Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry

Experimental Parasitology 123 (2009) 162–167

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Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry Yanhai Wang *,1, Zhe Cheng 1, Xiaofeng Lu, Chongti Tang Parasitology Research Laboratory, School of Life Science, Xiamen University, Xiamen 361005, China

a r t i c l e

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Article history: Received 30 December 2008 Received in revised form 16 June 2009 Accepted 18 June 2009 Available online 24 June 2009 Keywords: Echinococcus multilocularis Protoscolex Two-dimensional electrophoresis Proteomics Mass spectrometry Immunoproteomics Vaccine

a b s t r a c t Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5–10), followed by MALDITOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis, is considered to be one of the most lethal helminthic diseases in humans (Eckert and Deplazes, 2004). The infiltrative, tumor-like growth of the E. multilocularis metacestode usually affects the liver of intermediate hosts such as rodents or humans. The protoscoleces (PSC), a third larval stage in rodents, are eventually produced by the germinal layer within the metacestode after several weeks of development. PSC possess the potential to differentiate into adult tapeworms within the gut of the definitive host (Craig, 2003; McManus et al., 2003; Eckert and Deplazes, 2004) or develop into new hydatid cysts. Although rarely produced in humans (Gottstein and Hemphill, 1997), PSC have been used as a general source of metacestode proteins for human AE immunodiagnosis and vaccine research. Several proteins originally identified Abbreviations: 2-DE, two-dimensional electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDI-TOF MS, matrix-assisted laser desorption and ionization-time of flight mass spectrometry; MW, molecular weight; pI, isoelectric point; PMF, peptide mass fingerprinting; PSC, protoscolex/ protoscoleces; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA, trichloroacetic acid. * Corresponding author. Address: Parasitology Research Laboratory, School of Life Science, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China. Fax: +86 592 218 3040. E-mail address: [email protected] (Y. Wang). 1 These authors should be regarded as co-first authors. 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.06.014

in this larval developmental stage, such as Em18, Em16 and Em13, were well characterized for serodiagnostic purposes (Carmena et al., 2007). In the past two decades, research on AE infection has focused on identification and characterization of immunologically important proteins, especially potential immunodiagnostic or vaccine candidates (Carmena et al., 2007; Zhang and McManus, 2006). Proteomic analysis has been proposed as a powerful tool for investigating proteins of parasites, including those without the support of a genome project (Barrett et al., 2000). Recent improvements in the techniques of 2-DE (two-dimensional electrophoresis) and mass spectrometry, in combination with accumulating database resources, have made it possible not only to elucidate the protein expression profile in E. multilocularis, but also to discover new candidates for immunodiagnosis or for vaccine and drug targets in human AE disease. Previous work has shown that proteomic analysis can be performed prospectively for a broad analysis of the metacestode proteins of Echinococcus granulosus, the causative agent of cystic echinococcosis (Chemale et al., 2003). In this paper, we describe for the first time the identification of prominent proteins of E. multilocularis PSC from a 2-DE gel by peptide mass fingerprinting (PMF) and antigenic proteins by immunoproteomics. This work will facilitate the investigation of major proteins expressed in PSC larval stage and their functions in the parasite/host interaction. Defining the abundance and immunogenicity of PSC proteins may lead to the discovery of new antigens and potential therapeutical targets for human AE.

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2. Materials and methods

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in 2 ll 0.1% (v/v) trifluoroacetic acid (TFA) prior to MALDI-TOF MS analysis.

2.1. Parasite and PSC purification 2.4. MALDI-TOF MS analysis and database search E. multilocularis (China isolate) metacestodes were obtained from mice (KM strain) experimentally infected by intraperitoneal transplantation of metacestode tissue (Tang et al., 2004, 2006). PSC were isolated as described before (Brehm et al., 2003). Briefly, parasite tissues were carefully isolated from the host and homogenized. The homogenate was subsequently filtered once through 150 lm pore nylon mesh, separating PSC from large pieces of metacestode tissue. The filtrate was then passed through a 50 lm pore nylon mesh, separating the PSC from single cells and small cell clumps. PSC were washed off the nylon mesh with sterile phosphate-buffered saline (PBS), separated from equally sized metacestode vesicles manually, and washed five times with PBS and once with sterile water. PSC were finally collected by centrifugation at 2000g and then immediately used or stored in liquid nitrogen. 2.2. Two-dimensional electrophoresis (2-DE) Isoelectric focusing (IEF) rehydratation buffer was prepared with 7 mol/L Urea, 2 mol/L Thiourea, 4% CHAPS, 100 mM DTT, 20 mM Tris/HCl, pH 8.0, 2% Ampholine pH 3.5–10, and 1% (v/v) protease inhibitor cocktail (Sigma, USA). The purified PSC were lysed by incubating with the IEF rehydratation buffer at room temperature for 60 min. The destruction of the PSC structure was checked by visual inspection under the light microscope. The lysate was centrifuged at 15,000 g for 60 min at 15 °C and the supernatant was collected. Protein concentration was determined using BCATM Protein Assay Kit (Pierce, USA). Protein samples were immediately used for 2-DE analysis or frozen at -80 °C until required. The electrophoretic separation of proteins was performed essentially as described previously (O’Farrell, 1975; Görg et al., 1998). Isoelectric focusing (IEF) was performed with immobilized pH-gradient strips (IPG-strips, pH 3.5–10, 18 cm from Pharmacia Biotech. or laboratory-made). Protein solution was loaded on the IPG-strips and IEF was conducted overnight for a total of 14,000 Vh using a Bio-Rad IEF cell (Bio-Rad Laboratories, USA) and the following voltage steps: 2 h at 200 V, 2 h at 400 V, and 16 h at 800 V. Before the second dimension electrophoresis, strips were equilibrated for 25 min by incubating/rocking in equilibration buffer containing 0.06 M Tris/HCI, pH 6.8, 2% sodium dodecyl sulfate (SDS), 1% DTT and 20% glycerol. The second dimension was performed on a 12% SDS–PAGE for 8 h at 25 mA per gel. Gels were stained with colloidal Coomassie Blue G-250 (Bio Basic Inc., Canada) when 400 lg of proteins were loaded per strip or with silver nitrate when 120 lg was loaded per strip. 2-DE gels were scanned using Umax scanner software and analyzed with ImageMaster SoftwareTM (Amersham Pharmacia Biotech), including determination of normalized volume corresponding to the area and intensity of all protein spots detected on 2-DE gel. 2.3. In situ tryptic digestion of proteins Protein spots were excised from gels and subjected to in situ digestion with trypsin according to Fountoulakis and Langen (1997). Briefly, the gel pieces were sliced and rinsed with 50% (v/ v) acetonitrile (ACN) and 0.1 M NH4HCO3 for removal of salts and sodium dodecyl sulfate (SDS). Reduction of cysteine with DTT, and subsequent alkylation of free cysteine with iodoacetamide was then performed. In situ tryptic degradation was performed overnight at 37 °C, followed by three subsequent extractions of peptides. The pooled extracts were lyophilized and reconstituted

The peptide extraction (30–100 ppm) with equivalent matrix solution was loaded onto the MALDI-TOF target for MALDI-TOF MS analysis according to previously described procedures (Fountoulakis and Langen, 1997; Kussmann et al., 1997). MALDI-TOF MS was calibrated using trypsin auto-digestion peptide signals and matrix ion signals, with 4-cyano-4-hydroxycinnamic acid (HCCA) used as the matrix. All MALDI-TOF MS analysis was performed by a fuzzy logic feedback control system (Reflex III MALDI-TOF system Bruker, Karlsruhe, Germany) equipped with delayed ion extraction. Proteins were identified by peptide mass fingerprinting (PMF) using the MASCOT program (http://www.matrixscience.com), searching against the National Center for Biotechnology Information non-redundant (NCBInr) database. One missed cleavage per peptide and a mass tolerance of ±0.8 Da were allowed. Met-oxidation was set as a variable modification. In the present study, a global Mascot score greater than 61 was considered to be significant (P < 0.05). All proteins were identified as those with the top MASCOT score. 2.5. Immunoblot analysis PSC Proteins separated by 1-DE and 2-DE were transferred to nitrocellulose membranes (Amersham Biosciences) for 2 h at 200 mA in transfer buffer (48 mM Tris/HCl, pH 7.4, 39 mM glycine, and 20% methanol) at 4 °C. The membrane was blocked for 60 min with 5% skim milk in Tris-buffered saline (TBS) at 37 °C. After rinsing three times for 5 min with TBS, the membrane was incubated with mouse anti-E. multilocularis serum, at a dilution of 1:100 in 0.05% Tween 20 in TBS (TTBS) containing 5% skim milk for 1 h at 25 °C on a gentle shaker. Mouse antiE. multilocularis serum was prepared in advance as follows: 20 KM strain mice (6- to 8-week-old, 10 male and 10 female) were infected with E. multilocularis by intraperitoneal transplantation of freshly prepared metacestode vesicles (Dai and Gottstein, 1999); serum samples were collected from each of the mice at 26 weeks after infection and then were mixed as a pool of anti-E. multilocularis serum; serum samples were stored in 20 °C before use. The membrane was rinsed three times for 10 min, and incubated with the goat anti-mouse-IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Sigma, USA) at a dilution of 1:10,000 in TTBS containing 5% skim milk for 1 h at 37 °C. After three more washes with TTBS and one wash with 50 mM Tris/HCl buffer (pH 7.4), the blots were developed using dimethylaminoazobenzene (DAB) substrate. 3. 3 Results and discussion 3.1. 2-DE analysis of E. multilocularis PSC proteins To establish a sensitive and quantitative 2-DE protocol, we assessed the results when various amounts of PSC protein and different gel staining methods were used in the assay. The separation by 2-DE of 120 lg of solubilized PSC proteins stained with silver nitrate permitted the detection of around 500 spots using 3.5–10 linear immobilized pH-gradient strips (Fig. 1). A protein load of 400 lg per IPG strip (pH 3.5–10) was optimal for Coomassie Blue G-250 stained gels, which led to the detection of around 200 spots (Fig. 2). In these two methods, most protein spots on the gels were located between pH 4 and 9 with the molecular weight between 25 and 100 kDa. Five batches of E. multilocularis PSC protein were sep-

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3.2. Identification of E. multilocularis PSC proteins by MALDI-TOF MS analysis

Fig. 1. Two-dimensional electrophoresis (2-DE) analysis of Echinococcus multilocularis protoscolex proteins. 120 lg of protoscolex proteins were separated in the first dimension on an 18 cm IPG strip (pH 3.5–10). The second dimension of electrophoresis was performed on a 12% separating gel and then stained with silver nitrate.

One hundred prominent protein spots of various isoelectric points (pI) and molecular weights (MW) were selected for MALDI-TOF MS analysis. Twenty-five protein spots were subsequently identified by their peptide mass fingerprinting, corresponding to 14 different Echinococcus proteins (Fig. 2 and Table 1). All proteins listed in Table 1 are the highest ranked candidates that were unambiguously identified in the Mascot search. Most proteins presented at least five matched peptides with more than 20% sequence coverage, allowing us to confirm the correct identification of proteins. In some cases, some spots were related to similar parasite proteins, including spot 28 (related to Taenia solium actin) and spot 59 (related to Taenia solium oncosphere protein Tso22c). Nevertheless, the spectra of all identified proteins were inspected individually to ensure the quality of data. The matched peptides essentially showed the strongest peaks. A group of cytoskeletal proteins appeared to be very abundant in the E. multilocularis PSC stage (Fig. 2), including actin, tropomyosin, paramyosin, beta-tubulin and actin-binding protein (Table 1). Helminth cytoskeletal proteins have been claimed to be vaccine candidates (e.g. tropomyosin and paramyosin) or targets for anthelmintic attack (e.g. beta-tubulin) (Sereda et al., 2008; Wu et al., 2005; Roos et al., 1995). Another major group of proteins were related to heat shock proteins, including HSP70 (spot 47 and 48), GRP78 (spot 24) and HSP20 (spot 58). HSP70s are considered as inducible protective proteins critical for parasite survival and immuno-reactive proteins important in parasitic infection (Maresca and Kobayashi, 1994). Three proteins involved in metabolism, E. multilocularis glutathione transferase (GST) (spot 65), thioredoxin peroxidase (TPx) (spot 66) and transketolase (spot 74 and 75), were also identified. The detoxification and antioxidation by GSTs and TPx in parasitic helminthes may be particularly utilized, in an adaptive manner, to survive host-induced stress (Sheehan et al., 2001; Vibanco-Pérez and Landa-Piedra, 1998; Berggren et al., 2001). Furthermore, proteins with homology to hydatid disease diagnostic antigen P-29 (spot 34 and 38) and E. granulosus 14-3-3 protein (spot 14) also appeared to be very abundant in the PSC larval stage. Locations of some proteins on 2-DE gel in this study (e.g. tropomyosin, paramyosin, heat shock proteins, antigen P-29 and thioredoxin peroxidase) are very similar to those reported in an earlier proteomic analysis of E. granulosus PSC (Chemale et al., 2003). Interestingly, the presence of different pI variants of HSP70 and antigen P-29 (Fig. 2) is also consistent with the 2-DE analysis of E. granulosus, possibly owing to the same post-translational modification. Thus, we conclude that the pattern of expression and posttranslational modification of these proteins are highly conserved in these two species. 3.3. Identification of immunogenic proteins

Fig. 2. 2-DE analysis of Echinococcus multilocularis protoscolex proteins. Isoelectric focusing electrophoresis was performed with 400 lg of proteins for 14 KVh in a pH range of 3.5–10. SDS–PAGE was performed on a 12% separating gel and then stained with Coomassie Blue Colloidal G-250. The numbers indicate the identified protein spots that are listed in Table 1.

arated by 2-DE and the average position and number of the spots was determined with ImageMaster software. The protein patterns were very similar, thereby confirming the high reproducibility of our 2-DE protocol for E. multilocularis PSC protein detection and proteomic analysis.

In order to construct a systematic reference map of the immunogenic proteins in E. multilocularis PSC stage, immunoblot analysis of PSC 1-DE and 2-DE gels (pH 3.5–10) was performed using anti-E. multilocularis mice sera. About 15 bands reacted with sera on the 1-DE gel and more than 50 protein spots were positively detected on the 2-DE map (Fig. 3). When the same immunoblot was probed with sera from naive mice, no proteins were detected (data not shown). By comparison with Coomassie Blue-stained 2-DE gels, 15 immuno-reactive spots were identified, corresponding to 12 abundant proteins previously identified by their PMFs (Table 1). Analysis of the immunoblots indicated that most of the cytoskeletal proteins appeared to be antigenic, including paramyosin, tropomyosin, beta-tubulin and actin-binding protein (Fig. 3 and Table 1). HSPs (HSP70 protein, GRP78, and putative HSP20 related

Table 1 Results of MALDI-TOF MS analysis of Echinococcus multilocularis protoscolex proteins. Calculated pI

Coverage Scorea Matched peptides

Protein name and species

Protein IDb

1

42199

4.47

29%

68

8

Putative calreticulin [Echinococcus granulosus]

AAX73173

3 6 9 14 15 23 24 28 31 32 33 34 38 39 47 48 53

37800 32967 32967 27928 49813 98682 71631 41718 49813 50178 41776 27080 27080 31789 72509 72509 41324

5.36 4.65 4.65 4.91 4.75 5.21 5.09 5.3 4.75 4.75 5.29 5.65 5.65 6.67 5.66 5.66 5.74

42% 54% 42% 31% 42% 21% 31% 37% 48% 29% 32% 43% 37% 47% 36% 26% 39%

79 115 90 70 82 58 74 80 95 68 57 95 67 62 80 42 80

9 15 11 9 9 15 10 9 24 19 8 10 7 8 9 6 9

58 59 65 66

35690 38588 25454 21462

5.92 5.92 6.74 6.07

23% 41% 47% 37%

64 55 80 50

9 16 11 10

Similar to Actin-87E isoform 2 [Tribolium castaneum] Tropomyosin [Echinococcus multilocularis] Tropomyosin [Echinococcus multilocularis] Putative 14-3-3 protein [Echinococcus granulosus] Tubulin beta-2 chain [Echinococcus multilocularis] Paramyosin [Echinococcus granulosus] 78 kDa glucose-regulated protein [Echinococcus multilocularis] Actin [Taenia solium] Tubulin beta-2 chain [Echinococcus multilocularis] Tubulin beta-1 chain [Echinococcus multilocularis] Actin, muscle A2 [Bombyx mori] Hydatid disease diagnostic antigen P-29 [Echinococcus granulosus] Hydatid disease diagnostic antigen P-29 [Echinococcus granulosus] Similar to caspase-3, partial [Strongylocentrotus purpuratus] Heat shock cognate 70 kDa protein [Echinococcus granulosus] Heat shock cognate 70 kDa protein [Echinococcus granulosus] Actin-binding and severin family group-like protein [Echinococcus granulosus] Putative HSP20 related protein [Echinococcus multilocularis] Oncosphere protein Tso22c [Taenia solium] Glutathione transferase [Echinococcus multilocularis] Thioredoxin peroxidase [Echinococcus multilocularis]

74 75 90

72575 72575 98822

6.53 6.53 5.28

14% 21% 21%

52 63 74

7 9 16

Transketolase [Echinococcus multilocularis] Transketolase [Echinococcus multilocularis] Paramyosin [Taenia saginata]

a b

Function annotation

Calcium ion binding/cytoskeleton organization/cellular process regulation XP_975870 Cytoskeleton structure/cell mobility Q95PU1 Actin binding /cytoskeleton structure Q95PU1 Actin binding /cytoskeleton structure AAX73175 Protein domain specific binding/signal transduction Q9NFZ6 Microtubule constituent/GTP binding P35417 Filament structural component/motor activity Q24895 ATP-binding/stress response P68555 Cytoskeleton structure/cell mobility Q9NFZ6 Microtubule constituent/GTP binding Q9NFZ7 Microtubule constituent/GTP binding P07837 Cytoskeleton structure/cell mobility Q9U8G7 Protein binding/immunological activity Q9U8G7 Protein binding/immunological activity XP_792621 Cysteine-type peptidase activity/apoptosis Q24789 ATP-binding/stress response Q24789 ATP-binding/stress response AAK15753 Actin binding CAD12371 AAW88553 CAA59739 BAC11863 CAD45181 CAD45181 Q8T305

ATP-binding/stress response Putative immuno-reactive Glutathione transferase activity/metabolism Peroxidase activity/antioxidant activity/oxidative stress response Transferase activity/calcium ion binding Transferase activity/calcium ion binding Filament structural component/motor activity

Y. Wang et al. / Experimental Parasitology 123 (2009) 162–167

Nominal mass (Da)

Spot No.

Scores greater than 61 were significant (p < 0.05). NCBI protein accession number.

165

166

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Fig. 3. Western blot analysis of Echinococcus multilocularis protoscolex proteins. M: Marker; (A) SDS–PAGE; (B) Western blot analysis of 1-DE gels; (C) Western blot analysis of 2-DE gels. Numbers in (C) are identical to those described in Fig. 2 and Table 1.

protein) were also recognized by the serum derived from mice infected with E. multilocularis, suggesting the potential of these proteins as immunodiagnostic or vaccine candidates for AE infection. Proteins homologous to E. granulosus hydatid disease diagnostic antigen P-29 and the 14-3-3 protein were detected as well in the immunoblots. Calreticulin, a calcium-binding protein that can potentially interact with host receptors and signaling machinery (Nakhasi et al., 1998; Ferreira et al., 2004), was also found to be immuno-reactive (Fig. 3) and highly expressed in the PSC (Fig. 2). Further investigations into calreticulin are needed, in particular with regard to its impact on E. multilocularis larval infectivity and its potential role as vaccine candidate and drug target. 4. Conclusion Proteome analyses have been employed successfully for constructing reference maps of parasite proteins. However, the development of proteomic studies in Echinococcus spp. has been hampered by the lack of extensive protein sequence data for these parasites. This work presents a proteomic analysis of E. multilocularis protoscoleces proteins by 2-DE and mass spectrometry. A number of abundant and immunogenic proteins were identified by our approach. Though not all selected proteins could be identified, further advances can be expected from MALDI-TOF/TOF tandem mass spectrometry, in combination with the Echinococcus EST project currently being carrying out at the Sanger Institute. Acknowledgments We are very grateful to Professor Xuan-xian Peng for his kind help in 2-DE analysis. This work was supported by the Natural Science Fund of China, Grant 30471514, Program for New Century Excellent Talents in Fujian Province University (2007) and Project of Innovation Foundation of Xiamen University (2005). References Barrett, J., Jefferies, J.R., Brophy, P.M., 2000. Parasite proteomics. Parasitology Today 16, 400–403.

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