Acta Tropica 150 (2015) 79–86

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Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae Ruo Dan Liu, Jing Cui ∗ , Xiao Lin Liu, Peng Jiang, Ge Ge Sun, Xi Zhang, Shao Rong Long, Li Wang, Zhong Quan Wang ∗ Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou 450052, China

a r t i c l e

i n f o

Article history: Received 5 March 2015 Received in revised form 14 June 2015 Accepted 4 July 2015 Available online 13 July 2015 Keywords: Trichinella spiralis Intestinal infective larvae Muscle larvae Surface proteins proteome

a b s t r a c t The critical step for Trichinella spiralis infection is that muscle larvae (ML) are activated to intestinal infective larvae (IIL) and invade intestinal epithelium to further develop. The IIL is its first invasive stage, surface proteins are directly exposed to host environment and are crucial for larval invasion and development. In this study, shotgun LC-MS/MS was used to analyze surface protein profiles of ML and IIL. Totally, 41 proteins common to both larvae, and 85 ML biased and 113 IIL biased proteins. Some proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Gene ontology analysis revealed that proteins involved in generation of precursor metabolites and energy; and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process were enriched in IIL at level 4. Some IIL biased proteins might play important role in larval invasion and development. qPCR results confirmed the high expression of some genes in IIL. Our study provides new insights into larval invasion, host-Trichinella interaction and for screening vaccine candidate antigens. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Trichinella spiralis is a tissue-dwelling parasitic nematode infecting many kinds of carnivores and omnivores, and is the main aetiological agent of trichinellosis. Humans acquired this disease by the ingestion of raw or insufficiently cooked meat containing the T. spiralis muscle larvae (ML). T. spiralis ML are released in the stomach and activated to “intestinal infective larvae (IIL) by intestinal content or bile after 0.9 h post-infection (hpi) (Campbell, 1983). Then, the IIL invade, occupy and migrate through host’s intestinal epithelium cells (IECs) where they molt and develop to adults and reproduce newborn larvae (Liu et al., 2013). Therefore, ML are activated to IIL in intestines and invade intestinal mucosa to further develop is the critical step for establishing Trichinella infection in host (Ren et al., 2013). The IIL is the first invasive stage during the process of Trichinella infection. Since the parasite cuticle is directly in contact with the host environment, the cuticle surface of parasitic nematodes is antigenic in

∗ Corresponding authors at: Department of Parasitology, Medical College, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, PR China. Fax: +86 371 66997182 E-mail addresses: [email protected] (J. Cui), [email protected] (Z.Q. Wang). http://dx.doi.org/10.1016/j.actatropica.2015.07.002 0001-706X/© 2015 Elsevier B.V. All rights reserved.

many infected hosts (Pritchard et al., 1985). In a number of experimental systems antibodies are produced against surface molecules and mediate antibody dependent cell mediated cytotoxic reactions. T. spiralis surface proteins include the cuticle proteins themselves and the excretory–secretory (ES) proteins which were incorporated on the cuticle (Ortega-Pierres et al., 1984; Pritchard et al., 1985). They are directly exposed to the host’s immune system, are the main target antigens which induce the immune responses, and may play an important role in the invasion, development and immune escape process of T. spiralis larvae. In recent years, proteomics has been a powerful technique for detection and identification of specific proteins from different species of the genus Trichinella or different samples of T. spiralis (Robinson et al., 2007). Many studies have focused on the ES and somatic proteins with the aim of identifying key molecules correlated with the development process of the T. spiralis larvae (Bien et al., 2012; Robinson and Connolly, 2005). Previous proteomics studies showed that microenvironmental factors influence protein secretion by T.spiralis L1 in vitro and correlated this with infectivity to mice (Bolas-Fernandez et al., 2009). However, up to now, the comparative proteomic analysis of surface proteins of T. spiralis ML and IIL has not been reported. In the present study, we utilized the shotgun LC-MS/MS approach in combination with bioinformatics analysis to identify

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and characterize the surface protein expression profiles of T. spiralis ML and IIL. These data are expected to provide valuable hints for uncovering the molecular mechanism of the invasion of IECs by T. spiralis larvae and better understanding the interaction between parasite and host. 2. Materials and methods 2.1. Parasite and experiment animals T. spiralis isolate (ISS534) used in this study was collected from domestic pigs in Nanyang, Henan Province, China. Specific pathogen-free (SPF) six-week-old male Kunming mice were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, China). All animal procedures reported herein were reviewed and approved by the Life Science Ethics Committee of Zhengzhou University (Permission No. SYXK 2011-0001) 2.2. Collection of ML and IIL T. spiralis ML was recovered from infected mice at 42 days post infection (dpi) by artificial digestion with 1% pepsin (1:3,000) and 1% hydrochloric acid (Gamble et al., 2000; Li et al., 2010). After recovery, a part of ML were directly pooled for extracting surface proteins, the other part were orally inoculated into 50 mice, with 5000 ML per mouse (Ren et al., 2013). The infected mice were euthanized at 6 h post infection, the small intestine was opened longitudinally, and washed three times in ice-cold normal saline, then cut into 2 cm long fragments with sharp scissors and cultured in normal saline at 37 ◦ C for 2.5 h. Then, IIL larvae released from small intestine to normal saline were collected by Baermann’s method as previously described (Gamble et al., 2000). 2.3. Preparation of surface proteins from ML and IIL Surface proteins from ML and IIL were prepared with 0.25% hexadecyl trimethyl ammonium Bromide (Sigma, USA) and 2% sodium deoxycholate (Sigma, USA) (Pritchard et al., 1985; Cui et al., 2013). The supernatant was obtained by centrifugation at 4 ◦ C, 11,000 × g for 20 min, and dialyzed against deionized water at 4 ◦ C for 2 days. Then the supernatant contained surface proteins were concentrated through Amicon Ultra-3Centrifugal Filter Unit (MW cut off 3 kDa) at 4 ◦ C, 5000 × g. The surface proteins were stored at −80 ◦ C before use. Totally, larval surface protein samples were prepared as three biological replicates, mixed and subjected to the following SDS-PAGE and LC-MS/MS. 2.4. SDS-PAGE Protein samples (20 ␮g) were separated by SDS-PAGE on 5% stacking gels and 12% resolving gels (83 × 73 × 1.0 mm) in a minivertical electrophoresis system (Bio-Rad Laboratories, USA) (Wang et al., 2011). After electrophoresis, the gel was stained with 0.25% Coomassie brilliant blue (CBB) R-250 (Sigma, USA) for 4 h, and then bleached with the eluate. 2.5. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) The CBB-stained SDS-PAGE gel lane was manually cut into 7 slices depending on protein molecular weight (MW). Each slice was diced into 1 mm × 1 mm pieces, and then subjected to in-gel tryptic digestion (Wang et al., 2012). After digestion, Peptide mixtures were separated by high-performance liquid chromatography (HPLC) followed by tandem MS analysis.

2.6. Protein identification and annotation All MS/MS spectra were searched against the NCBI T. spiralis protein database (33,133 sequences, 6/10/2014, http://www.ncbi. nlm.nih.gov/) by using SEQUEST algorithm. Multiple peptide identifications were generally returned by SEQUEST for each MS/MS spectrum and for each parention change state. The protein identification criteria used in our study was based on Delta CN (≥0.1) and Xcorr (one charge ≥ 1.9, two charges ≥ 2.2, three charges ≥ 3.75) (Washburn et al., 2001). 2.7. Bioinformatic analysis MW and isoelectric points (pI) were calculated using the online compute pI/MW tool (http://web.expasy.org/compute pi/). The online tools (http://www.cbs.dtu.dk/services/TMHMM/) and (http://www.cbs.dtu.dk/services/SignalP/) were used to analyze the presence of a signal peptide and a transmembrane domain. Bioinformatic Analysis InterProscan software (http://www.ebi.ac. uk/Tools/pfa/iprscan/) was used to perform protein sequences searches to identify signatures (Zdobnov and Apweiler, 2001). Then the matched terms were subjected to GO categories using the Web Gene Ontology Annotation Plot (WEGO) (http://wego.genomics. org.cn/cgi-bin/wego/index.pl) (Ye et al., 2006). 2.8. Quantitative real-time PCR (qPCR) Select six genes from the identified proteins, then further determine the transcription level by qPCR. Total RNAs were extracted from ML and IIL using TRIzol reagent (Takara, Japan) and then reverse-transcribed into cDNA by using a PrimeScript® RT reagent kit with gDNA Eraser (Takara). Using the Primer 5.0 software to designed the gene-specific primers (listed in Table 1). The q-PCR was performed in 7500 fast real-time PCR system (Applied Biosystems, USA) according to the following references (Liu et al., 2013). 2.9. Statistical analysis A Fisher’s exact test calculated by SPSS version 17.0 (SPSS Inc., Chicago, IL) was used to assess significant differences between ML and IIL surface protein samples (P < 0.05) based on the number of proteins per GO category in each sample (Zhang et al., 2013). A two-sample t-test calculated was used to assess relative expression levels significant differences between ML and IIL (P < 0.05). 3. Results 3.1. Global analysis of surface proteome Surface proteins from ML and IIL were separated by SDS-PAGE and subjected to shotgun LC-MS/MS analysis (Fig. 1). A total of 287 ML surface proteins and 402 IIL surface proteins were identified by searching the databases of T. spiralis in NCBI. Removing redundant sequences, ML and IIL surface protein data were clustered into 126 and 154 unique proteins, of which 82 (65.1%) and 99 (64.3%) proteins were annotated by InterProscan software. A comparison of the identified proteins between ML and IIL was shown in Fig. 2. Overall, 41 common identified proteins constituted 32.5% of total proteins in ML and 26.6% in IIL (Table 2). Of total proteins in different larvae, 85 (67.5%) were ML biased and 113 (73.4%) were IIL biased (Table 3). Out of 41 common identified proteins, 19 (46.3%) proteins possessed a signal peptide and 11 (26.9%) possessed a transmembrane domain. In 113 IIL biased proteins, 23 (20.3%) proteins contained a signal peptide and 24 (21.2%) contained a transmembrane domain.

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Table 1 Primers used in qPCR assays. Gene

GenBank accessionnumber

Primer sequence

Product size (bp)

Peptidyl-prolyl cis–trans isomerase B (PPIB)

XM 003374497.1

257

Putative copper/zinc superoxide dismutase (SODC)

XM 003381953.1

Putative protein kinase domain protein (PKDP)

XM 003380891.1

Adult-specific DNase II-10 (DNase II)

AY963704.1

Putative Low-density lipoprotein receptor domain class A (LDLRA)

XM 003371765.1

Secretin receptor(SR)

XM 003375966.1

G3PDH(Reference)

AF452239

F 5 -TTACCAATGGCGATGGCACT-3 R 5 -ACGATCGCTAGGACCAGTCT-3 F 5 -GGGTTGCATTTCTGCTGGTG-3 R 5 -GGCTTTCGTCATCACCTCCT-3 F 5 -CGCGGTTTACGAATCCGAAC-3 R 5 -TGTTGGCCAGTTCAGCTAGG-3 F 5 -CGGCGGCTTTGTTCAGAAAC-3 R 5 -ACATATCCGCAAAATAAACTGCTCA-3 F 5 -CGAACACCGAATTCCATCGC-3 R 5 -ACGACCACAACTACACGTCC-3 F 5 -GAGGTGGAATACAGCTGCGA-3 R 5 -AGAACTCGTTTCGGCTGGTT-3 F 5 -AGATGCTCCTATGTTGGTTATGGG-3 R 5 -GTCTTTTGGGTTGCCGTTGTAG-3

239 233 87 119 156 196

Table 2 A part of the identified common surface proteins from T. spirslis ML and IIL with annotations. T. spiralis protein Accession name number

Putative gi|339236167 onchocystatin gi|162535 43 kD secreted glycoprotein gi|339258142 Hypothetical protein Tsp 04680 Serine protease gi|168805931 gi|339246491 Collagen alpha-2(IV) chain Serine protease gi|13242031 precursor P49 antigen, partialgi|162542 Enteropeptidase gi|339241229 Putative dedicator gi|316972932 of cytokinesis protein 5 Putative scavengergi|339245959 receptor cysteine-rich domain protein a

Theoretical Mr/pIa

Signal peptide

Transmembrane ML domain

IIL

No. of unique peptides

Cover percent (%)

No. of unique peptides

Cover percent (%)

21.80/8.41

No

No

6

26.56

6

29.69

37.70/5.95

Yes

No

8

31.98

7

25.58

36.31/4.75

No

No

7

22.56

3

10.37

35.21/5.97 158.06/7.69

Yes No

No No

5 2

28.12 1.96

2 2

8.95 1.59

71.62/8.83

Yes

No

11

17.39

2

3.00

37.70/5.95 70.11/7.17 107.27/5.81

No No No

No No No

3 9 3

12.06 13.60 4.53

6 4 3

20.06 6.88 5.69

363.35/7.81

No

Yes

2

0.59

2

0.53

Theoretical molecular mass (kDa) and pI.

Fig. 1. Separation of surface proteins from T. spiralis ML and IIL by SDS-PAGE. Each gel lane was cut into 7 slices. M, protein marker with low molecular weights; lane 1, muscle larvae; lane 2, intestinal infection larvae.

Fig. 2. Venn diagram of the number of identified surface proteins from ML and IIL of T. spiralis. The overlap shows the number of common-expressed proteins between ML and IIL. The number in parentheses denotes the proteins with annotation from the InterProscan software.

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Table 3 A part of the identified surface proteins which are highly expressed in T. spirslis IIL with annotations.

a

T. spiralis protein name

Accessionnumber

TheoreticalMr/pIa

Putative low-density lipoprotein receptor domain class A Enolase

gi|339253180

499.09/5.38

gi|316968557

Fructosebisphosphate aldolase class-I Adult-specific DNase II-3 Adult-specific DNase II-1 Adult-specific DNase II-11, pattial Adult-specific DNase II-8 Adult-specific DNase II-9 Adult-specific DNase II-10 Deoxyribonuclease II family protein Putative ATP synthase F1, delta subunit

Cover percent(%)

Signal peptide

Transmembrane domain

Molecular function

0.45

No

Yes

Calcium ion binding; protein binding

53.36/5.87

17.28

Yes

No

gi|339250594

41.11/6.61

15.08

No

No

gi|63095175

38.45/7.58

46.70

Yes

No

gi|63095171

38.06/7.58

38.33

Yes

No

gi|63095191

34.48/8.75

32.91

No

No

gi|63095185

38.18/8.07

27.30

Yes

No

gi|63095187

38.18/8.06

22.13

Yes

No

gi|63095189

38.25/6.52

27.30

Yes

No

gi|339245551

36.71/9.04

11.71

No

Yes

gi|316973880

67.90/8.84

2.19

No

No

Putative protein kinase domain protein

gi|316976081

195.74/5.38

1.16

No

Yes

Triosephosphate isomerase

gi|316968042

30.78/8.04

9.35

No

No

Peptidyl-prolyl cis–trans isomerase B Putative copper/zinc superoxide dismutase Secretin receptor

gi|339245823

22.47/8.99

8.87

Yes

No

gi|339233768

18.96/5.88

26.11

No

No

Magnesium ion binding; phosphopyruvate hydratase activity Catalytic activity; fructosebisphosphate aldolase activity Deoxyribonuclease II activity Deoxyribonuclease II activity Deoxyribonuclease II activity Deoxyribonuclease II activity Deoxyribonuclease II activity deoxyribonuclease II activity Deoxyribonuclease II activity Protontransporting ATP synthase activity, rotational mechanism Protein kinase activity; protein tyrosine kinase activity; ATP binding; transferase activity, transferring phosphoruscontaining groups Catalytic activity; triose- phosphate isomerase activity Peptidyl-prolyl cis–trans isomerase activity Superoxide dismutase activity; metal ion binding

gi|339240177

67.59/9.2

3.90

No

No

Binding

Theoretical molecular mass (kDa) and pI.

3.2. Theoretical two-dimensional distribution of identified proteins For both ML and IIL, the MW ranged from 7.38 kDa to 1393.83 kDa with the most proteins distributed between 10 kDa and 160 kDa. The pI ranged from 4.17 to 12.21, with about 96% of proteins distributed between 4 and 10. MW and pI distribution of larva-biased proteins were similar to the distribution of total proteins (Fig. 3). 3.3. Gene ontology annotation A total of 82 proteins from ML were linked to 254 GO terms and 99 proteins from IIL were linked to 289 GO terms. Of 254 GO categories in ML samples, 57 were common and 197 were unique; in IIL samples, 232 were unique GO categories (Fig. 4). To further under-

stand the functions of the proteins identified, annotation terms were classified into cellular component, molecular function and biological process according to GO hierarchy using WEGO (Fig. 5). After determining significant differences for all levels of GO, we observed no significant differences between the two larvae at level 2 in three hierarchical structures, but 19 significant differences were detected at higher levels (Table 4). These differentially annotated categories might contain shared and unique GO terms. In the biological process ontology, a large proportion of proteins were related to metabolic process (GO:8152, 53.7% of 82 annotated ML surface protein, 49.5% of 99 annotated IIL surface proteins) and cellular process (GO:9987, 42.7%, 52.5%). Besides, cellular component organization (GO:16,043), pigmentation (GO:43,473), localization (GO:51,179), establishment of localization (GO:51,234) and biological regulation (GO:65,007) were also showed active. Moreover, higher levels (above level 2) had 12 differentially anno-

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Table 4 Differential GO categories of identified surface proteins from ML and IIL of T. spiralis.a X. level

G0 ID

Term

ML number 1

ML number 2

IIL number 1

Biological process 3

GO:0006807

60

15

65

28

0.039

4

GO:0006139

60

15

65

28

0.039

5 3 4

GO:0006259 GO:0009056 GO:0009057

60 60 60

7 22 20

65 65 65

19 11 7

0.026 0.015 0.002

5 6 3 4

GO:0043285 GO:0030163 GO:0044237 GO:0006091

60 60 60 60

20 18 26 0

65 65 65 65

7 7 41 6

0.002 0.013 0.032 0.028

4 2 7 Molecular Function 4 5

GO:0019538 GO:0009987 GO:0006508

Nitrogen compound metabolic process Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; DNA metabolic process Catabolic process Macromolecule catabolic process Biopolymer catabolic process Protein catabolic process Cellular metabolic process Generation of precursor metabolites and energy Protein metabolic process Cellular process Proteolysis

60 60 60

28 35 18

65 65 65

16 53 7

0.014 0.006 0.013

72 72

18 18

95 95

7 7

0.002 0.002

6 4 5 6

GO:0004175 GO:0017171 GO:0008236 GO:0004252

Peptidase activity Peptidase activity, acting on l-amino acid peptides Endopeptidase activity Serine hydrolase activity Serine-type peptidase activity Serine-type endopeptidase activity

72 72 72 72

18 16 16 16

95 95 95 95

6 6 6 6

0.001 0.003 0.003 0.003

GO:0008233 GO:0070011

IIL number 2

E-value

a The GO categories that are statistically significantly different between the two samples are shown at all levels of GO under biological processes and molecular functions. Number 1 refers to the number of the proteins that fall within each GO category at the first level while number 2 means that the number of proteins which are classified into the differential GO subcategories. Significantly different (p < 0.05) categories, which are shown, are calculated using Fisher’s exact test.

Fig. 3. Theoretical two-dimensional (MW, pI) distribution of identified surface proteins which are highly expressed in ML or IIL of T. spiralis.

in IIL and macromolecule catabolic process (GO:0009057) and protein metabolic process (GO:0019538) were enriched in ML. Considering the molecular function category, seven subcategories were assigned, of which the activities of binding (GO:5488, more than 50% in both ML and IIL) and catalytic activity (GO:3824, more than 45% in both) were the two major molecular function categories. Most of the assigned binding activity could be assigned to protein binding (GO:5515), nucleotide binding (GO:166), nucleoside binding (GO:1882), nucleic acid binding (GO:3676) and ion binding (GO:43,167). The majority of proteins in catalytic activity group are related to hydrolase activity (GO:16,787). In addition, 6 differentially annotated categories were all enriched in ML at higher levels (above level 2): peptidase activity (GO:0008233), peptidase activity, acting on L-amino acid peptides (GO:0070011), endopeptidase activity (GO:0004175), serine hydrolase activity (GO:0017171), serine-type peptidase activity (GO:0008236), serine-type endopeptidase activity (GO: 0004252). For the cellular components category, proteins mapping to cell (5623), cell part (GO:44,464), macromolecular complex (GO:32,991) and organelle (GO:43,226) related GO terms were the most abundant. No significant differences were observed between the two samples at any level in cellular components category.

3.4. Relative quantitative mRNA expression analysis Fig. 4. Venn diagram of the number of gene ontology terms from ML and IIL samples, including all matches from biological process, molecular function, and cellular component. The number in parentheses denotes the total number of GO categories.

tated categories, 6 of which were enriched in ML and 6 of which were enriched in IIL. At level 4, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (GO:0006139), and generation of precursor metabolites and energy (GO:0006091) were enriched

To further examine the gene expression levels of some interesting proteins, six genes (PPIB, SODC, PKDP, DNase II, LDLRA and SR) of IIL were selected (Fig. 6). The expression level of SODC, DNase II, LDLRA and SR in IIL was significantly higher than that in ML (p < 0.05), which was approximately consistent with our proteomic analysis results. Unexpectedly, the gene expression of PPIB and PKDP seems inconsistent with their protein expressions. The PPIB and PKDP in IIL were expressed at low level.

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Fig. 5. Gene ontology categories for identified surface proteins of ML and IIL of T. spiralis. The identified proteins were classified into cellular component, molecular function, and biological process by WEGO according to their GO signatures. The number of genes denotes that of proteins with GO annotations. The left-hand shows its proportion in total genes of related organs with GO terms.

Fig. 6. qPCR comparison of the expression levels of the selected six genes in T. spiralis larvae. Total RNAs from muscle larvae (ML) and intestinal infective larvae (IIL) were subjected to qPCR as described in “Section 2.” The fold change in the target genes, normalized to glyceraldehyde 3-phosphate dehydrogenase (G3PDH) and relative to the expression in the larvae, was calculated using comparative Ct (2−DCt ) method. *Significant difference of gene expression compared to controls. PPIB: peptidyl-prolyl cistrans isomerase B; SODC: putative copper/zinc superoxide dismutase; PKDP: putative protein kinase domain protein; DNase II: adult-specific DNase II-10; LDLRA: putative low-density lipoprotein receptor domain class A; SR: secretin receptor.

4. Discussion Previous studies showed that ML can’t invade the IECs cultured in vitro model unless they are exposed to the intestinal milieu or bile and activated into the IIL (Ren et al., 2013; Wang et al., 2012). Therefore, comparison of surface protein components of T. spiralis ML and IIL and study on their molecular function could provide

attractive information to elucidate the mechanism of parasite invasion, immune evasion and identify possible molecular targets for vaccine. In this study, we used a shotgun proteomic approach to identify proteins on the cuticle surface proteins of ML and IIL of T. spiralis. Overall, surface protein data of ML and IIL were clustered into 126 and 154 unique proteins, of which 41 common identified proteins. Compared with their physicochemical characteristics,

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most of the proteins are middle-to-low molecular weight, most of the pI distributed between 4 and 10. These results might elucidate physiological differences between the ML and IIL. T. spiralis ML persist in a hostile environment of hosts for decades without experiencing detectable damage (Campbell, 1983). ML undergoes physiological adaptations, so as to evade host immune system attack and adapt to a new living environment. The parasite strategy is immune escape, in addition to the exploitation of host endocrine and immune signals in which the surface proteins are important (Nagano et al., 2009). In our study, putative scavenger receptor cysteine-rich domain protein and putative onchocystatin were common identified in ML and IIL. Potential roles of scavenger receptors include uptake and transport of LDLs, phospholipids and cholesterol from host for use in the synthesis and maintenance of membranes, for growth and development (Acton et al., 1994), and host-immune evasion by accumulation and display of host-lipids on their surface (Furlong et al., 1992). Identification of this protein on the cuticle surface of ML and IIL suggested that it might be important for T. spiralis development and survival. Filarial cystatin of O. volvulus (onchocystatin) belongs to a superfamily of cystatins. Cystatins in parasitic nematodes not only have the unique inhibition activity on cysteine proteases but also modulate the host immune response and have an important role in the moulting and immune evasion from host response and the adaptation to parasitism (BolasFernandez et al., 2009; Klotz et al., 2011). Onchocystatin might have a role in the regulation of parasite cysteine proteases during molting or hatching of the worms (Lustigman et al., 1992). Recombinant T. spiralis cystatin-like protein plays an important role in Trichinella resistance to rapid expulsion (Liu et al., 2014). The expression of putative onchocystatin might indicate that it have a role in Trichinella escaping immune attack. In this study, the identified surface proteins of ML and IIL were further gene ontology annotation to provide a comprehensive understand. In the biological process ontology, a large proportion of proteins were related to metabolic process and cellular process. GO category comparisons indicated that generation of precursor metabolites and energy (GO:0006091) and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (GO:0006139) were enriched in IIL at level 4. Proteins in generation of precursor metabolites and energy (GO:0006091) subcategory including enolase, triosephosphate isomerase, fructose- bisphosphate aldolase class-I, ATP6 15606 ATP synthase F0 subunit 6, putative ATP synthase F1, delta subunit, and oxoglutarate dehydrogenase. Enolase is an enzyme which participates in the glycolytic pathway. Enolase can promote the larval migration through tissues by plasminmediated proteolysis such as degradation of host’s extracellular matrix (Jolodar et al., 2003). In addition, Enolase of parasites enhances the activation of plasminogen, and plasminogen mediated by enolase contributes to larval migration in tissues (Nakada et al., 2005). Therefore, the glycolytic process might be more important for IIL than ML, and the enolase may play some role in parasite invasion to the host tissue through combination with plasminogen. The category of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (GO:0006139) was enriched in IIL. IIL samples had several biased proteins in this subcategory including adult-specific DNase II-1,3,8,9,10,11, putative ATP synthase F1, delta subunit, and oxoglutarate dehydrogenase et al. Recently, it has been shown that extracellular nucleotides are the key regulators of molecular signals in tissue damage, which signal through purinergic receptors (Gounaris and Selkirk, 2005). Evidence is provided that Nucleotide-metabolizing enzymes catalyze the degradation of extracellular nucleotides, with a potential physiological role in the regulation of purinergic signaling (Gounaris, 2002). In vertebrates, DNase II play differential roles in arious development processes and DNA degradation during cell death, and they have important regulative action in organismal homeostasis (Evans and Aguilera,

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2003). In C. elegans, DNase II, which is involved in apoptotic DNA degradation and development, is important for regulative action in DNA degradation (Lai et al., 2009). In T. spiralis, Plancitoxin-1-Like DNase II Gene has been cloned and characterized, and they might remove damaged DNA during the growth and development of T. spiralis (Liao et al., 2014). In order to confirm that ML and IIL surface proteins identified by shotgun LC-MS/MS are differentially expressed, the transcription levels were further determined by qPCR. The results of qPCR showed that the expression of four genes from six genes of IIL were up-regulated compared with that of ML, which was approximately consistent with our proteomic analysis results. The expression of PPIB and PKDP genes seems inconsistent with their protein expressions. This inconsistency between transcription and protein expression levels might be caused by the differences between transcriptome and proteome. For a better understanding of differential expression of the proteome between the surface proteins of ML and IIL, the quantitative proteomics approaches need to be used in the future research. 5. Conclusions The present study provided a reliable catalog and a complete analysis of the proteome expression profiles of the surface proteins of T. spiralis ML and IIL. Some identified proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Some IIL highly expressed GO categories might consist of T. spiralis proteins associated with invasion and development. These results provide valuable information for further understanding the parasite-host interaction, the larval development and invasion of intestinal epithelium, and for screening vaccine candidates and drug targets against T. spirslis infection. Acknowledgements The authors wish to thank Shanghai Applied Protein Technology Co. Ltd. for the technology support. This work was supported by National Natural Science Foundation of China (No. 81471981, 81271860). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica.2015. 07.002 References Acton, S.L., Scherer, P.E., Lodish, H.F., Krieger, M., 1994. Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J. Biol. Chem. 269, 21003–21009. Bien, J., Nareaho, A., Varmanen, P., Gozdzik, K., Moskwa, B., Cabaj, W., Nyman, T.A., Savijoki, K., 2012. Comparative analysis of excretory-secretory antigens of Trichinella spiralis and Trichinella britovi muscle larvae by two-dimensional difference gel electrophoresis and immunoblotting. Proteome Sci., 10. Bolas-Fernandez, F., Dea-Ayuela, M.A., Connolly, B., Robinson, M.W., 2009. Micro-environmental conditions modulate protein secretion and infectivity of the Trichinella spiralis L1 larva. Vet. Parasitol. 159, 236–239. Campbell, W.C., 1983. Trichinella and Trichinosis. Plenum Press, New York, pp. 75. Cui, J., Liu, R.D., Wang, L., Zhang, X., Jiang, P., Liu, M.Y., Wang, Z.Q., 2013. Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry. Parasite Vectors 6, 355. Evans, C.J., Aguilera, R.J., 2003. DNase II: genes, enzymes and function. Gene 322, 1–15. Furlong, S.T., Thibault, K.S., Rogers, R.A., 1992. Fluorescent phospholipids preferentially accumulate in sub-tegumental cells of schistosomula of Schistosoma mansoni. J. Cell Sci. 103, 823–830 (Part 3). Gamble, H.R., Bessonov, A.S., Cuperlovic, K., Gajadhar, A.A., van Knapen, F., Noeckler, K., Schenone, H., Zhu, X., 2000. International Commission on Trichinellosis: recommendations on methods for the control of Trichinella in

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