100 mM Tris pH 8.0, 0.2% (v/v) Triton X-100, 500 mM urea) and stirred for 30 min at room temperature. Insoluble material containing the recombinant protein was pelleted by centrifugation at 27,000 × g for 10 min. The pellet was resuspended with 250 ml Urea Wash Buffer, stirred and pelleted in the same previous conditions. This procedure was repeated 3 times. The washed pellet was then resuspended in 40 ml of 8 M urea, 100 mM Tris pH 8.5, 32 mM ␤-mercaptoethanol and stirred overnight at 4 ◦ C to solubilise the recombinant material. Residual insoluble material was removed by centrifugation at 27,000 × g for 30 min. 2.3. Refolding of recombinant BbiCPL1 Refolding of recombinant BbiCPL1 was optimized by testing more than 100 different buffer combinations in a microplate format as described previously (Sijwali et al., 2001a). Buffers differed in pH, the concentration of redox couple (reduced glutathione (GSH) to oxidized glutathione (GSSG)), aggregation suppressors (l-arginine, KCl, Triton X-100, and polyethylene glycol), and co-solvents (glycerol and sucrose). Refolding was initiated by 100-fold rapid dilution (Hirose et al., 1989) of ice-cold reduced-denatured protein to a final protein concentration of 0.1 mg/ml in 300 ␮l of ice-cold refolding buffer followed by incubation at 4 ◦ C for 16 h. Refolding efficiency was evaluated by assaying 50 ␮l of each refolding reaction for hydrolysis of Z-Phe-Arg-AMC as described in Section 2.4 (in 100 mM sodium acetate, pH 4.6), and the efficiency for each reaction was defined as the percentage of the maximum difference of hydrolysis achieved in this experiment. For large-scale refolding, recombinant BbiCPL1 was rapidly diluted to a final protein concentration of 0.1 mg/ml in 2 l of ice-cold optimized refolding buffer (20 mM Tris pH 11.0, 1 mM EDTA, 5 mM GSH, 0.5 mM GSSG) and incubated at 4 ◦ C for 16 h. The resulting soluble matter was concentrated 10 times using an Amicon stirred cell (Millipore, Watford, UK) fitted with a 10 kDa cut-off membrane. To allow the processing to an active enzyme, the concentrated preparation was acidified by

the addition of one-tenth volume of 1 M sodium acetate buffer pH 4.6 (final pH of approximately 5.0) and the sample was incubated at 4 ◦ C for 20 h. Insoluble material was removed by centrifugation at 27,000 × g for 30 min. The resulting soluble protein was further concentrated 8 times using an Amicon stirred cell fitted with a 10 kDa cut-off membrane and then stored in 50% (v/v) glycerol at −20 ◦ C. Protein concentration was determined by a colorimetric method (Bradford, 1976) with a Coomassie protein assay kit (Pierce) according to the manufacturer’s instructions. Western blot analysis was performed as previously described (Martins et al., 2011). 2.4. Activity and inhibition assays BbiCPL1 activity was assayed fluorimetrically as the hydrolysis of the substrates Z-Phe-Arg-AMC (Sigma–Aldrich, St. Louis, USA), Z-Leu-Arg-AMC, Z-Leu-Leu-Arg-AMC and Z-Val-Val-ArgAMC (Bachem Holding AG, Bubendorf, Switzerland) were followed over time as previously described (Martins et al., 2011). A defined quantity of enzyme solution (8 ␮l corresponding to a final concentration of 5.13 nM) was assayed in triplicate in 100 mM sodium acetate (pH 5.5) containing 50 ␮M fluorogenic substrate and 1 mM dithiothreitol (DTT) in a final volume of 0.2 ml (or with changes in fluorogenic substrate concentration, pH or reductant as described). For the inhibition assays, the activity of BbiCPL1 was assayed in triplicate in the absence or presence of inhibitors and the respective solvent dimethyl sulfoxide (DMSO) as a control. An excess of the inhibitors was assayed according to the manufacturer described range of working concentrations. DMSO was assayed at the maximum concentration obtained when used in assays with inhibitors. The activity of BbiCPL1 against gelatine was determined by zymography as described elsewhere (Martins et al., 2011) with minor changes. Activity of samples separated in a 12.5% SDS-PAGE, polyacrylamide gel was developed by overnight incubation at 37 ◦ C in 100 mM sodium acetate (pH 5.5), 1 mM DTT, before staining and de-staining. 2.5. Enzyme kinetic assays BbiCPL1 enzyme concentration was determined by active site titration with E-64 (Barrett and Kirschke, 1981). Rates of hydrolysis of fluorogenic peptide substrates were determined in triplicates and in the presence of a constant BbiCPL1 enzyme concentration (5.13 nM). The observed rates in relative fluorescence units (RFU) per second were converted to molar concentration per second using a conversion factor calculated by the slope of a standard linear curve of RFU versus concentration of AMC. The kinetic constants Km and kcat were determined using the GraphPad Prism 5.0 program (GraphPad Software, San Diego, CA, USA). The derived kinetic constants Km and kcat always had estimated errors of <15%. 3. Results The full length BbiCPL1 gene was previously cloned and expressed as a fusion protein with GST, and the expressed soluble fusion protein was purified by affinity chromatography (Martins et al., 2011). This procedure yielded very low quantities of active enzyme for biochemical characterization. Using the same expression construct, we proceeded with the purification and refolding of the insoluble fraction of the recombinant GST-BbiCPL1, which constituted the bulk of the expressed fusion protein. Insoluble inclusion bodies were washed with a urea buffer and later solubilised with 8 M urea (Fig. 1A). Afterwards, the denatured and reduced GST-BbiCPL1 protein was refolded in more than 100 buffer testing conditions in a microplate format. Aggregation suppressors (l-arginine, KCl, Triton X-100, and polyethylene glycol), and co-solvents (glycerol and sucrose) had no positive influence in the

T.M. Martins et al. / Acta Tropica 121 (2012) 1–5

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Fig. 1. Expression, purification and gel substrate activity of recombinant BbiCPL1. The expression of the 78.7 kDa GST-BbiCPL1 was evaluated by 12.5% SDS-PAGE (A). Uninduced E. coli BL21 cells and induced with IPTG for 3 h. Inclusion bodies containing the denatured fusion GST-BbiCPL1 protein were purified by urea washes and later solubilised with 8 M urea. GST-BbiCPL1 was refolded, and later acidified to a processed BbiCPL1 mixture with GST, and the BbiCPL1 mixture with GST was then stored in 50% glycerol. Activity was evaluated by gelatine zymography (B) in a one month old “refolded” sample at pH 9.0 and in a 50% glycerol “stored” BbiCPL1. The positions of molecular weight markers are labeled in kDa. Black arrow indicates the GST-BbiCPL1 fusion protein and white arrows indicate activity. Western blot (C) of GST and GST-fusion proteins.

refolding efficiency. The best conditions for the refolding of denatured GST-BbiCPL1 to an active enzyme were obtained with basic pH 11.0 and a GSH:GSSG ratio of 5:0.5 (Table 1). The refolded protein samples showed a near to exponential increase in activity during the initial time period of analysis, suggesting that upon acidification to pH 5.0, the refolded pro-enzyme processed itself into a mature and more active enzyme. A truncated form of BbiCPL2, another B. bigemina cathepsin Llike cysteine protease, containing the last 257 amino acid residues (for protein sequence see Martins et al., 2011) was also expressed, purified and refolded using the same procedure used for BbiCPL1 (data not shown). Enzymatic activity of this truncated form of BbiCPL2 was not detected in tests carried out in parallel with BbiCPL1 thus serving as a negative control to exclude activities of bacterial protein contaminants. Large-scale refolding of BbiCPL1 was carried out by 100-fold rapid dilution of the denatured protein in optimized buffer (20 mM Tris, 1 mM EDTA, 5 mM GSH and 0.5 mM GSSG, pH 11.0). The profile of the refolded protein material did not suffer significant alterations in comparison with the denatured proteins as observed by SDSPAGE analysis (Fig. 1). The refolded pro-enzyme was then acidified to promote auto-activation. The conditions for the auto-activation of BbiCPL1 were optimized with respect to pH (4.0–6.0) and temperature (4–37 ◦ C). The best pH and temperature were defined as the condition at which the maximum of activity in the acidified samples was obtained after 2 h and 4 h of incubation and were pH 5.0 and 4 ◦ C, respectively. Low temperatures are routinely used to prevent protein agglomeration. Despite this, and at all temperatures tested, a significant amount of refolded protein precipitated

almost immediately with acidification, and at lower values than the optimum pH 5.0, the amount of precipitated material increased significantly. Part of the precipitated material is composed of E. coli contaminating proteins, as these are not observed in SDS-PAGE analysis after acidification (Fig. 1A). Upon acidification, a change in the mobility of the estimated 78.7 kDa GST-BbiCPL1 was observed in the SDS-PAGE analysis (Fig. 1A). The auto-activation was confirmed by gelatinase activity gel, as activity was observed as a colorless band at approximately 30 kDa, the expected molecular weight of the mature BbiCPL1 (determined from analysis of the protein sequence and from comparisons with related proteases). The auto-activation of BbiCPL1 is a multi-step process, as an intermediate product with gelatinase activity at 43 kDa was observed in an old refolding sample (Fig. 1B), stored at 4 ◦ C in refolding buffer (pH 9.0). The majority of the protein observed at approximately 30 kDa (Fig. 1A) was later confirmed as being the GST (27 kDa) since it could be removed by affinity purification and detected by western blot (Fig. 1C). Although the removal of GST did not alter the initial activity velocity of the mature BbiCPL1, it changed the long term storage stability as it was observed a rapid loss of enzyme activity overtime in assays with the peptide Z-Phe-Arg-AMC. Cysteine proteases are usually relatively unstable enzymes which are prone to autocatalytic degradation, oxidation, and protein denaturation (Brömme et al., 2004). The more stable protein mixture of GST and mature BbiCPL1 (mixture with 5.6% of active enzyme; final yield of about 450 ␮g active enzyme per litre of culture) was then used in all subsequent kinetic experiments. BbiCPL1 had typical properties of a papain-family cysteine protease, with acidic pH optimum against peptide substrates and

Table 1 Optimization of the refolding of BbiCPL1. Effect of pH and GSH:GSSG ratio (concentrations in mM) on the efficiency of the refolding of BbiCPL1 in 20 mM Tris. Activity of the hydrolysis of Z-Phe-Arg-AMC for each reaction was normalized against a maximum of 100 to express refolding efficiency. (Activity, % of maximum)

pH

GSH:GSSG

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

10:1 10:0.5 5:1 5:0.5 2.5:0.5 1:0.5 0.5:0.5 0:0

0.0 0.3 0.1 0.2 0.9 3.6 2.8 1.1

0.3 0.8 24.4 30.4 31.6 18.7 11.6 3.1

35.7 44.8 54.6 62.7 51.5 31.7 20.8 6.1

56.5 64.9 72.5 72.0 61.3 39.2 25.6 9.4

68.3 74.3 77.8 86.0 72.7 45.8 30.5 13.9

90.5 89.6 91.6 96.7 80.1 51.1 32.9 17.5

83.2 88.1 84.1 96.8 77.2 49.1 34.1 19.5

92.3 98.0 94.8 100.0 87.0 54.7 39.1 20.7

83.3 87.5 89.4 99.0 76.7 53.5 37.0 20.3

89.7 97.3 98.6 99.5 85.3 57.2 39.9 28.2

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Fig. 2. Effect of pH, reductant concentration, and inhibitors on activity of BbiCPL1. The activity of recombinant BbiCPL1 was assayed against 50 ␮M Z-Phe-Arg-AMC in 100 mM sodium chloroacetate (pH 3.0), sodium acetate (pH 3.5–5.5), sodium phosphate (pH 6.0–7.0) or Tris (7.5–9.0) and 1 mM DTT (A) and in 100 mM sodium acetate pH 5.5, with different concentrations of DTT (B). For inhibition assays, recombinant BbiCPL1 was added to 50 ␮M Z-Phe-Arg-AMC in 100 mM sodium acetate pH 5.5 and 1 mM DTT, in the absence or presence of E-64 (1 ␮M), ALLN (100 ␮M), leupeptin (100 ␮M), cystatin (10 ␮g/ml), DCI (50 ␮M), PMSF (1 mM), AEBSF (1 mM), pepstatin (8 ␮M), EDTA (1 mM), 1,10-phenanthroline (0.5 mM) or DMSO 1% (v/v) (C). Release of AMC was continuously monitored for 30 min, slopes of fluorescence over time were calculated, and results were expressed as percentage of maximum activity for each assay. Error bars indicate standard deviation of triplicate samples.

requirement for a reducing environment for maximum activity (Fig. 2A and B). BbiCPL1 showed a broad range of pH activity, with more than 30% of maximum activity from pH 4.0 to pH 9.0. The ability of the mature form of BbiCPL1 to cleave a variety of peptide substrates was examined (Table 2). BbiCPL1 preferred substrates with valine at the P2 position with a P2 rank order of Val > Leu > Phe in contrast with Falcipain-2 and -3 from P. falciparum that preferred Leu > Phe > Val (Sijwali et al., 2001b). The higher specificity constant (kcat /Km ) for the substrate with valine at the P2 position was a result of increased substrate turnover (kcat ), as BbiCPL1 showed apparent higher affinity (lower Km ) for the interaction with substrates with leucine at the P2 position. The interaction of BbiCPL1 with inhibitors of cysteine proteases and other classes was also analysed (Fig. 2C). As is typical of cysteine proteases, BbiCPL1 was potently inhibited by E-64, ALLN, leupeptin

Table 2 Kinetic parameters for substrate hydrolysis by recombinant BbiCPL1. Substrate

Km (␮M)

kcat (s−1 )

kcat /Km (s−1 M−1 )

Z-Phe-Arg-AMC Z-Leu-Arg-AMC Z-Leu-Leu-Arg-AMC Z-Val-Val-Arg-AMC

59.0 14.6 10.4 22.7

0.306 0.333 0.245 0.681

5.19 × 103 2.27 × 104 2.37 × 104 3.00 × 104

and cystatin. Inhibitors of other classes of proteases only had a minor effect or no effect on the activity of BbiCPL1. 4. Discussion The recombinant form of BbiCPL1 showed characteristic properties of a papain-family cysteine protease, including hydrolysis of typical papain-family peptide substrates (Table 2), an acidic pH optimum, requirement for a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors (Fig. 2). Despite all these typical properties of a papain-family cysteine protease, BbiCPL1 has unusual features such as the nature of the S2 pocket and high activity at basic pH. The nature of the S2 pocket and in particular the residue present in the hollow end of the pocket is essential to the substrate specificity of clan CA enzymes. The S2 pocket is usually constituted by hydrophobic residues, but the key residue (residue 205 in papain) present at the bottom is not conserved (Sajid and McKerrow, 2002). In the related apicomplexan parasite, P. falciparum, the cysteine proteases Falcipain-2 and -3 have in this critical position the polar residues Asp234 and Glu243, respectively (Kerr et al., 2009). BbiCPL1 has a bulky hydrophobic phenylalanine residue at the end of the S2 pocket (Martins et al., 2011), an unusual feature in common with the human cathepsin W and with the CP1 protease from the cellular slime mold Dictyostelium discoideum (Brinkworth

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et al., 2000). In view of that, BbiCPL1 showed a P2 rank order of Val > Leu > Phe in contrast with Falcipain-2 and -3 that preferred Leu > Phe > Val (Sijwali et al., 2001b). The preference of BbiCPL1 for the small residue valine at P2 , suggests a narrow S2 pocket in agreement with the presence of the bulky hydrophobic phenylalanine residue at the end of the S2 pocket, and therefore possible restrictions in substrate specificity. Two potent inhibitors of BbiCPL1 were previously found to effectively inhibit the in vitro growth of B. bovis (Okubo et al., 2007). Intracellular processing by Falcipain-2 and -3 was also shown to be inhibited by the same two compounds E-64d and ALLN (Dahl and Rosenthal, 2005). These Falcipains have been implicated in essential roles such as host haemoglobin degradation (Sijwali et al., 2001b), and rupture of infected erythrocytes (Dua et al., 2001; Hanspal et al., 2002). Invasion of erythrocytes by B. bovis was inhibited by E-64d and not by ALLN (Okubo et al., 2007), suggesting other cysteine protease than the B. bovis ortholog of BbiCPL1 to be involved in this role. BbiCPL1 was recently found to be also potently inhibited by artemisinin-vinyl sulfone hybrid molecules (Martins et al., 2010) that effectively inhibited the growth of P. falciparum cultured parasites (Capela et al., 2009). Some of these inhibitors are more potent against BbiCPL1 than to Falcipain-2 (up to 25 fold difference). This opens the possibility of antimalarial strategies based on similar compounds to be used against human babesiosis. At pH 7.5, Falcipain-2 with around 40% of maximum activity is more active against peptide substrates than Falcipain-3 with around 5% (Sijwali et al., 2001b). Accordingly, Falcipain-2 cleaves cytoskeletal proteins (pH optimum of 7.5–8.0) and may facilitate erythrocyte rupture by mature schizonts (Dua et al., 2001; Hanspal et al., 2002). BbiCPL1 also had considerable activity against peptide substrates at slightly basic pH 7.5 (>80% maximum activity). This raises the question if the high activity of BbiCPL1 at basic pH is an adaptation to the absence in intraerythrocytic Babesia of an acidic environment such as the plasmodial food vacuole, or if this enzyme participates in the degradation of host proteins in cytosol environment where a slightly basic optimum pH would be ideal. Recently, BbiCPL1 and the B. bovis ortholog (named bovipain-2) were immunolocalized to an undefined intracellular organelle and to the cytoplasm of infected erythrocytes (Mesplet et al., 2010). This suggests that BbiCPL1 may have a role in host cytosol environment and is in agreement with the observed and unusual high activity at neutral pH for this class of enzymes. At the same time a role in an intracellular organelle is also suggested. Pseudo food vacuoles are formed by enclosing host cytoplasm in intraerythrocytic Babesia (Kawai et al., 1999a). In addition, Babesia spp. induce severe ultrastructural changes in the erythrocyte membrane shape, with tubular structures extending from the surface of the infected erythrocyte membrane directly to the parasites, and it was suggested that Babesia spp. come into direct contact with the host plasma and may obtain nutrients from both outside and inside host cells (Braga et al., 2006; Kawai et al., 1999a,b). The feeding mechanisms of Babesia spp., and the possible involvement of cysteine proteases in this process must still be studied in detail. The availability of recombinant BbiCPL1 may aid in the elucidation of this subject, and in addition, will serve as a tool to screen specific inhibitors against babesiosis in large-scale studies.

Acknowledgements Tiago Martins would like to acknowledge Fundac¸ão para a Ciência e a Tecnologia (FCT), Portugal, for his PhD grant (SFRH/BD/19059/2004) under POCI2010 and the European Social Fund.

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