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  • Open Access

Trichinella britovi muscle larvae and adult worms: stage-specific and common antigens detected by two-dimensional gel electrophoresis-based immunoblotting

Parasites & Vectors201811:584

https://doi.org/10.1186/s13071-018-3177-x

  • Received: 27 July 2018
  • Accepted: 28 October 2018
  • Published:

Abstract

Background

Trichinella britovi is the second most common species of Trichinella that may affect human health. As an early diagnosis of trichinellosis is crucial for effective treatment, it is important to identify sensitive, specific and common antigens of adult T. britovi worms and muscle larvae. The present study was undertaken to uncover the stage-specific and common proteins of T. britovi that may be used in specific diagnostics.

Methods

Somatic extracts obtained from two developmental stages, muscle larvae (ML) and adult worms (Ad), were separated using two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. The positively-visualized protein spots specific for each stage were identified through liquid chromatography-tandem mass spectrometry (LC-LC/MS).

Results

A total of 272 spots were detected in the proteome of T. britovi adult worms (Ad) and 261 in the muscle larvae (ML). The somatic extracts from Ad and ML were specifically recognized by T. britovi-infected swine sera at 10 days post infection (dpi) and 60 dpi, with a total of 70 prominent protein spots. According to immunoblotting patterns and LC-MS/MS results, the immunogenic spots recognized by different pig T. britovi-infected sera were divided into three groups for the two developmental stages: adult stage-specific proteins, muscle larvae stage-specific proteins, and proteins common to both stages. Forty-five Ad proteins (29 Ad-specific and 16 common) and thirteen ML proteins (nine ML-specific and four common) cross-reacted with sera at 10 dpi. Many of the proteins identified in Ad (myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin) are involved in structural and motor activity. Among the most abundant proteins identified in ML were 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. Heat-shock protein, intermediate filament protein ifa-1 and intermediate filament protein B were identified in both proteomes.

Conclusions

To our knowledge, this study represents the first immunoproteomic identification of the antigenic proteins of adult worms and muscle larvae of T. britovi. Our results provide a valuable basis for the development of diagnostic methods. The identification of common components for the two developmental stages of T. britovi may be useful in the preparation of parasitic antigens in recombinant forms for diagnostic use.

Keywords

  • Trichinella britovi
  • Adult worm
  • Muscle larvae
  • 2-DE
  • Mass spectrometry
  • Immunoblotting

Background

Trichinellosis is an important food-borne parasitic worldwide zoonosis caused by nematodes belonging to the genus Trichinella and is known to have high socioeconomic and medical significance. Humans typically acquire trichinellosis through the consumption of raw or improperly-processed meat of either farmed or wild animals containing infective muscle larvae (ML) of Trichinella [13]. The entire life-cycle of the parasite takes place in a single host. Trichinella displays three major antigenic stages: muscle larvae (ML), adult worms (Ad), and newborn larvae (NBL). Muscle larvae ingested with animal-derived meat are released into the host stomach upon the activation of digestive enzymes; they then migrate to the epithelial cells of the small intestine where they molt and transform into adult worms (Ad) within 48 hours post-infection (pi). Newborn larvae (NBL) are released after five days post-infection (dpi) and move through the lymphatic vessels to reach the striated muscle, where they grow and develop into encapsulated and non-encapsulated forms [4, 5]. All developmental stages of Trichinella elicit a protective immune response, as well as antigens which can be used for serological detection of Trichinella spp. infection. Several reports note that the Trichinella antigens produced by adult worms, new-born larvae and muscle larvae are stage-specific [68]. Our previous study indicated that together with stage-specific proteins, T. spiralis produces species-specific and common proteins for each developmental stage [911]. Although a few Trichinella antigens have been fully characterized, the complex interactions between the parasite and the host’s immune system are not yet fully understood [1216]. Thus, there is still a need to find other parasite proteins which may play an important role during the establishment of infection, which influence immune evasion strategies or modulate the host response. Recent studies have shown that a serine protease inhibitor released by T. spiralis may allow it to escape immune attack, and is related to the survival and colonization of the parasite in the hosts [17]. Identification of these proteins is not only important for understanding parasite-host interrelations, but is also a key factor in the development of serological diagnostic methods for species-specific differentiation and for detecting early-stage infection.

The combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometry has been widely used to characterize the protein profiles of various Trichinella species [9, 1821]. When used together with immunoblotting, the techniques enable the identification of the proteins that induce immune response and which could be used for immunodiagnosis. This immunoproteomics tool has previously been used to determine both the characteristics of immunogenic proteins and the serological response directed against parasites, such as Schistosoma japonicum [22], Toxoplasma gondii [23], Ascaris lumbricoides [24] and Taenia solium [25]. As T. spiralis is considered the main etiological agent of most human infections and deaths, most studies have focused only on the identification of potentially immunogenic proteins expressed by T. spiralis stages [20, 2629]. Although T. spiralis is commonly used as a representative species of the genus Trichinella, T. pseudospiralis, T. nativa and the T8 genotype, have also been described as being valuable sources of information regarding the parasite proteins needed for the development of immunological diagnostics [18, 19, 30].

Over the years, numerous cases with trichinellosis have been attributed to T. britovi, considered the second-most common species of Trichinella and one that may affect human health [3136]. Although the clinical and biological features observed during human infection caused by T. spiralis and T. britovi are different, it is not possible to attribute these features to a single species because the number of infective larvae is unknown. Trichinella spiralis infections are typically more severe than those caused by T. britovi, and the main distinctions between the two types of infections were that patients infected with T. spiralis displayed a longer duration of parasite-specific IgG, increased CPK levels, and a more severe intestinal symptomatology than those infected with T. britovi. This could be due to the fact that the fecundity of T. britovi females is lower than those of T. spiralis [36]. Our previous proteomic study of the excretory-secretory proteins of T. britovi muscle larvae found that the 5'-nucleotidase and serine protease may be potential proteins for diagnosis [9]. Currently, little is known about the protein profile shared by all developmental stages of T. britovi. Therefore, there is a need for more information about common and stage-specific T. britovi proteins to aid the development of species-specific diagnostics, and to better understand the adaptation of T. britovi to a parasitic niche and its host-parasite relationship.

The aim of the present study was to identify the T. britovi proteins that may be used in specific diagnostics. Somatic antigen extracts obtained from two developmental stages of T. britovi, muscle larvae (ML) and adult worms (Ad), were separated by two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. In addition, any positively-visualized proteins specific for each stage were further identified by liquid chromatography-tandem mass spectrometry (LC-LC/MS).

Methods

Experimental animals and collection of T. britovi adult worms and muscle larvae

The T. britovi nematodes had been maintained by several passages in male C3H mice at the Institute of Parasitology, PAS. To generate ML and Ad forms of T. britovi, the mice were orally infected with a dose of 700 ML T. britovi. ML were collected 42 days post-infection (dpi), and Ad were collected at 4 dpi. Muscle larvae of T. britovi were recovered by HCl-pepsin digestion from the previously-infected mice [37]. The recovered ML were subsequently purified several times with water through succeeding steps of sedimentation in cylinders. After the final sedimentation, the ML were collected into 1.5 ml tubes. The larval pellet was extensively washed three times in phosphate-buffered saline (PBS) supplemented with antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin). The adult worms were collected from the small intestine of C3H mice (3–4 months-old). Briefly, after recovery, the intestines were washed with sterile water with the use of a syringe, cut longitudinally and crosswise into 1–2 cm pieces, placed on a mesh in a conical dish filled with RPMI 1640 medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) supplemented with 25 mM HEPES, 2 mM L-glutamine, antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin) and incubated for three hours at 37 °C. Any Ad worms located on the bottom of the dish were then collected into 15 ml tubes, and washed three times with PBS supplemented with antibiotics. The T. britovi stages were then stored at -70 °C before protein extraction and proteomic analysis.

Protein extraction

The same protein sample preparation procedure was used for both T. britovi stages. After thawing, the collected T. britovi ML and Ad were again extensively washed three times in PBS and then suspended in a lysis buffer (8 M Urea, 4% CHAPS, 40 mM Trizma base), supplemented with protease inhibitor cocktail (Roche, Berlin, Germany). The protein extract was then homogenized in glass Potter-homogenizer and disintegrated by sonication three times for 10 s. The lysis extract was clarified by centrifugation at 14,000× g at 4 °C for 15 min. The supernatant was collected, placed in new 1.5 ml tubes, and protein concentration was measured with the use of a NanoDrop-1000 Uv/Vis Spectrometer (NanoDrop Technologies, Wilmington, USA). The proteins were frozen at -70 °C for further analysis.

Two dimensional gel electrophoresis (2-DE)

Three replicates of T. britovi protein samples were run in parallel on three immobilized pH-gradient IPG strips (RioRad, Hercules, USA). The 100 μg samples of previously prepared protein extracts from T. britovi Ad and ML were purified with the 2-D Clean-Up Kit (GE Healthcare, New Jersay ,USA) in accordance with the manufacturer’s protocol. After the final centrifugation step, the protein pellets were rehydrated overnight in 250 μl of 2-D Starter Kit Rehydration/Sample Buffer (BioRad, Hercules, USA) and loaded onto a 7 cm pH 3-10 IPG strips (BioRad, Hercules, USA) for first dimension separation. The protein samples were separated in accordance with their pI values through isoelectric focusing (IEF) using a Protean IEF Cell (BioRad) device at 20 °C as follows: first step 15 min at 250 V; second step rapid ramping to 4000 V for two hours; and third step for 15,500 Vhrs (current limit of 50 μA/IPG strip). After focusing, the strips were submitted for two steps of equilibration, the first for 25 min in ReadyPrep 2-D starter Kit Equilibration Buffer I, containing DTT (BioRad, USA), and the second for 25 min in ReadyPrep 2-D Starter Kit Equilibration Buffer II containing iodoacetamide (BioRad, USA) instead of DTT. The two-dimensional SDS-PAGE was run using 12% acrylamide separating gels and 4% polyacrylamide stacking gels in a Mini-PROTEAN Tetra Cell electrophoresis chamber (BioRad, USA) at 200 V for approximately 50 min. The PageRuler Unstained Protein Ladder (Thermo Fisher Scientific, Massachusetts, USA) was loaded onto each gel as a weight marker. All gels were separated in the same conditions.

Silver staining and 2-DE immunoblotting

After 2-DE electrophoresis gels were silver-stained using PlusOne Silver Staining Kit (GE Healthcare) in accordance with manufacturer’s protocol, while those used for 2-DE immunoblotting were not stained. The obtained gels were scanned with ChemiDoc MP system (BioRad, USA) and analyzed in Image Lab 5.2.1. software (BioRad, USA).

In addition, proteins from unstained gels were transferred onto Immuno-Blot polyvinylidene fluoride (PVDF) membranes (BioRad) by a wet transfer system (BioRad, USA) at 95 V for one hour in cool conditions. The PVDF membranes with the Ad and ML proteins were blocked in Pierce Protein-Free T20 (TBS) Blocking Buffer (Thermo Fisher Scientific) for one hour at room temperature. Following this, the PVDF membranes were incubated overnight at 4 °C with T. britovi-infected pig sera (dose of 20,000 ML) diluted 1:100, at 10 dpi and 60 dpi. Adult worm proteins transferred onto the membrane were treated with antisera taken at 10 dpi while the ML proteins were treated with antisera from 10 dpi and 60 dpi. The secondary antibody HRP-conjugated goat anti-pig IgG were diluted 1:35 000 (Sigma-Aldrich, Louis, USA). The uninfected sera were used as parallel negative controls. The negative control experiment used the same method as mentioned above. The immunoreactive proteins were visualized on a film using a Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Walthman, USA) according to the provided instruction. Reproducibility of the immune recognition was verified by repeating the immunoblot at least three times.

LC-MS/MS

Spots of interest visible on the films were gently excised from compatible silver-stained gels and analyzed by liquid chromatography coupled to a mass spectrometer in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Samples were concentrated and desalted on a RP-C18 pre-column (Waters), and further peptide separation was achieved on a nano-Ultra Performance Liquid Chromatography (UPLC) RP-C18 column (Waters, BEH130 C18 column, 75 μm i.d., 250 mm long) in a nanoACQUITY UPLC system, using a 45 minute linear acetonitrile gradient. The column outlet was directly coupled to an Electrospray ionization (ESI) ion source of a Orbitrap Velos type mass spectrometer (Thermo Scientific, Waltham, USA), operating in a regime of a data-dependent MS to MS/MS switch with HCD-type peptide fragmentation. An electrospray voltage of 1.5 kV was used.

Bioinformatics

Raw data files were pre-processed with Mascot Distiller software (version 2.4.2.0, MatrixScience). The obtained peptide masses and fragmentation spectra were matched to the National Center Biotechnology Information (NCBI) non-redundant database (115,488,495 sequences/ 42,334,050,411 residues), with a Nematoda filter (748,652 sequences) using the Mascot search engine (Mascot Daemon v. 2.4.0, Mascot Server v. 2.4.1, MatrixScience). The following search parameters were applied: enzyme specificity was set to trypsin, peptide mass tolerance to ± 30 ppm and fragment mass tolerance to ± 0.1 Da. The protein mass was left as unrestricted, and mass values as monoisotopic, with one missed cleavage being allowed. Alkylation of cysteine by carbamidomethylation as fixed, oxidation of methionine was set as a variable modification. Protein identification was performed using the Mascot search engine (MatrixScience), with a probability-based algorithm. The expected value threshold of 0.05 was used for the analysis, which means that all peptide identifications had less than a one-in-20 chance of being a random match. All proteins identified in the MASCOT search were subsequently assigned to the UniProtKB database (https://www.uniprot.org/) and QuickGO (http://www.ebi.ac.uk/QuickGO/) and classified in gene ontology (GO) in accordance with its molecular function, biological process and cellular component information.

Results

2-DE and immunoblot analysis of Ad and ML proteins of T. britovi

To identify species-specific parasite antigens, extracts of T. britovi Ad and ML were separated by IEF on 7 cm, pH 3–10 strips. Figures 1a and 2a represent one of the three replicated silver-stained proteome gels used for further analysis. The proteomes of Ad and ML presented 261 and 272 spots, respectively, with a pH range of 3–10 and molecular weight (MW) ranging from 10 kDa to 250 kDa (Figs. 1a, 2a). The results of the 2-DE immunoblot of the Ad and ML extracts are given in Figs. 1b and 2b, c. Approximately 31 Ad-immunoreactive protein spots and nine ML protein spots were positively recognized by T. britovi-infected swine sera at 10 dpi. Sera taken from pigs at 60 dpi recognized 30 ML protein spots. Potentially immunogenic proteins migrated with a MW between 10 and 150 kDa (Figs. 1b, 2b, c). These immunoreactive spots matched to the corresponding protein spots on silver stained gels, and were selected for further LC-MS/MS identification. No protein reacted to uninfected swine sera (Figs. 1c, 2d).
Fig. 1
Fig. 1

An image of 2-DE separations and immunoblot analysis of somatic antigen extract of T. britovi adult worms (Ad). a 2-DE gels were stained with a silver stain. b 2D-immunoblot of Ad proteins were probed with infected pig sera at 10 dpi. c 2D-immunoblot of Ad proteins probed with uninfected swine sera. Matched spots selected for subsequent LC-MS/MS analysis are marked

Fig. 2
Fig. 2

An image of 2-DE separations and immunoblot analysis of somatic antigen extract of T. britovi muscle larvae (ML). a 2-DE gels were stained with a silver stain. 2D-immunoblot of ML proteins were probed with infected pig sera at 10 dpi (b) and at 60 dpi (c). d 2D- immunoblot of ML proteins probed with uninfected swine sera. Matched spots selected for subsequent LC-MS/MS analysis are marked

LC-MS/MS analysis of antigenic proteins of T. britovi specific for adult worms

The protein data obtained in the present study were compared against deposited protein sequences available for other Trichinella spp. The obtained MS/MS datasets were therefore searched against the NCBI database with the Mascot search engine, and the samples detected as Trichinella spp.-specific were selected based on score, matches and sequence coverage data. Thirty-one of the positive spots recognized by the T. britovi-infected serum samples taken at 10 dpi were matched and located on the silver-stained gels and then subjected to LC-MS/MS analysis (Table 1). The results revealed the presence of 45 proteins with potential antigenic character, among which 29 were specific only for the adult stage of T. britovi. Five of these antigenic proteins were present in more than one spot (Table 2 ), and most of the analyzed spots contained more than one protein. The highest number of proteins were identified from spot number 22, containing five proteins, spots 6, 7 and 19 containing four proteins, and spots 8, 23, 28, 30 and 31 containing three proteins (Table 1). Only one protein was present in nine spots (nos 2, 4, 11, 12, 13, 14, 24, 25 and 29). No protein set was found in spot no. 21. Several of the immunogenic proteins specific for adult worms were matched to myosin, actin-depolymerizing factor 1, isoforms a/b, heat-shock cognate 71 kDa protein, stress-70 protein, Rho GDP-dissociation inhibitor 1, paramyosin or serine/arginine-rich splicing factor 1 (Tables 1 and 2).
Table 1

Results of LC-MS/MS analysis of Trichinella britovi adult worms (Ad) selected spots which reacted with pig sera collected at 10 dpi

Spot

NCBIprot accession no.

MSa

MPb

Seqc

SC (%)d

emPAIe

Mr(kDa)/pIf

Description

1

KRZ04300.1

746

11

10

15

0.82

71.823/6.58

Transketolase, partial

KRY23714.1

363

5

5

8

0.33

75.949/8.54

Succinate dehydrogenase (ubiquinone) flavoprotein subunit, mitochondrial, partial

2

KRY10810.1

817

13

11

16

1.08

70.999/6.60

Transketolase

3

KRY09282.1

1067

18

17

25

1.86

73.168/6.07

Intermediate filament protein ifa-1

KRY23083.1

1015

16

16

22

1.19

76.326/6.17

Stress-70 protein, mitochondrial, partial

4

KRY23083.1

405

6

6

10

0.32

76.326/6.17

Stress-70 protein, mitochondrial, partial

5

KRY23083.1

247

3

3

5

0.18

76.326/6.17

Stress-70 protein, mitochondrial, partial

KRZ06996.1

99

2

2

2

0.12

74.844/5.78

Intermediate filament protein ifa-1, partial

6

KRY11608.1

1889

36

26

40

4.74

75.526/6.24

Intermediate filament protein B

KRY09282.1

1883

31

27

38

4.10

73.168/6.07

Intermediate filament protein ifa-1

KRY16427.1

1772

33

22

22

1.78

108.386/6.14

Heat-shock cognate 71 kDa protein, partial

CAA73574.1

1612

30

21

31

3.15

71.860/5.77

Heat-shock protein 70

7

KRY58599.1

1946

38

26

23

1.54

131.529/6.88

Heat-shock cognate 71 kDa protein, partial

KRY11608.1

1933

37

23

38

5.03

75.526/6.24

Intermediate filament protein B

CAA73574.1

1773

32

25

36

3.87

71.860/5.77

Heat-shock protein 70

KRY09282.1

1503

25

21

29

2.95

73.168/6.07

Intermediate filament protein ifa-1

8

CAA73574.1

1633

35

22

33

3.42

71.860/5.77

Heat-shock protein 70

KRY11608.1

1094

18

17

25

1.62

75.526/6.24

Intermediate filament protein B

KRZ06996.1

906

13

12

17

0.98

74.844/5.78

Intermediate filament protein ifa-1, partial

9

KRY21440.1

646

12

9

16

0.97

62.134/5.04

Calreticulin

KRY19442.1

329

5

5

15

0.81

35.572/9.61

Y-box factor -like protein

10

KRY21426.1

507

8

7

27

1.80

29.821/4.64

Myosin light chain kinase, smooth muscle

AET09716.1

215

3

3

15

0.78

22.620/4.54

Tropomyosin, partial

11

KRZ13803.1

412

6

5

17

1.07

35.565/8.61

32 kDa beta-galactoside-binding lectin lec-3 (Galectin)

12

KRY20423.1

465

8

6

21

1.48

33.995/7.69

32 kDa beta-galactoside-binding lectin (Galectin)

13

KRX13351.1

62

1

1

4

0.15

29.477/8.21

RNA-binding protein rnp-1

14

KRX46812.1

497

7

6

32

2.26

22.941/7.07

Peroxiredoxin-2, partial

15

KRZ77496.1

378

6

4

3

0.08

23.7876/6.48

Dedicator of cytokinesis protein 1

KRY20040.1

140

3

2

10

0.97

18.977/6.97

Heat-shock protein beta-1

16

KRY20040.1

445

12

6

34

3.69

18.977/6.97

Heat-shock protein beta-1

KRX20324.1

323

6

4

28

1.33

19.886/8.12

OV-16 antigen, partial

17

KRY20040.1

405

11

5

30

3.01

18.977/6.97

Heat-shock protein beta-1

KRX20324.1

369

6

4

28

1.43

19.886/8.12

OV-16 antigen, partial

18

KRX47621.1

701

19

9

53

7.94

18.951/6.97

Heat-shock protein beta-1

KRX19442.1

106

2

2

10

0.57

18.490/8.74

Transcription factor BTF3 -like protein 4

19

KRX16844.1

358

11

6

30

2.86

18.894/6.32

Alpha-crystallin B chain

KRY20040.1

297

6

4

26

2.06

18.977/6.97

Heat-shock protein beta-1

KRZ07637.1

278

3

3

14

0.75

23.026/6.12

Stromal cell-derived factor 2

KRZ08373.1

75

2

2

9

0.47

22.324/7.68

Peroxiredoxin-2

20

KRX18074.1

398

8

6

9

0.74

54.209/4.92

BAG family molecular chaperone regulator 2, partial

KRZ17076.1

165

3

2

10

0.75

22.920/5.45

Rho GDP-dissociation inhibitor 1

21

Unidentified

22

KRY31449.1

1224

26

15

20

1.11

91.608/5.31

Transitional endoplasmic reticulum ATPase -like protein 2

KRZ08767.1

1011

13

13

7

0.27

234.755/5.91

Myosin-4, partial

KRZ03705.1

287

3

3

4

0.13

101.672/5.38

Paramyosin

KRY00202.1

206

4

3

5

0.20

92.727/5.26

Heat-shock 70 kDa protein 4L

KRY14731.1

172

3

3

3

0.10

131.892/6.63

CAP-Gly domain-containing linker protein 1

23

KRY18882.1

994

15

12

14

0.79

108.605/5.51

LIM domain and actin-binding protein 1

KRZ08767.1

345

6

6

3

0.12

234.755/5.91

Myosin-4, partial

KRZ13693.1

302

5

5

4

0.20

125.148/6.31

Integrin alpha pat-2

24

KRZ06959.1

252

3

3

11

0.61

28.479/7.74

Triosephosphate isomerase, partial

25

KRZ12367.1

299

4

4

24

1.19

24.385/7.01

GTP-binding nuclear protein Ran

26

KRX15368.1

656

14

10

33

3.52

34.025/6.29

32 kDa beta-galactoside-binding lectin, partial

KRY59871.1

115

2

2

12

0.32

30.478/9.08

Serine/arginine-rich splicing factor 1, partial

KRX23478.1

101

2

2

4

0.27

35.530/6.82

Protein MEMO1, partial

27

KRX41818.1

376

5

5

21

1.13

29.431/5.58

Putative phosphomannomutase

KRY00151.1

137

3

2

4

0.25

59.844/5.54

ATP synthase subunit beta, mitochondrial

KRY00848.1

114

2

2

2

0.09

108.425/6.27

Heat-shock 70 kDa protein, partial

28

KRX20997.1

368

6

6

8

0.40

76.565/8.34

Guanine nucleotide-binding proteinalpha-12 subunit, partial

KRX16428.1

294

6

5

23

1.65

26.141/5.80

V-type proton ATPase subunit E

KRZ10894.1

177

3

3

9

0.58

27.709/7.55

GrpE -like protein 1, mitochondrial

29

KRY01036.1

394

8

5

27

2.16

22.274/6.88

Actin-depolymerizing factor 1, isoforms a/b, partial

30

KRY01036.1

440

8

5

27

2.12

22.274/6.88

Actin-depolymerizing factor 1, isoforms a/b, partial

KRY17912.1

295

4

4

18

1.63

17.440/6.18

Uncharacterized protein T12_13420

KRY00151.1

143

4

2

4

0.33

59.844/5.54

ATP synthase subunit beta, mitochondrial

31

KRY01216.1

278

5

5

21

1.85

19885/5.43

Ubiquitin-conjugating enzyme E2 G1, partial

KRY21297.1

199

4

3

14

1.15

21.790/8.89

Peptide methionine sulfoxide reductase MsrB

KRY15966.1

185

4

3

10

0.44

34.259/6.31

Hypothetical protein T12_8663

aMascot score

bMatched peptide

cSequence

dSequence coverage (%)

eExponentially modified protein abundance index

fExperimental nominal mass (kDa) and isoelectric point

Table 2

Alphabetical list of stage-specific antigenic proteins of adult worms of T. britovi, which reacted with pig sera collected at 10 dpi, together with spot number information. Identification by LC-MS/MS

Protein name

Spot number

Actin-depolymerizing factor 1, isoforms a/b, partial

29, 30

BAG family molecular chaperone regulator 2, partial

20

Calreticulin

9

CAP-Gly domain-containing linker protein 1

22

GrpE-like protein 1, mitochondrial

28

Guanine nucleotide-binding protein alpha-12 subunit, partial

28

Heat-shock 70 kDa protein 4L

22

Heat-shock cognate 71 kDa protein, partial

6, 7

Hypothetical protein T12_8663

31

Integrin alpha pat-2

23

LIM domain and actin-binding protein 1

23

Myosin-4, partial

22, 23

Myosin light chain kinase, smooth muscle

10

Paramyosin

22

Peptide methionine sulfoxide reductase MsrB

31

Putative phosphomannomutase

27

Rho GDP-dissociation inhibitor 1

20

RNA-binding protein rnp-1

13

Serine/arginine-rich splicing factor 1, partial

26

Stress-70 protein, mitochondrial

3, 4, 5

Stromal cell-derived factor 2

19

Succinate dehydrogenase (ubiquinone) flavoprotein subunit, mitochondrial, partial

1

Transitional endoplasmic reticulum ATPase -like protein 2

22

Transketolase /partial

1, 2

Triosephosphate isomerase, partial

24

Ubiquitin-conjugating enzyme E2 G1, partial

31

Uncharacterized protein T12_13420

30

Y-box factor -like protein

9

V-type proton ATPase subunit E

28

LC-MS/MS analysis of antigenic proteins of T. britovi specific for muscle larvae

Nine ML protein spots cross-reacting with T. britovi infected swine sera were identified by MS analysis at 10 dpi, and 30 spots were found at 60 dpi (Tables 3 and 4).
Table 3

Results of LC-MS/MS analysis of Trichinella britovi muscle larvae (ML) selected spots which reacted with pig sera collected at 10 dpi

Spot

NCBIprot accession No.

MSa

MPb

Seqc

SC (%)d

emPAIe

Mr(kDa)/pIf

Description

24

a

KRY11608.1

1765

36

26

36

4.01

75.526/6.24

Intermediate filament protein B

KRY09282.1

1457

30

22

30

2.92

73.168/6.07

Intermediate filament protein ifa-1

b

KRY11608.1

1903

40

29

39

5.11

75.526/6.24

Intermediate filament protein B

KRY09282.1

1541

33

23

33

3.30

73.168/6.07

Intermediate filament protein ifa-1

c

KRY11608.1

930

14

13

19

1.24

75.526/6.24

Intermediate filament protein B

KRY09282.1

2328

66

34

47

7.99

73.168/6.07

Intermediate filament protein ifa-1

29

 

XP_003373575.1

1206

52

15

41

4.74

42.210/5.30

Actin-5C

30

 

XP_003373575.1

527

9

8

25

1.47

42.210/5.30

Actin-5C

 

KRY50178.1

415

8

6

15

0.89

46.783/5.44

Hypothetical protein T03_17187

 

KRZ06996.1

160

3

3

4

0.12

74.844/5.78

Intermediate filament protein ifa-1, partial

 

KRZ09733.1

323

5

5

5

0.28

96.031/6.00

Mitochondrial-processing peptidase subunit beta, partial

 

KRX47705.1

293

4

4

3

0.14

150.442/6.28

Serine protease 30

31

 

KRZ02603.1

1083

28

14

34

2.98

50.922/6.01

Enolase, partial

 

KRY13126.1

544

10

9

20

1.47

48.623/5.41

26S protease regulatory subunit 7

32

 

KRY18793.1

883

19

14

30

3.45

54.997/5.00

Protein disulfide-isomerase 2

 

OUC40875.1

749

17

10

26

2.73

48.387/4.87

Putative Tubulin/FtsZ family, GTPase domain protein

 

KRY00151.1

655

14

8

17

1.31

59.844/5.54

ATP synthase subunit beta, mitochondrial

33

 

KRX41020.1

1127

27

16

27

1.92

72.856/5.09

Heat-shock 70 kDa protein C, partial

 

KRY00702.1

364

6

5

9

0.46

68.894/5.08

V-type proton ATPase catalytic subunit A

34

 

Unidentified

aMascot score

bMatched peptide

cSequence

dSequence coverage (%)

eExponentially modified protein abundance index

fExperimental nominal mass (kDa) and isoelectric point

Table 4

Results of LC-MS/MS analysis of Trichinella britovi muscle larvae (ML) selected spots which reacted with pig sera collected at 60 dpi

Spot

NCBIprot accession No.

MSa

MPb

Seqc

SC (%)d

emPAIe

Mr(kDa)/pIf

Description

1

KRX47621.1

732

22

10

53

13.42

18.951/6.97

Heat-shock protein beta-1

KRX19442.1

133

2

2

10

0.64

18.490/8.74

Transcription factor BTF3 -like protein 4

2

KRX14469.1

241

3

3

2

0.06

247.333/6.85

Dedicator of cytokinesis protein 1

KRZ13097.1

165

5

3

13

1.75

19.090/5.43

Heat-shock protein beta-1, partial

3

KRY20040.1

376

8

6

34

4.92

18.977/6.97

Heat-shock protein beta-1

KRX20324.1

310

5

4

28

1.64

19.886/8.12

OV-16 antigen, partial

4

KRX16844.1

662

60

10

66

10.33

18.894/6.32

Alpha-crystallin B chain

KRY20040.1

396

8

7

35

4.78

18.977/6.97

Heat-shock protein beta-1

KRY18783.1

109

2

2

9

0.18

25.119/6.44

Stromal cell-derived factor 2

5

KRX46812.1

546

11

8

41

4.02

22.941/7.07

Peroxiredoxin-2, partial

6

KRX46812.1

409

8

6

28

2.07

22.941/7.07

Peroxiredoxin-2, partial

KRZ12367.1

292

4

4

19

1.02

24.385/7.01

GTP-binding nuclear protein Ran

KRX18658.1

226

3

3

15

0.73

23.516/6.92

ATP synthase subunit d, mitochondrial

7

Unidentified

8

Unidentified

9

KRZ13803.1

406

7

5

17

1.11

35.565/8.61

32 kDa beta-galactoside-binding lectin lec-3 (Galectin)

10

KRZ13803.1

584

9

7

24

1.58

35.565/8.61

32 kDa beta-galactoside-binding lectin lec-3 (Galectin)

KRY30017.1

304

6

5

17

0.82

34.995/8.74

Putative 3-hydroxyacyl-CoA dehydrogenase

11

KRZ13161.1

105

2

2

6

0.23

42.112/7.12

Glutamine synthetase

12

KRY11984.1

432

7

7

15

0.90

49.560/6.59

Poly-cysteine and histidine-tailed protein

KRX28313.1

364

7

6

14

1.01

45.667/6.09

Calponin -like protein OV9M, partial

KRX47308.1

240

3

3

3

0.14

107.151/6.52

Deoxyribonuclease-2-alpha

13

KRY01407.1

324

4

4

10

0.46

51.099/5.91

Cuticlin-1, partial

14

KRY01407.1

319

4

4

10

0.48

51.099/5.91

Cuticlin-1, partial

KRY00848.1

166

3

3

2

0.15

108.425/6.27

Heat-shock 70 kDa protein, partial

15

CBX25713.1

322

5

5

14

1.14

32.896/4.65

Tropomyosin, partial

16

KRY09099.1

476

8

8

22

1.47

38.218/5.20

Hypothetical protein T12_13379, partial

KRX15676.1

174

3

3

7

0.43

35.904/5.46

40S ribosomal protein SA, partial

17

KRY09099.1

381

6

6

16

1.20

38.218/5.20

Hypothetical protein T12_13379, partial

KRZ15717.1

217

4

3

9

0.46

39.852/5.63

Guanine nucleotide-binding protein subunit beta-1, partial

KRY18502.1

203

4

3

3

0.26

65.700/4.95

Microtubule-associated protein RP/EB family member 3, partial

KRX15059.1

126

3

2

5

0.52

36.189/5.00

Disorganized muscle protein 1

18

KRX21567.1

504

11

6

21

1.66

40.427/5.49

Pyruvate dehydrogenase E1 component subunit beta, mitochondrial

19

KRX15059.1

667

23

9

28

2.96

36.189/5.00

Disorganized muscle protein 1

KRZ03570.1

403

6

6

18

1.20

34.457/4.75

Tropomyosin

20

XP_003378934.1

1001

20

12

46

6.93

28.294/4.83

14-3-3 protein zeta

AET09716.1

248

4

4

19

1.09

22.620/4.54

Tropomyosin, partial

KRX19348.1

159

3

3

13

0.56

28.034/4.82

Toll-interacting protein

21

KRZ50222.1

917

16

14

3

0.17

449.723/6.87

Propionyl-CoA carboxylase alpha chain, mitochondrial

22

KRZ50222.1

1081

18

17

4

0.19

449.723/6.87

Propionyl-CoA carboxylase alpha chain, mitochondrial

23

KRY09873.1

557

9

9

2

0.09

441.173/6.76

Propionyl-CoA carboxylase alpha chain, mitochondrial

24

KRY45949.1

1999

45

29

42

6.80

73.429/6.07

Intermediate filament protein ifa-1

KRY09282.1

1720

36

26

36

4.01

75.526/6524

Intermediate filament protein B

25

KRX15368.1

437

7

7

20

1.80

34.025/6.29

32 kDa beta-galactoside-binding lectin, partial

KRZ10402.1

91

2

2

4

0.33

35.568/6.67

Protein MEMO1, partial

KRZ78587.1

42

3

1

2

0.11

48.445/5.59

Secernin-3

26

KRY07641.1

341

6

5

18

1.14

38.033/6.38

1,5-anhydro-D-fructose reductase

27

KRY07641.1

239

4

4

12

0.63

38.033/6.38

1,5-anhydro-D-fructose reductase

28

Unidentified

29

XP_003373575.1

1255

39

15

41

4.48

42.210/5.30

Actin-5C

AET09716.1

168

2

2

11

0.45

22.620/4.54

Tropomyosin, partial

KRY38295.1

160

3

3

6

0.26

54.444/6.39

Secernin-3

KRZ06996.1

232

3

3

4

0.19

74.844/5.78

Intermediate filament protein ifa-1, partial

30

KRY50178.1

993

18

15

40

4.11

46.783/5.44

Hypothetical protein T03_17187

XP_003373575.1

527

9

8

25

1.47

42.210/5.30

Actin-5C

KRZ06996.1

363

6

6

9

0.47

74.844/5.78

Intermediate filament protein ifa-1, partial

KRX47705.1

293

4

4

3

0.14

150.442/6.28

Serine protease 30

KRZ09733.1

323

5

5

5

0.28

96.031/6.00

Mitochondrial-processing peptidase subunit beta, partial

KRZ17128.1

256

4

4

9

0.51

46.607/5.26

Putative histone-binding protein Caf1

KRY13378.1

250

5

5

9

0.54

54.969/5.66

Rab GDP dissociation inhibitor alpha

aMascot score

bMatched peptide

cSequence

dSequence coverage (%)

eExponentially modified protein abundance index

fExperimental nominal mass (kDa) and isoelectric point

LC-MS/MS analysis revealed the presence of 13 immunoreactive ML proteins recognized by sera at 10 dpi samples, nine of which were stage-specific (Table 5). In the samples at 60 dpi, 39 proteins were recognized by sera, with only 25 being stage-specific (Table 6). One protein recognized by sera at 10 dpi was present in two spots (nos 29, 30) (Table 5) and seven proteins recognized by sera at 60 dpi were present in more than one spot (Table 6). The highest number of proteins, i.e. seven, were observed in spot number 30, followed by four proteins in spots 17 and 29, and three proteins in spots 4, 6, 12, 20, 30 and 32 (Table 4 ). The remaining spots contained fewer than three proteins (Tables 3 and 4). Only spot no 34 contained no proteins recognized by sera at 10 dpi, while at 60 dpi, three spots contained no recognized proteins (7, 8 and 28) (Tables 3 and 4).
Table 5

Alphabetical list of stage-specific antigenic proteins of muscle larvae of T. britovi, which reacted with pig sera collected at 10 dpi, together with spot number information. Identification by LC-MS/MS

Protein name

Spot number

26S protease regulatory subunit 7

31

Actin-5C

29/30

Enolase, partial

30

Hypothetical protein T03_17187

30

Protein disulfide-isomerase 2

32

Putative Tubulin/FtsZ family, GTPase domain protein

32

V-type proton ATPase catalytic subunit A

33

Mitochondrial-processing peptidase subunit beta, partial

30

Serine protease 30

30

Table 6

Alphabetical list stage-specific antigenic proteins of muscle larvae of T. britovi, which reacted with pig sera collected at 60 dpi, together with spot number information. Identification by LC-MS/MS

Protein name

Spot number

1,5-anhydro-D-fructose reductase

26, 27

14-3-3 protein zeta

20

40S ribosomal protein SA, partial

16

Actin-5C

29, 30

ATP synthase subunit d, mitochondrial

6

Calponin -like protein OV9M, partial

12

Cuticlin-1, partial

13, 14

Deoxyribonuclease-2-alpha

12

Disorganized muscle protein 1

17, 19

Glutamine synthetase

11

Guanine nucleotide-binding protein subunit beta-1, partial

17

Hypothetical protein T03_17187

30

Hypothetical protein T12_13379, partial

16, 17

Microtubule-associated protein RP/EB family member 3, partial

17

Mitochondrial-processing peptidase subunit beta, partial

30

Poly-cysteine and histidine-tailed protein

12

Propionyl-CoA carboxylase alpha chain, mitochondrial

21, 22, 23

Putative 3-hydroxyacyl-CoA dehydrogenase

10

Putative histone-binding protein Caf1

30

Pyruvate dehydrogenase E1 component subunit beta, mitochondrial

18

Rab GDP dissociation inhibitor alpha

30

Secernin-3

25, 29

Serine protease 30

30

Stromal cell-derived factor 2

4

Toll-interacting protein

20

The following immunogenic proteins specific for the ML stage were identified in the 10 dpi serum samples: 26S protease regulatory subunit 7; actin-5C; enolase; protein disulfide-isomerase 2; V-type proton ATPase catalytic subunit A; and serine protease 30 (Table 5). The following were identified in the 60 dpi samples: 14-3-3 protein zeta; 40S ribosomal protein SA; calponin-like protein OV9M; propionyl-CoA carboxylase alpha chain; Rab GDP dissociation inhibitor alpha; secernin-3; serine protease 30; Toll-interacting protein (Table 6). Finally, the following proteins were identified in both the 10 and 60 dpi samples: actin 5C; serine protease; intermediate filament protein (IFA-1); and mitochondrial-processing peptides subunit beta (Tables 5 and 6).

LC-MS/MS analysis of antigenic proteins common for both stages of T. britovi

Although some proteins were found to be specific for both the Ad and ML stages of T. britovi, most were common to both stages (Table 7). The following proteins appeared in both proteomes, and were most frequently identified from multiple spots: heat-shock protein beta-1 (present in five spots - Ad 10 dpi, four spots - ML 60 dpi); intermediate filament protein IFA-1; partial (present in five spots - Ad 10 dpi, three spots - ML 60 dpi, four spots - ML 10 dpi); intermediate filament protein IFA-1 (present in five spots - Ad 10 dpi, three spots - 10 dpi and one spot - ML 60 dpi); peroxiredoxin-2/partial (present in three spots - Ad 10 dpi, two spots - ML 60 dpi); tropomyosin (present in one spot - Ad 10 dpi, four spots - ML 60 dpi); and heat-shock 70 kDa protein (present in four spots - Ad 10 dpi, one spot - ML 60 dpi) (Tables 1, 3, 4 and 7). The presence of these different isoforms could be attributed to differences in amino acid sequence, alternative splicing or post-translational modifications. The dominant proteins for both stages were identified as heat-shock protein 70 kDa, heat-shock protein beta-1, intermediate filament B and IFA-1 (Table 7).
Table 7

Alphabetical list of antigenic proteins, common for both adult worms (Ad) and muscle larvae (ML) stages T. britovi recognized by sera at 60 dpi and 10 dpi, together with spot number information. Identification by LC-MS/MS

Protein name

Spot number Ad T. britovi

Spot number ML T. britovi

10 dpi

10 dpi

60 dpi

32 kDa beta-galactoside-binding lectin lec-3 (Galectin)

11

9, 10

32 kDa beta-galactoside-binding lectin, partial (Galectin)

12, 26

25

Alpha-crystallin B chain

19

4

ATP synthase subunit beta, mitochondrial

27, 30

32

Dedicator of cytokinesis protein 1

15

2

GTP-binding nuclear protein Ran

25

6

Heat-shock 70 kDa protein, partial

6, 7, 8, 27

14

Heat-shock protein beta-1

15, 16, 17, 18, 19

1, 2, 3, 4

Intermediate filament protein B

6, 7, 8

24 a,b,c

24

Intermediate filament protein ifa-1, partial

3, 5, 6, 7, 8

24a/b/c, 30

24, 29, 30

OV-16 antigen, partial

16, 17

3

Peroxiredoxin-2, partial

14, 19, 30

5, 6

Protein MEMO1, partial

26

25

Transcription factor BTF3 -like protein 4

18

1

Tropomyosin, partial

10

15, 19, 20, 29

V-type proton ATPase subunit E

28

33

Gene ontology (GO) analysis

The gene ontology (GO) database was used to identify the antigenic proteins of the Ad and ML stages according to their molecular function, cellular component and biological process.

For the T. britovi adult stage, the proteins were classified according to molecular function (39), cellular components (21) and biological process (21). Seven subcategories of molecular function were determined, the most abundant of which were binding (24) and catalytic activity (18); however, structural molecule activity (6), molecular function regulation (3), transporter activity (2), signal transducer (1) or peroxiredoxin activity (1) subcategories were also observed. Eight subcategories for cellular component were determined, the most numerous being the cell part subcategory (18); however, intracellular organelle part (7), macromolecular complex (7), organelle (6), membrane part (5), intermediate filament (4), membrane (3) or cell (1) subcategories were also observed to a lesser extent. Seven subcategories of biological process were determined. The most abundant were assigned to the cellular process (16) and the metabolic process (11) subcategories, while the remainder were assigned to biological regulation (5), localization (3), transport (2), response to oxidative stress (1), cell adhesion (1) or cellular component organization (1) (Fig. 3a-c). Based on the gene ontology analysis, the potentially antigenic proteins of T. britovi muscle larvae which reacted with both 10 dpi and 60 dpi pig sera, were categorized according to molecular function (35), cellular component (24) or biological process (18). Six subcategories for molecular function were determined. The most abundant were binding (20), and catalytic activity (20), whereas structural molecule activity (6), transmembrane transporter activity (3), molecular function regulation (2) or peroxiredoxin activity (1) were visibly less numerous. Eight cellular component subcategories were determined, with the most numerous subcategory being cell part (18), followed by intracellular organelle part (9), macromolecular complex (7), polymeric cytoskeletal fiber (6), membrane part (5), membrane (3), organelle (3) and cell (1). Four subcategories for biological process were determined. The cellular process (15) subcategory was the most numerous, followed by metabolic process (11), biological regulation (5) and localization (5) (Fig. 3a-c).
Fig. 3
Fig. 3

Comparison of Gene Ontology (GO) database analysis outcomes for T. britovi muscle (ML) and adult (Ad) larvae identified proteins. The proteins were categorized according to molecular function (a), cellular component (b) and biological process (c)

Discussion

Recent reports indicate most cases of Trichinella britovi infection occur amongst patients unaware of eating improperly cooked meat products [3234, 38]. Early diagnosis of trichinellosis is crucial, as anthelmintic drug treatment is much more effective if administered during the initial phases before muscle larvae become encapsulated [39].

In trichinellosis, the interaction between the parasite and the host is influenced by the Trichinella life-cycle, which includes a range of stage-specific antigens, immune evasion strategies and modulatory effects on host responses. The combination of immunoblot analysis and proteomic techniques, such as the two-dimensional gel electrophoresis and mass spectrometry used in the present study, is a comprehensive approach to identifying Trichinella proteins [40]. Although most proteomic studies have focused on the identification of proteins characteristic of T. spiralis life-cycle stages, same of them were dedicated to other Trichinella species/genotypes including T. pseudospiralis, T. nativa, T. papuae and T8 [18, 19, 21, 26, 41].

However, further effort is still needed to identify the T. britovi proteins that may play an important role in understanding host-parasite interactions, and to develop immunological diagnostic methods. Only two papers have addressed the identification of antigenic proteins from T. britovi, the second-most common species of Trichinella that may affect human health [9, 30]. Dea-Ayuela & Bolaz-Fernandez [30], using 2-DE immunoblot, identified the T. britovi proteins that likely belong to the Trichinella TSL-1 group of antigens: enolase; P49 antigen; and actins. These proteins play a part in parasite invasion and migration through the host cells. Other studies based on the immunoproteomics of the excretory-secretory systems of T. britovi muscle larvae identified a range of proteins, including various glycoproteins (gp43, p49), serine-protease and 5'-nucleotidase [9], that play a role in the development and migration of NBL in host tissue and in the regulation of the immune response by modulating nucleotide levels during infection [42].

The purpose of the present study, therefore, was to identify the T. britovi-specific immunodominant proteins present in adult worms and muscle larvae. The crude protein extracts of both stages were separated by 2-DE, subjected to immunoblot analysis with sera from animals infected with T. britovi (at 10 dpi and 60 dpi), and identified by LC-MS/MS. A previous immunoproteomic study performed on T. spiralis antigens showed that 64 proteins from adult worm crude extract were recognized by sera from pigs and mice infected with T. spiralis at 7 dpi, but only seven proteins in muscle larvae crude extract were detected using sera from T. spiralis-infected mice and pigs at 5 dpi and 45 dpi, respectively [11, 15, 27].

In the present study, the immunogenic spots recognized by the various pig T. britovi-infected sera were divided into three groups according to immunoblotting pattern and LC-MS/MS results: adult (Ad) stage-specific proteins; muscle larvae (ML) stage-specific proteins; and proteins common to both developmental stages. Forty-five proteins in the Ad samples (29 stage-specific for Ad and 16 common) and 13 proteins in the ML samples (9 stage-specific for ML and 4 common) cross-reacted with sera at 10 dpi, while 39 proteins in the ML samples (25 stage-specific for ML and 14 common) reacted with the sera taken at 60 dpi.

Additionally, to further understand the functions of the T. britovi proteins, these proteins were categorized according to the GO into biological processes, molecular function and cellular components. The results reveal the presence of a range of proteins known to be antigens involved in the mechanisms of invasion of host tissue and cells, larval migration or molting, immune modulation, metabolic processes in other helminths: actin; heat-shock proteins; paramyosin; 14-3-3-protein; myosin; serine protease; enolase; poly-cysteine and histidine-tailed protein; and deoxyribonuclease-2-alpha [21, 26, 27, 4345]. Of these proteins, the following were common for both tested T. britovi stages: 32 kDa beta galactoside-binding lectin lec-3 (Galectin); heat-shock 70 kDa protein; heat-shock protein beta-1; intermediate filament protein IFA-1; intermediate filament protein B; GTP-binding nuclear protein Ran; OV-16 antigen; protein MEMO1; transcription factor BTF3-like protein 4; tropomyosin; and peroxiredoxin-2. These have previously been found to be present and active throughout the parasite development process; however, they were present in varying amounts, as indicated by the observed dissimilarities in spot intensities.

Adult T. britovi are frequently found to contain proteins involved in structural and motor activity, such as myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin. These cytoskeleton proteins with an actin binding function, are responsible for cellular component organization and actin filament depolymerization, thus facilitating the parasite growth and development processes. Some of them, including actin-depolymerizing factor 1 and paramyosin, were identified in the ML stage but not the early stage of Trichinella development [11, 46]. One of these, carleticulin, belongs to the carleticulin family of proteins, which are involved in the protein folding process, and were recently reported to facilitate T. spiralis immune evasion by interacting with the first component of the human classical complement pathway, C1q [47]. In addition to its role in muscle length and stability determination, paramyosin also possess immunomodulatory functions. The surface-exposed paramyosin is thought to act as a protective agent during the host inflammatory processes by inhibiting the complement activation cascade and membrane attack complex (MAC) formation [48]. However, V-type proton ATPase subunit E, a member of the ATPase protein family, is activated at a wide pH range and possesses interesting properties under certain biochemical conditions. ATPases are involved in metabolite movements, purging of toxins and energy generation for metabolic processes; they also take part in the environmental response [49, 50] and hence are thought to be involved in the nematode immune response course. Most of the analyzed T. britovi antigens are derived from the muscle stage of the larvae. GO analysis of the obtained results showed that some of the proteins participate in various cellular and metabolic processes mostly associated with the synthesis and degradation of macromolecules (nucleotides, proteins) which play an important role in the invasion and development of Trichinella in the host [10, 26, 28, 51]. The most frequently identified immunodominant antigens of ML T. britovi recognized by infection sera include 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. For example, the actin-5c protein (recognized by sera at 10 dpi/60 dpi), known to bind ATP molecules (GO), has previously been identified with the use of early and late infection sera [26, 52]. This protein is related to the invasion of a parasite into the intestinal epithelial cells and plays a critical role in larval development [53]. Serine protease 30, with peptidase and hydrolase activities, was recognized by sera at 10 dpi/60 dpi. The protein belongs to serine protease family, along with enzymes that take part in digestion, blood coagulation and fibrinolysis processes. It is involved in host tissues and cell invasions, and plays a pivotal role in nematode molting [54]. Additionally, deoxyribonuclease 2-alpha of the deoxyribonuclease II family was identified, which plays an important role in Trichinella invasion, development and survival [55]. The 60 dpi sera also identified the 14-3-3 protein. This is a key regulator of multiple biological processes, including signal transduction, cell differentiation and cell survival, it is also known to induce humoral and cellular immune response and has been tested as a potential vaccine target [56, 57]. The GO analysis revealed that some of the isolated proteins possess catalytic, ligase, hydrolase and peptidase activities, and are responsible for ATP and glutamine synthesis processes; these include ATP-synthase subunit d, glutamine synthase and propionyl-CoA carboxylase alpha chain, all of which were recognized in the 60 dpi sera. GO analysis also showed mitochondrial-processing peptidase (MPP) subunit beta, secernin-3 protein and the previously mentioned serine protease 30 to demonstrate proteolytic and peptidase activity [58]. Microtubule-associated protein RP/EB family member 3 and cuticlin-1, classified as a cellular component belonging to the ML proteome and recognized by sera at 60 dpi, possesses a microtubule binding function. In Caenorhabditis elegans, cuticlin-1 contributes to the formation of extracellular envelopes, thereby protecting the organism from the environment [59].

It is important to note that in accordance with previous studies [11, 60, 61], the 10 dpi sera in the present study identified the protein enolase in crude ML extract. Bernal et al. [61] revealed that enolase plays a part in many processes, including fibrinolysis and degradation of the extracellular matrix, through the activation of plasminogen (a proenzyme of the serine protease plasmin). Moreover, this enzyme may contribute to tissue migration during all T. spiralis developmental stages [59]. Dea-Ayuela & Bolas-Fernandez [30] confirmed that enolase the immunoreactive property using a combination of 2D-immunoblot and MS. Our findings also confirm the presence of a common proteins for both T. britovi stages which was recognized by sera from pigs at 10 dpi and 60 dpi. One particularly well-studied group of proteins comprises the heat-shock proteins (Hsps), which are known to assist the parasite in tissue invasion and intracellular survival, as well as protect it against injury or stress conditions arising as a result of host immune response stimulation [62]. This is consistent with earlier results which identified Hsps as being a common to the adult and muscle larvae stages [10, 26, 51, 55, 63], and were recognized by sera at 15 dpi and 45 dpi [11]. The present GO analysis demonstrated that the identified Hsp proteins present oxidoreductase and structural molecule activity, and that they are located on ribosomes and take part in the translation processes, suggesting that they participate in host cellular stress and immune responses, as well as in the regulation of gene expression and parasite development [27, 64].

Our findings also indicate that the heat-shock protein beta identified in both the Ad and ML proteomes belongs to the small heat-shock proteins (sHsp), which are considered to be an important focus of research in the fight against parasitic diseases [65]. Wu et al. [66] reported that sHsp likely play a role in enhancing the survival of the T. spiralis muscle larvae under conditions of chemical and physical stress, as well as in the development of larvae. Wang et al. [64] suggested that recombinant Hsp70 is an immunogenic protein released by parasites and that it is exposed to the host immune system during infection.

Intermediate filament protein (IFA-1) and intermediate filament protein B were identified in both T. britovi proteomes. These are members of the diverse family of intermediate filaments; these are cytoskeletal components of animal cells which contribute to their mechanical strength and facilitate growth [67]. In nematodes, they allow epidermal elongation in the larvae, worm growth and muscle stability maintenance [68]. Peroxiredoxin-2 has antioxidant and oxidoreductase activity, participates in cellular oxidant detoxification processes and preserves cell redox homeostasis. It therefore plays a crucial role during the host immune response by protecting parasites from endogenous and host-derived ROS, and is possibly involved in cellular signaling [69].

The present study examined somatic extracts taken from adult worms (AW) and muscle larvae of T. britovi. Some of the proteins present in these somatic extracts might not be excretory-secretory (E-S) proteins, and they cannot be exposed to the host immune system and induce the specific antibody response. Hence, some of the identified proteins may have less sero-diagnostic value, or perhaps no significance at all. Nevertheless, in the process of Trichinella infection, the E-S antigens produced by the AW and ML are directly exposed to the immune system and elicit the production of specific anti-Trichinella antibodies by the host. Immunoproteomics studies have identified the early diagnostic antigens associated with the E-S proteins of T. spiralis AW and ML in animal or patient sera during early infection, and the recombinant 31 kDa antigen from T. spiralis ML E-S proteins has been proved to be valuable for early diagnosis of trichinellosis [70, 71]. Hence, further diagnostic antigens for T. britovi infection may be identified by future studies on the E-S antigens of AD and ML with early infection sera.

Few proteomic studies examine T. britovi exclusively or compare the findings with those of different Trichinella spp. [9], and those that have been performed focus on the characterization of mitochondrial genomes [72]. This approach results in the acquisition of a narrow range of knowledge regarding the nuclear genomic or transcriptomic data associated with this parasite, and this narrow focus presents a serious obstacle in the identification of its proteins, and the understanding of their precise function during parasite invasion. Therefore, many proteins are not represented in existing studies, and their precise function can only be assumed on the basis of indirect resemblance analysis.

Conclusions

To our knowledge, the present study describes the first immunoproteomic identification of the antigenic proteins of adult worm and muscle larvae of T. britovi. The somatic extracts from adult worms and muscle larvae of T. britovi were specifically recognized by T. britovi-infected pig sera at 10 dpi and 60 dpi; a total of 70 prominent protein spots were thus identified, and these were found to contain 45 adult worm and 52 muscle larvae proteins. Adult worms and muscle larvae of T. britovi produce proteins (both stage-specific and common proteins) with antigenic properties, some of which have been identified in other helminths as potential diagnostic targets and vaccine candidates. The presence of common and stage-specific proteins for both investigated T. britovi stages was confirmed; these included heat-shock proteins, intermediate filament protein IFA-1, 32 kDa beta-galactosidase-binding lectin, peroxiredoxin-2 or 14-3-3 protein, actin-5C, paramyosin, intermediate filament protein B, calreticulin, deoxyribonuclease-2-alpha, enolase, serine protease. These proteins were related to many significant molecular functions, cellular components and biological processes of the parasite, suggesting that the somatic proteins of these two developmental stages may induce a humoral immune response, making them potential antigens for the development of diagnostic methods for T. britovi infection.

Abbreviations

2-DE: 

Two-dimensional electrophoresis

Ad: 

Adult worms

dpi: 

Days post-infection

ELISA: 

Enzyme-linked immunosorbent assay

ES: 

Excretory-secretory

IEF: 

Isoelectric focusing

LC-MS/MS: 

Liquid chromatography-tandem mass spectrometry

ML: 

Muscle larvae

MW: 

Molecular weight

NBL: 

Newborn larvae

pI: 

Isoelectric point

PVDF: 

Polyvinylidene fluoride membrane

Declarations

Acknowledgements

We are grateful to K. Nöckler (Federal Institute for Risk Assessment, Berlin, Germany) for providing experimentally Trichinella-infected sera.

Funding

Financial support for this study was provided by the National Science Centre Poland (grant UMO-2015/18/E/NZ6/00502).

Availability of data and materials

The data supporting the conclusions of this article are included within the article. The datasets generated during the present study have been deposited in the ProteomeXchange Consortium repository under the accession number PXD011215.

Authors’ contributions

JB designed and supervised the experiments. JB and SG performed the experiments, analyzed the data and drafted the manuscript. BM contributed in the data analysis and manuscript preparation. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All experimental procedures used in the present study had been pre-approved by the First Local Ethical Committee for Scientific Experiments on Animals in Warsaw, Poland (resolution no.: 020/2016, 23 March 2016).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Witold Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warsaw, Poland

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