Parasitol Res (2014) 113:4335–4348 DOI 10.1007/s00436-014-4107-2

ORIGINAL PAPER

Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella Renqiang Liu & Xueting Ma & Aijun Liu & Lei Zhang & Jianping Cai & Ming Wang

Received: 20 April 2014 / Accepted: 26 August 2014 / Published online: 25 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in birds. Cysteine protease is a major virulence factor in parasitic protozoa, and it may be a suitable chemotherapeutic target and vaccine candidate molecule. A 100 amino acid (aa.) partial sequence of cathepsin L, which is a cysteine protease, was reported by Katrib et al. (Ac. No. CDJ41293) (2012). A 219 aa. sequence was reported by Reid et al. (Ac. No. AFV92863) (2013). However, the open reading frame (ORF) was not reported. In this study, a full sequence of a cathepsin-L-like peptidase in Eimeria tenella (EtcatL) was obtained and its biochemical characterizations and expression profiles were analyzed across different stages of the parasite’s life cycle. Results showed that the EtcatL gene encodes a protein 470 aa. in length, with 47 and 49 % identity to Toxoplasma gondii and Eimeria acervulina. Considering the close phylogenetic relationship, TgcatL (PDB. ID 3F75) was selected for use as a template for homology modeling with quality factors of 90.9. Gelatin SDS-PAGE showed it to exert protease R. Liu : A. Liu : M. Wang (*) National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Haidian District, Beijing 100193, China e-mail: [email protected] X. Ma : J. Cai (*) State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institude, Chinese Academy of Agricultural Sciences, No. 1 Xujiaping, Chengguan District, Lanzhou, Gansu Province, China e-mail: [email protected] L. Zhang : M. Wang Key Laboratory of Veterinary Bioproduction and Veterinary Medicine of the Ministry of Agriculture, Zhongmu Institutes of China Animal Husbandry Industry Co., Ltd, No. 156 Beiqing Road, Haidian District, Beijing 100095, China

activity at ≈38 and ≈26 kDa. Further analysis showed the kinetic parameters of the recombinant peptidase to be Km =8.9 μM and Vmax =5.7 RFU/s μM at pH 5.5 containing 10 mM dithiothreitol (DTT) in the reaction matrix, and the IC50 value of E64 was 65.32±3.02 nM. The recombinant protein was active from 25 to 50 °C, with optimal activity at 42 °C. The RT-PCR and Western blot results showed it to be expressed mainly at the endogenous stages and the initial phase of the sporulation. The protective experiment showed that chickens immunized with 100 and 200 μg rEtcatL had reduction of weight loss values 48.7 and 57.9 % those of infected controls, respectively. Their reduction of lesion scores (RLS) were 25.0 and 47.2 % that of control chickens, and relative oocyst production (ROP) was 39.6 and 15.5 % that of control chickens. These results indicate that the EtcatL can be used as an effective immunogen, and further studies are needed to enhance its potential as a vaccine candidate molecule. Keywords Eimeria tenella . Cysteine protease . Cathepsin L . Enzyme kinetics . Expression profile

Introduction Avian coccidiosis, a parasitic disease caused by many different species of eimerian protozoa, can give rise to tremendous economic losses in the poultry industry (Shirley et al. 2005). Currently, control of this economically important disease still relies mainly on prophylactic medication in animal feed. However, the increasingly common emergence of drugresistant Eimeria strains and public concerns on the anticoccidial residues have highlighted the need for new approaches. Parasite-derived proteases have important roles in parasitic pathogenesis. They are involved in host invasion, evasion of

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the immune system, and nutrient uptake (McKerrow et al. 2006). Some of them have been shown to be novel targets for antiparasite agents and vaccine candidate molecules (Dalton et al. 2003; Teixeira et al. 2011). These proteases can be classified into six broad groups, including serine protease, threonine protease, cysteine protease, aspartate protease, glutamic acid proteases, and metalloproteases. One of the most studied proteases is the cysteine protease from clan CA, family C1. It contains cathepsins B, L, and C. In apicomplexan parasites, cysteine proteases of this family are particularly prominent. Several studies have shown the crucial roles of cysteine cathepsins (Dou and Carruthers 2011; Kim 2004; Klemba and Goldberg 2002). It was found that the cathepsin L in Plasmodium falciparum (P. falciparum) was necessary for hemoglobin digestion during erythrocyte infection (Rosenthal et al. 1988; Rosenthal et al. 1989). In Toxoplasma gondii (T. gondii), the cathepsin L (TgCPL) is conductive to maturation of TgCPB and essential for processing microneme protein, although recent studies have shown that TgCPB does not undergo self-maturation as maturase for rhoptry proteins (Dou et al. 2013; Parussini et al. 2010). Some studies have been performed on the serine proteases, aspartyl proteases, and other proteases in Eimeria tenella (E. tenella) (Jean et al. 2000; Jean et al. 2001; Laurent et al. 1993). However, the study in cysteine protease was few (Fuller and McDougald 1990). Recently, it was reported that the E. tenella can express five cysteine proteases (Katrib et al. 2012; Rieux et al. 2012), including one cathepsin B, one cathepsin L, and three cathepsin Cs, but only the full sequence of cathepsin B has been obtained and characterized (Rieux et al. 2012; Schaeffer et al. 2012). Only partial sequences of the cathepsin L in E. tenella has been reported, 100 amino acids (aa.) by Katrib in 2012 and 219 aa. by Reid in 2013 (Ac. Nos. CDJ41293 and AFV92863). Reid et al. also published a cathepsin L sequence encoding 582 aa. of Eimeria acervulina (Ac. No. CDI79476) in 2013, but there are no research data available yet. In this study, the full sequence of cathepsin-Llike cysteine peptidases from E. tenella was obtained and the biochemical properties of the recombinant protein were characterized.

Material and methods Parasites Parental Houghton strain E. tenella was provided by Martin W. Shirley (Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berks, UK) and maintained by passage every 3 months in susceptible 14-day-old Arbor Acre (AA) broiler chickens at the College of Veterinary Medicine, China Agricultural University, Beijing, China, using established protocols (Long et al. 1976).

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Chickens One-day-old AA broiler chickens obtained from the Huadu Broiler Hatchery (Being, China) were reared in a coccidia-free laboratory, housed in cages, and provided with free access to food and water. Ethics statement Experimentation with animals was approved by the Animal Ethics Committee of China Agricultural University (approval number 201206078) and followed the Regulations of Experimental Animals of Beijing Authority. Sporulation time course experiment The unsporulated oocysts were harvested from infected caeca, purified, and incubated at various times in 2.5 % potassium dichromate at 25 °C. The samples were ultrasonicated and kept in the sodium dodecyl sulfate (SDS) loading buffer (50 mM Tris–HCl, pH 6.8, 2 % SDS, 0.1 % bromophenol blue, 5 mM EDTA, 10 % glycerol, 1 % 2-mercaptoethanol) or ground in liquid nitrogen and kept in TRIzol (Invitrogen). Sporozoites were prepared from cleaned sporulated oocysts and were purified by chromatography with columns packed with nylon wool and DE-52 cellulose (Schmatz et al. 1984). Merozoite collection For merozoite (MZ) collection, ten broiler chickens (2 weeks of age) were killed 112 h after infection and MZ were isolated as described previously (Schwarz et al. 2010). The gametocytes were also collected and purified as described in standard protocols (Abdul Hafeez et al. 2006). Finding the coding fragment for EtcatL The T. gondii cathepsin-L-like protein (TgcatL) sequence was used as a query to search E. tenella whole genome database (http://www.genedb.org/Homepage/Etenella) using the miniBLAST program included in the web server. ETH_ 00033530 was found to contain a coding fragment for the conserved domain of peptidase_C1 superfamily homologue to TgcatL. This fragment was used as the template for cloning the EtcatL gene. Cloning of EtcatL Total RNA was isolated from the sporulated oocysts. Briefly, 1×106 sporulated oocysts were resuspended in TRIzol (1 mL) (Invitrogen), one volume of 0.5-mm glass beads was added to the sample, which was vortexed it for 30-s intervals until 90 % of the oocysts had been disrupted by confirmation under

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bright field microscopy. Then, total RNA was isolated by chloroform extraction and isopropanol precipitation. Firststrand cDNA was synthesized from the total RNA using a SMART RACE. cDNA Amplification Kit (Clontech). Primers were designed based on the identified fragment encoding the partial EtcatL fragment. The sequence of forward primer was 5′-cgggagagctgccggagtcggtgg-3′, and the reverse primer was 5′-aagccgcgttgcccccagccctctc-3′. The amplification reaction was done with a thermal cycling profile of 94 °C (5 min), 35 cycles at 94 °C (20 s), 60 °C (20 s), and 72 °C (1 min), followed by a final extension at 72 °C (10 min). The amplified gene fragment was purified and inserted into pEASY-T1 vector (Transgen). Based on this fragment, a pair of specific primers (SPf: 5′-ggaaagaagacgcaaaaccacaaag-3′ and SPr: 5′ccgcggcggacataagcggaaaa-3′) were designed to gain the 3′and 5′-ends of the gene by using a SMART RACE. cDNA Amplification Kit (Clontech) according to the manufacturer’s instructions. Then, the products were purified and cloned into pEASY-T1 vector (Transgen) for sequencing. The sequence data were analyzed using the DNAStar software and TMpred (http://embnet.vital-it.ch/software/TMPRED_form.html) for transmembrane domain identification. Expression and purification of recombinant EtcatL in E. coli For the expression of soluble recombinant EtcatL (rEtcatL), the protein was truncated slightly; the transmembrane and extracellular domains were removed. EtcatL was amplified with primers 5′-gttgaattcccagtgagtgtttatgactgg-3′ and 5′acaggatccttatgaaggcgttgaagg-3′. Restriction enzyme sites, EcoRI and BamHI were incorporated into the 5′-end of the primer, respectively. The PCR products were purified and subcloned into the pET28a(+) vector (Invitrogen) for expression of the enzyme with an N-terminal His tag and then transformed into DH5α. A positive transformant was selected, and the plasmid was transformed into competent E. coli Transetta (DE3) cells (Transgen). The expression of the recombinant protein was induced by adding isopropyl-1-thio-βD-galactopyranoside (IPTG) to a final concentration of 0.5 mM and analyzed on Coomassie-stained SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels. The cells were harvested at 16 h after incubation with IPTG at 16 °C, and then the pellet of cells from 2,000 mL of culture were resuspended in 120 mL of 50 mM Tris–HCl, pH 8.0. The supernatant fraction from the sonicated cell was placed in a His-bind column (Novagen). The column was washed twice with wash buffer (50 mM Tris–HCl, pH 8.0, 40 mM imidazole, 0.5 M NaCl), and the recombinant protein was eluted in eight fractions with elution buffer (50 mM Tris–HCl, pH 8.0, 200 mM imidazole, 0.5 M NaCl). The pooled fractions were dialyzed, and the yield and purity of the protein were analyzed on Coomassie-stained SDS-PAGE gels. The concentration of protein was determined with a BCA protein assay kit

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(Cwbiotech), and the activity of the recombinant protein was evaluated using a gelatin zymography assay as described below. Gelatin zymography assay One part sample was mixed with one part Tris-glycine SDS b u ff e r ( 0 . 1 m M Tr i s – H C l , p H 6 . 8 , 0 . 1 m M 2 mercaptoethanol, 4 % SDS, 10 % glycerol, and 0.1 % bromophenol blue) and allowed to incubate for 10 min at room temperature. Then, the sample was run on 8 % polyacrylamide gels containing 0.1 % gelatin (w/v) (SigmaAldrich). After electrophoresis at 4 °C, the gels were rinsed three times in 0.25 % Triton X-100 for 30 min to remove SDS and allow protein renaturation. Then, the gel was incubated in the zymography developing buffer (100 mM NaOAc, 10 mM DTT, pH 5.5) at 37 °C for 36 h. The gels were stained with 0.1 % Coomassie Brilliant Blue R-250. Enzyme kinetics and inhibitory test The activity of rEtcatL was determined by assessing its ability of cleavage of a fluorogenic substrate, Z-Phe-Arg-AMC (Hasnain et al. 1993). In brief, the rEtcatL was preincubated for 30 min at 37 °C with 160 μL of reaction buffer containing 50 mM sodium acetate (pH 5.5), 5 mM EDTA, and 10 mM dithiothreitol (DTT). The enzyme reaction was initiated by the addition of 80 μL of different amount of Z-Phe-Arg-AMC (Sigma) for 15 min at 37 °C. The appearance of 7-amino-4methylcoumarin (AMC) was measured using the excitation wavelength at 350 nm and emission at 465 nm. The thermal activity of rEtcatL was investigated by preincubating rEtcatL at 20, 28, 37, 42, 45, and 50 °C in the reaction buffer for 30 min and evaluating the activity as described above. Data were analyzed using a Michaelis-Menten model with the equation velocity=Vmax ×[substrate]/(Km +[substrate]) to determine the enzyme kinetic parameters. For evaluation of the inhibitory kinetic features of EtcatL, a known inhibitor, E64, was selected for assay (Barrett et al. 1982). The 50 % inhibitory concentration (IC50) values of E64 of recombinant EtCatL was measured in a fluorescence endpoint assay using Z-F-R-AMC as a substrate (Selzer et al. 1999). The assay was carried out in black 96-well plates. Assays for EtCatL were performed at 37 °C in a 50 mM sodium acetate buffer at pH 5.5 containing 10 mM DTT, 5 mM EDTA, and 5 μM Z-F-R-AMC. Prior to the addition of substrate, different concentrations of the inhibitor ranging from 0.1 to 200 nM were preincubated for 10 min with the enzyme to allow the establishment of the enzyme-inhibitor complex. The reaction was started by the addition of the substrate and stopped after a 30-min reaction at 37 °C, the enzyme activity was measured from the increase of fluorescence using excitation at 350 nm with emission at 465 nm. All

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values used were mean values from at least three independent assays to produce statistically significant results. Molecular modeling of EtcatL Homology modeling of the mature domain of E. tenella cathepsin L was performed with MODELLER v9.5 (Sali and Blundell 1993). TgcatL (Protein Data Bank (PDB.) ID 3F75) was selected as a template for homology modeling. Alignment of the target sequence with 3F75 (chain A) sequence was performed using ALIGN2D in MODELLER. Once alignment of target template had been performed, auto-model class was used to calculate a 3D model of the target completely automatically in MODELLER. The structure of EtcatL generated by MODELLER was improved by energy minimization using the Discovery Studio 2.5 (Accelrys Inc., San Diego, CA, USA). After energy minimization, the structure was submitted to the website of the Structural Analysis and Verification Server (http://nihserver.mbi.ucla.edu/SAVES/), and the Procheck module was used to evaluate the quality of the models. The ligand E64 was docked into the activity pocket of the receptor EtcatL using AUTODOCK 4.0 (Morris et al. 1998). The Lamarckian genetic algorithm (LGA) was used to search for the optimized conformation. During the docking, the search was conducted in a grid with 60 points per dimension. Each grid spacing was 0.0375 nm. The maximum number of generations and maximum number of energy evaluations were set to 2.7×105 and 2.5×106, respectively. The conformation with lowest binding free energy of all docking results was selected and hypothesized to be a representative binding mode of E64 and EtcatL. Energy minimization was performed to produce a more meaningful complex conformation for analysis. The complex structure for analysis was minimized with steepest descent and conjugate gradient by Discovery Studio 2.5. Phylogenetic analysis To explore the phylogenetic relationships involving cathepsin L, a rooted Bayesian (BI) phylogenetic tree with 47 sequences was inferred from the amino acid sequences using MrBayes v3.2.2 (http://mrbayes.csit.fsu.edu/) under the best-fit model WAG + I + G (Huelsenbeck and Ronquist 2001; Huelsenbeck et al. 2001). The best-fit model (WAG + I + G) for amino acid substitution was selected using ProtTest v2.4 with discrete gamma distribution in four categories (Abascal et al. 2005). All parameters (gamma shape=1.189; proportion of invariants=0.046) were estimated. A total of 2,000,000 generations were run with a burn-in of 500 trees. The tree was visualized using a FigTree program v1.4.0 (http://tree.bio.ed.ac.uk/ software/figtree/).

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Preparation of rEtcatL antibodies and Western blotting To prepare the antibodies, female BALB/c mice (6 weeks old) were immunized subcutaneously with purified rEtcatL (100 μg/mice) emulsified with complete (day 0) or incomplete (day 14) Freud’s adjuvant. On days 21 and 28, equal amounts of proteins were immunized intraperitoneally to boost the antibody titer. Mice were killed on day 35, and sera were pooled. For immunoblotting, parasite extracts collected during different periods were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked overnight with 5 % fat-free milk in PBS containing 0.05 % Tween-20 (PBST), incubated with a 1:1,000 dilution of the antibody in 5 % nonfat milk for 1 h at room temperature (RT), washed five times with PBST, incubated with goat anti-mouse IgG conjugated with HRP at 1:2,000 dilution in 5 % nonfat milk for 1 h at RT, and washed five times with PBST. The reaction was developed using electrochemiluminescence (ECL), and the reaction signal was detected by exposure to X-ray film. qRT-PCR analysis For quantitative real-time PCR (qRT-PCR), cDNA was synthesized from four different parts of the parasite’s life cycles. The threshold cycle (Ct) values were set up automatically. qRT-PCR was carried out in a 20-μL reaction volume containing 20 μL 2× SYBR™ Green PCR Master Mix (ABI), 1.0 μL cDNA, each primer (10 μM), and 7 μL PCR-grade water. Primer used in the assay included the following: Factin [5′-CACCACCgCCgAgAAAgA-3′], Ractin [5′-gAACAAC ATTgCCgTAgAgg-3′], FcatL [5′-CCAgTgAgTgTTTA TgACTggA-3′], and RcatL [5′-TAAgAgTgACCT TgTTggTTgTg-3′]. Thermal cycling and fluorescence detection were performed using an Applied Biosystems 7500 Real-Time PCR System. Amplification was performed using initial denaturation at 94 °C for 10 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 40 s, with a final extension at 72 °C for 10 min. All data are given here as the level of EtcatL mRNA relative to that of β-actin mRNA and expressed as a mean±SD (Xu et al. 2008). Values were compared using one-way ANOVA and Tukey’s multiple comparison test at P

Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella.

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in birds. Cysteine protease is a major virulence factor in parasitic...
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