Identification of candidate antigens by 2-DE Immunoblotting for diagnosis of Toxoplasma gondii infection in chickens and rabbits

Experimental Parasitology 204 (2019) 107723

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Identification of candidate antigens by 2-DE Immunoblotting for diagnosis of Toxoplasma gondii infection in chickens and rabbits

T

Jing Wena,b, Jun Zoua, Xiaoxi Huanga, Hao Wenc, Tselmegd, Xun Suoa, Xianyong Liua,* a Key Laboratory of Animal Epidemiology of the Ministry of Agriculture & National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China b Key Laboratory of Clinical Diagnosis and Treatment Techniques for Animal Disease, Ministry of Agriculture and College of Veterinary Medicine, Inner Mongolia Agricultural University, Huhhot, 010018, China c Inner Mongolia Yili Industrial Group Co.,Ltd, Information Technology Center, Huhhot, 010018, China d College of Foreign Language, Inner Mongolia Agricultural University, Huhhot, 010018, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Toxoplasma gondii Immunoproteomics Diagnostic antigens 2-DE immunobloting Mass spectrometry

Toxoplasmosis, caused by apicomplexan parasite Toxoplasma gondii, is a common food-borne disease in humans. Undercooked meat is a potential source of T. gondii infection. As meat of chicken or rabbit is consumed worldwide, tools such as ELISA for the detection of infection of this parasite in rabbits and chickens are muchneeded. To search diagnostic antigens of T. gondii special for rabbits and chickens, we conducted two dimensional electrophoresis (2-DE), Western blotting and mass spectrometry (MS) with T. gondii tachyzoite proteins. When probed with rabbit or chicken anti-T. gondii sera, about 60 positive spots among over 500 visible protein spots were detected. In subsequent mass spectrometric analysis, microneme 4 (MIC4) and a putative rhoptry protein are of diagnositic value among the 13 spots selectively picked from the equivalent gel. This study encourages further validation of these candidate antigens for the development of immunologic tools for the detection of T. gondii infection in chickens and rabbits.

1. Introduction

Considering the risk of T. gondii infection in the husbandries of chickens and rabbits, the development of accurate and sensitive tools is urgent needed for the diagnosis of T. gondii. Though recombinant antigens, such as SAG1, GRA7 and GRA5 from T. gondii are employed in many ELISA kits for the detection of specific IgM or IgG antibodies in human serum samples, no commercial kit special for chicken or rabbit has been developed yet (Schares et al., 2017). In this study, we tried to find candidate antigens for the detection of T. gondii infection in chickens or rabbits by 2-DE and Western blot.

Toxoplasmosis is a globally distributed zoonosis and it is estimated that one-third of the world population were infected (Felin et al., 2017). Ingesting undercooked meat containing T. gondii represents one of the major sources of human toxoplasmosis, which leads to abortion or congenital disease baby born in pregnant women and death in AIDS and immunocompromised patients (Suzuki, 2002). As chicken and rabbit meat is consumed worldwide, the recent trend of consuming of free-ranging poultry and rabbit meat increases the possibility of T. gondii infection. Free-ranging poultry and livestock are easier to get infected from soil contaminated with T. gondii oocysts when they feed on the ground. In addition, free-ranging animals could also become infected through ingesting tissue containing bradyzoite and from leftover food (Sousa et al., 2016). A Previous study showed that chickens raised in door were 1.4% positive for T. gondii (Dubey et al., 2005); while another study showed that the infection rate of T. gondii in free-range chicken is from 27% to 100% in the United States (Dubey and Jones, 2008).

2. Materials and methods 2.1. Parasites and animals T. gondii RH strain was used in this study. AA broilers (Arbor Acres) and New Zealand White rabbits were purchased from Beijing Arbor Acres Poultry Breeding Co., Ltd. and Xinglong Experimental Animal Breeding Plant at Haidian District in Beijing, respectively. All experiments involving animals were performed in accordance

*

Corresponding author. Yuanmingyuan West Road, No. 2, Haidian District, Beijing, 100193, China. E-mail addresses: [email protected] (J. Wen), [email protected] (J. Zou), [email protected] (X. Huang), [email protected] (X. Suo), [email protected] (X. Liu). https://doi.org/10.1016/j.exppara.2019.107723 Received 21 February 2019; Received in revised form 4 June 2019; Accepted 3 July 2019 Available online 09 July 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.

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were stained with Coomassie Brilliant Blue R-350 for 15 min at 90 °C then distained with 1% acetic acid. Gels images were obtained at 300 bpi using a Hewlett Packard G4050 scanner and analyzed with PDQuest (Bio-Rad, Hercules, Calif). Protein spots resolved by 2-DE were transferred onto 0.45 μm PVDF membranes using a Trans-blot Semi-dry Transfer Cell™ (Bio-Rad, USA) in semi-dry transfer buffer (48 mM Tris, 39 mM glycine, pH 9.2, 20% methanol). The membranes were rinsed in methanol for 1 min and washed three times with PBST buffer pH 7.4 (with 0.5% v/v Tween). After blocked with 5% milk in PBS, pH 7.2 with 0.05% Tween-20 at 4 °C overnight, the membranes incubated with pooled chicken serum (1:104 ELISA titer) or rabbit serum (1:105 ELISA titer) at room temperature for 1 h. Then the membranes were washed three times for 15 min with PBST buffer again. Immunoreactive protein spots were detected using HRP labeled anti-chicken and anti-rabbit IgG diluted 1:10000 at room temperature for 1 h. After 3 washes in PBST for 15 min, the membranes were visualized with ECL reagents (Applygen Technologies Inc. Beijing. China). PDQuest software was used for matching of immunoreactive spots with protein spots visualized by CBB-R350 staining, which was compatible with subsequent MS analysis, and analyzing of protein spots on 2-D gels. We examined the repetitive of the gels and a reference gel was created by combining all of the spots resent in different gels into one image. The reference gel was then used for matching of corresponding protein spots across different gels. Protein gels were aligned with immunoblots to identify antigens of the same molecular mass and isoelectric point.

with the China Agricultural University Institutional Animal Care and Use Committee guidelines. These experiments were also approved by the Beijing Administration Committee of Laboratory Animals (No. 1114120800096). 2.2. Molecular weight and theoretical PI We downloaded the protein information about Toxoplasma gondii protein information from ToxoDB (www.Toxodb.com) and over 8000 toxoplasma gondii protein information such as molecular weight and theoretical PI (isoelectric point) were obtained. We arranged the protein using molecular weight as the horizontal coordinate and the theoretical PI as the vertical coordinate to get a computer 2D simulation map. 2.3. Preparation of antisera against T. gondii tachyzoites in chickens and rabbits Tachyzoites were propagated in Vero cell monolayers as previously described (da Costa-Silva et al., 2012). For the preparation of antisera from chickens and rabbits, 1 × 107 live tachyzoites were intravenously injected into an animal (3 SPF chickens and 3 rabbits). Before inoculation, we collected sera and performed western blot which were negative. Another 2 infections with the same amount of parasites were conducted at 1 week interval. Seven days after the final infection, animals were sacrificed and blood were collected for the separation of serum. The titers of the sera against T. gondii were monitored by standard ELISA assay with a commercial ELISA kit (Haitai, China) according to the instruction of the manufacturer. The manual of the kit shows it is for the diagnosis of animals, not specific for chicken or rabbit.

2.6. Protein analysis by MALDI-TOF/TOF-MS The in-gel tryptic digestion and protein analysis by MALDI-TOF/ TOF-MS were conducted according to a previous study (Sun et al., 2012). After image analysis, spots which corresponding to the immunogenic spots on the PVDF membranes were fixed and manually excised from stained 2-DE gels. Gels plugs were distained with 50% acetonitrile, and then dehydrated and rehydrated in 10 mM DTT/ 100 mM NH4HCO3 at RT. The gel pieces were further incubated in 55 mM iodoacetamide/100 mM NH4HCO3 in the dark at RT, washed with 50 mM NH4HCO3 and dried again. They were rehydrated in digestion buffer composed of 50 mM NH4HCO3, 5 mM CaCl2 and 12.5 ng/ μl of sequence-grade modified porcine trypsin (Promega, Madison, USA) and incubated on ice. After the excess liquid was removed, protein were subjected to overnight digestion at 37 °C. The supernatant containing the resultant peptides was recovered, extracted in 1:1 (v/v) mixture of 5% formic acid: acetonitrile, pooled and dried in a vacuum freeze drier. The peptides after vacuum freeze-drying were dissolved in 2 μl solution containing deionized water, acetonitrile and trifluoroacetic acid (93:5:2). This solution (0.5 μl) was mixed with 0.5 μl matrix (5 mg/ml α-cyano-4-hydroxycinnamic acid). And then the peptides were dried at RT. The tryptic peptide samples were sent to Shanghai Applied Protein Technology Co. for MALDI-TOF/TOF MS analysis. MS and MS/MS data for protein identification were obtained by using an ABI4800 Mass Spectrometer (Applied Biosystems, USA). Data interpretation was performed with the MS/Ms spectra data using the GPS Explorer™ v3.6 and the Mascot search engine v2.1 (http://www.matrixscience.com). The proteins were identified by searching ToxoDB (www.toxodb.org/toxo), Eukaryotic Pathogens Database Resource (http://eupathdb.org/ eupathdb), and the NCBI nr database (ftp://ftp.ncbi.nih.gov/blast/ db/FAST/nr.gz), assuming that peptides were non-isotopic. The search parameters were: confidence interval, 95%; cleavage enzyme, trypsin; fixed modification, Carbamidomethyl (C); variable modifications, oxidation (M); and maximum missed cleavage, 1. A peptide tolerance of 100 ppm, a fragment mass tolerance of ± 0.4 Da, and a peptide charge of 1 + were considered significant. Sequence coverage, matched peptides, probability score and protein score

2.4. Protein sample preparation for two-dimensional electrophoresis (2-DE) Purified tachyzoites (2 × 108) were suspended in lysis buffer containing 8 M urea, 4% (W/V) CHAPS, 2% (V/V) immobilized pH gradient strips (IPG) buffer (pH 3–10), 10 mM PMSF, 20 U/ml DNase I and 0.25 mg/ml RNase A. The suspension was subjected to three rounds of rapid freezing in liquid nitrogen and defrosting in 30 °C waterbath. Insoluble material was recovered by centrifugation at 16,000×g for 1 h at 4 °C. The concentration of the protein was determined using a 2DE quant kit (GE Healthcare, Sweden). 2.5. 2-DE and western blot The first dimensional separation or isoelectric focusing (IEF) was performed on the PROTEAN® IEF System (Bio-Rad, USA). Each nonlinear IPG strip (pH 3–10 and 17 cm in length) was rehydrated with an equal amount of 350 μg total protein premixed with a rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, 2.8 mg/ml DTT (added freshly before use), 0.5% (v/v) IPG buffer (pH 3–10) and bromophenol blue. Rehydration and isoelectric focusing were performed at 20 °C. Bio-Rad suggested focusing conditions: 50 v for 12 h (active rehydration), 250 v for 30 min, 1000 V for 1 h, 10000 v for 5 h and 10000 v for 60000 v. Except where stated, all electrophoretic equipment, gel analysis equipment and reagents for 2-DE were supplied by Bio-rad. When the isoelectric focusing was finished, the strips were taken out and stored in −20 °C. Before the second dimension electrophoresis, strips were freezing then equilibrated twice with SDS Equilibration Buffer (50 mM Tris-Hcl, 6M urea pH 8.8, 30% glycerol, 2% SDS, 0.002% bromophenol blue) for 15 min, adding 1% (w/v) DTT and 2.5% (w/v) iodoacetamide, respectively. The second dimensional separation was performed in 12.5% (w/v) homogeneous polyacrylamide gels (1 mm thickness × 18.5 cm width × 18.5 cm length), sealed with 0.5% (w/v) agarose. Proteins electrophoresed at 80 V for 30 min and then at 250 V until the bromophenol blue moved to 0.5 cm above the lower margin of the gel. Gels 2

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Fig. 1. In silico analysis of whole cell protein of T.gondii. Spots were plotted by the molecular weight against Isoelectric points.

3.2. Immunoreactive T. gondii proteins identified by MS

considered significant could be used for further manual validation. The analysis was done at least three times under identical experimental conditions.

To figure out which proteins react with anti- T. gondii sera of rabbits and chickens, we performed 2-DE western blot. Among the 500 protein spots visualized on a stained 2-DE gel, approximately 60 protein spots reacted with rabbit or chicken serum were found (Fig. 3A and B). Thirteen immunoreactive protein spots were selectively cut out from the preparative 2-DE gels and analyzed by MALDI-TOF/TOF MS based on matching across membranes, and the presence and the name of the protein were labeled (Fig. 3A and B). For the 13 immunogenic proteins identified, characters of them are listed in Table 1. The functional analysis of the proteome was constructed using the GO classifications listed on ToxoDB (Table 2).

3. Results 3.1. T. gondii proteins resolved by 2-D gel electrophoresis The in silico analysis show that resolved proteins of T. gondii are typically located between pI 4 to 10, and over 90% of the proteins molecular weight between 20 and 150 kDa, which showed a Gaussian distribution (Fig. 1). In our study, over 500 protein spots of T. gondii tachyzoites were resolved in each of the stained 2-D gels, with most of urea-soluble protein located between pH 4 and 9 (Fig. 2A). Other 3 replicates of 2-DE gels showed a similar staining (Fig. 2B I-IV).

4. Discussion and conclusion Combination

analysis

of

2-DE,

immunobloting

and

mass

Fig. 2. Reproducibility of the sample preparation, gel electrophoresis and staining. A, two-dimensional electrophoresis gel separations of proteins from 2 × 108 tachyzoites of T. gondii RH strain. Proteins were focused to their isoelectric points using Immobiline® DryStrip gels (17 cm, pH 3–10); B, each panel (I-III) represents the same enlarged region (dotted box) of replicate R350-stained gels. Each of the three gels was run independently on different days, using different batches of prepared tachyzoites and different batches of gels. A digital composite image (panel IV) of all three gels created using PDQuest 8.0 software is shown using spots present in all three replicate gels to produce an “averaged gel”.

3

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Fig. 3. T. gondii immunogenic proteins. The separated proteins were probed by rabbit sera (A) or chicken sera (B).

were screened by sero-detection with rabbit or chicken sera. As many immunoreactive proteins reported to date are not validated with more field samples, the future development of sero-diagnostic tools for rabbits or chickens should focus on the feasibility of these proteins resolved by 2-DE and MS. In summary, we identified several immunoreactive proteins of T. gondii with chicken or rabbit sera in a preliminary study. Further studies for the development of sero-diagnostic tools for detecting in meat product should take consideration of the specificity of the animal species.

spectrometric is an effective immunoproteomics approach for the identification of novel antigens in pathogens. This strategy has been successfully used for characterizing antigens of bacteria such as Francisella tularensis (Twine et al., 2006) and protozoa such as Neospora caninum (Lee et al., 2005) and Eimeria tenella (de Venevelles et al., 2004). Though many studies focused on the immunoreactive proteins of T. gondii, most of them were conducted with sera samples of model animals or humans. To date, studies toward the diagnostic antigens of T. gondii for rabbit or chicken are extremely limited. In this study, we used rabbit or chicken anti-T. gondii sera to probe immunoreactive proteins in this parasite. In a complex mixture of around 500 proteins, 13 were identified using mass spectrometry. ROP1 is considered as the classic diagnostic antigen for T. gondii, and had been used for the detection of T. gondii infection in animals such as ovine (Holec-Gąsior et al., 2014), swine and sheep (Ferra et al., 2015). In the present study, ROP1 were detected by both rabbits and chicken serum, indicating that ROP1 can be used as the common antigen for animal toxoplasmosis diagnosis. Among the proteins discovered, catalase (Nischik et al., 2001; Sun et al., 2012), MIC2-associated protein (Dautu et al., 2007), MIC4 (Lourenço et al., 2006), HSP (Calderwood et al., 2007) HSP70 (Aosai et al., 2002; Chu et al., 2014; Mohamed et al., 2003), HSP90 (Anderson et al., 2014; Roesch et al., 2012) and lactate dehydrogenase (Reichmann et al., 2001) are immune-related ones, suggesting these proteins are conserved antigens in different hosts of T. gondii. The immunoreactive proteins, such as fructose-1, 6-bisphosphate aldolase,

Conflicts of interest Authors declare that we don't have conflict of interest.

Acknowledgements This study was supported by the National Key Research and Development Program of China (2017YFD0500400), the Special Fund for Agro-scientific Research in the Public Interest (201303042), the National Natural Science Foundation of China (31172309) and the Open Project of the State Key Laboratory of Veterinary Etiological Biology (SKLVEB2011KFKT001). Jing Wen was also supported by Inner Mongolia Agricultural University Experimental Teaching Instrument and Specimen Preparation (YZ2015001).

Table 1 Immunogenic proteins of Toxoplasma gondii identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Protein name

NCBI ID

pI

MW (kDa)

Mascot

Protein score C.I. %

%Cov

No. of matched peptide

catalase fructose-1,6-bisphosphate aldolase heat shock protein, putative heat shock protein 70 heat shock protein 90 lactate dehydrogenase MIC2-associated protein M2AP [Toxoplasma gondii membrane-associated calcium-binding protein MIC4 rhoptry 1 rhoptry 1 Chain C, Crystal Structure Of The Toxoplasma Gondii Pyruvate Kinase N Terminal Truncated receptor for activated C kinase, RACK protein, putative

gi|5706732 gi|237839383 gi|221507275 gi|3850197 gi|237835295 gi|211965769 gi|237843035 gi|221488859 gi|262479201 gi|897823 gi|897823 gi|238537852

6.71 9.01 5.23 5.07 4.98 6.03 4.28 4.57 5.05 5.8 5.8 6

57.58 47.39 73.38 72.7 97.05 36.1 34.87 38.37 65.13 42.64 42.64 56.45

408 873 923 1010 580 814 549 802 323 64 91 433

100 100 100 100 100 100 100 100 100 98.73 99.99 100

26.7 51.0 43.1 38.7 23.5 60.0 40.1 37.6 14.1 10.6 12.8 36.0

7 20 26 27 20 16 8 19 7 4 7 14

gi|237843397

5.89

35.75

906

100

31.5

13

4

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Table 2 Bioinformatics analysis of immunogenic proteins of Toxoplasma gondii identified MALDI-TOF MS. Protein name

Singal IP

transmembrane domains

localization

functional

catalase

no

0

Cyto:12.5

fructose-1,6-bisphosphate aldolase heat shock protein heat shock protein 70 heat shock protein 90 lactate dehydrogenase M2AP membrane-associated calcium-bndiing protein

no yes no yes no yes no

1 1 0 1 0 0 0

Golg:14.0 E.R.:17.0 Cyto:19.5 E.R.:13.0 Cyto:16.0 Extr:31.0 E.R.:6.0

microneme protein (MIC4) Chain C, Crystal Structure Of The Toxoplasma Gondii Pyruvate Kinase N Terminal Truncated receptor for activated C kinase, RACK protein, putative rhoptry protein 1

yes no

0 0

Extr:23.0 Mito:30.0

cellular transport, transport facilitation and transport routes energy cell rescue, defense and virulence cell rescue, defense and virulence cell rescue, defense and virulence energy cell rescue, defense and virulence cellular transport, transport facilitation and transport routes cell rescue, defense and virulence energy

no no

0 0

Nucl:18.5 Extra:26.0

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