rTgOWP1-f, a specific biomarker for Toxoplasma gondii oocysts.

Toxoplasma gondii oocyst wall protein 1 (TgOWP1) integrates a family of seven proteins, consensually assumed as specific antigens of Toxoplasma gondii oocyst stage, located in the outer layer of the oocyst wall. Herein, we notice the expression of a recombinant antigen, rTgOWP1-f, derived from a fragment selected on basis of its structural homology with Plasmodium MSP1-19. Rabbit polyclonal antibodies anti-rTgOWP1-f evidence ability for specific identification of environmental T. gondii oocysts. We assume, rTgOWP1-f, as a possible biomarker of oocysts. In addition, we present findings supporting this vision, including the development of an immunodetection method for T. gondii oocysts identification.

Toxoplasma gondii infections are widely prevalent in humans and animals worldwide 1 . Humans become infected postnatally by ingesting tissue cysts from undercooked meat and consuming food or drink contaminated with oocysts [2][3][4][5] . The relative importance of transmission via tissue cysts versus oocysts is unknown 4 . Consensually, literature admits that oocysts can remain viable for long periods in environment and, in addition, they are resistant to chemical and physical treatment currently applied in water plants, including chlorination and ozone treatment 6,7 . However, the detection of Toxoplasma oocysts in water is complex, and no standardized methods are available. Recently, Heather Fritz and Patricia Conrad, 2018 8 , proposed a strategy for oocyst identification based on antibodies against a selected group of TyRP's and TgOWP2 proteins (US 2018/0017557A1). Herein, we propose a different approach: an immunofluorescence assay based on rabbit polyclonal antibodies against a selected sequence, rTgOWP1-f. The choice of TgOWP1 as a possible biomarker for environmental oocysts was based on its location in the outer layer wall of both sporulated and unsporulated oocysts 9,10 . However, a technical constraint was clear; TgOWP1 sequence and primary structure is complex, and presents additional difficulties in the expression of a homologue recombinant antigen in E. coli, because its low solubility and low expression 9 . To overcome the situation, we have submitted TgOWP1 sequence at structural searches by ExPASy workstation in order to identify fragments that may constitute targets to host immunological response. Curiously, we have remarked that several sequences within TgOWP1 gene present structural homology with Plasmodium merozoite surface protein 1 C-terminal 19-kDa fragment . This peptide is involved in the interaction of Plasmodium merozoites with red cells membranes, and it is highly immunogenic in malarial infections 11,12 . This finding was critical for our choice. We designed specific primers to amplify the portion of the gene coding the sequence referred as TgOWP1-f and presenting structural homology with MSP1-19. A recombinant homolog sequence was expressed in an E. coli vector and purified. Polyclonal antibodies against the recombinant protein, rTgOWP1-f, obtained after rabbit and mice immunization, evidence a clear-cut ability to identify T. gondii oocysts.

Results
Structural analysis of TgOWP1. TgOWP1 is a 499-amino acid protein, with a putative signal peptide sequence, followed by six type I (six-cysteine) domains and by a single four-cysteine type I domain at the C-terminus. Type II domains are absent in TgOWP1. The domain structure of TgOWP1 (Fig. 1a) was previously described 13 . Analysis of proteins containing sequences homologues to the TgOWPf with BLAST shows high identity values (> 90%) with proteins from T. gondii and with an oocyst wall protein of Hammondia hammondi (Fig. 1d). ExPASy workstation was utilized in the search for structural homologies, and highlighted the presence of two fragments with significant homology to the C-terminal sequence of Plasmodium merozoite surface protein I (MSP1-19) (Fig. 1c). The TgOWP1-f shows significant structural homology with P. yoelii MSP1-19 (sample 2mgp.1.A from ExPASy Structural database) 14 with values of Global Model Quality Estimation (GMQE) of 0.24 and Qualitative Model Energy Analysis (QMEAN) of −3.53, and a sequence identity of 21.13% (Fig. 1c). Structural analysis of the fragment TgOWP1, by Swiss Prot Modelling, and secondary structure tools 15 , suggest that the fragment has several extended strands separated by random coils (Fig. 1b)  the vectors pQE30, and pQE30H for immunological purposes 13,16 , and pQE30F 13,17 for production purposes (Fig. 2a,b). As expected, antigens production showed a significant increase for both tags, although more important when the F-tag was used (5.8 mg / L for rFTgOWP1-f, 0.9 mg / L for rTgOWP1-f and 2.7 mg / L for rHT-gOWP1-f). Purified proteins were analyzed by SDS-PAGE (Fig. 2b). Molecular weight was slightly higher than expected (15 kDa for rTgOWP1-f, 16 kDa for rHTgOWP1-f and 23 kDa for rFTgOWP1-f), probably associated to structural characteristic of TgOWP. rFTgOWP1-f antigen was used for serological assays to evaluate the presence of specific anti-TgOWP1-f antibodies.
Evaluation of TgOWP1-f immunogenicity. To confirm the development of antibodies against rTgOWP1-f, mice were immunized with soluble recombinant antigen without the use of adjuvant and ELISA evaluated their immunogenicity. The presence of specific antibodies against rTgOWP1-f was detected from the 3 rd week onwards (Fig. 3). A plateau was reached at day 35 after injection. Both antigens are immunogenic. However, the presence of the H-tag seems to increase the consistency of the level of antibodies against rTgOWP1-f. For production of specific antibodies against TgOWP1-f in rabbits, rHTgOWP1-f was used due to the increased levels of production.
Production of specific antibodies. In order to evaluate the specificity of the antibodies an immunoblotting assay was done using rTgOWP1-f under native (Fig. 4b) and denaturated conditions (Fig. 4a). Under native conditions, three bands with molecular weights compatibles with a monomer, a dimer and a trimer of rTgOWP1-f were observed, suggesting that rTgOWP1-f is able to form polymeric structures. Anti-rTgOWP1-f antibody recognized the native protein in oocysts (Fig. 5). However, does not recognized the protein in T. gondii tachyzoites.

Detection of T. gondii oocysts by immunofluorescence.
Immunofluorescence assay was performed to evaluate the ability of antibodies anti-rHTgOWP1-f to detect oocysts previously treated with NaOCl, as prior described 18 . The immunofluorescence assays were done with oocysts in suspension and visualized without any fixation process. The immunoserum recognized the wall of both sporulated and unsporulated oocysts (SI - figure  B and D). No significant fluorescence was observed with pre-immune and control negative serum (SI - figure E). In order to determine if the antibody is specific for oocysts, immunofluoresce assays were done with T. gondii

Discussion
The detection of Toxoplasma oocysts remains an issue for many years. Herein we describe a new recombinant TgOWP1 derived-fragment, rTgOWP1-f, as biomarker for environmental oocysts. The TgOWP1 selection was based on i) its presence in the oocyst wall protein 9 , ii) not detectable in tachyzoites 9 , iii) neither in tissue cysts 10 . Protein structure and function of TgOWPs family is not clear. Nevertheless, a potential role on the production of an extracellular matrix inducing heteropolymeric complexes stabilized by disulphide or di-tyrosine bridges has been suggested 9 . In addition, Santana et al., 2015 reported specific antibodies anti-TgOWP1 in naturally infected hosts 10 , and Sotiriadou and Karanis, 2008, suggested it as target gene for the detection of T. gondii oocysts in natural water samples 19 . The selection of this specific TgOWP1 sequence/fragment was based on its structural homology with a C-terminal sequence of Plasmodium merozoite surface protein I-19 (MSP1-19) and, critically, high diversity comparing with TgOWP family proteins or in similar proteins from Cryptosporidium spp, Besnoiti spp, Hammondia hammondi and Neospora caninum. Our findings indicated high immunogenicity of rTgOWP1-f and a strong ability to recognize T. gondii oocysts specifically. Unfortunately, Neospora caninum or Hammondia hammondi oocysts were not available to be included in the assay. The sensitivity and specificity of these antibodies is critical for the development of ImmunoMagnetic Separation/IFA assay similar to EPA Method 1623 20 . So far, three monoclonal antibodies were described: mAb 3G4 21 , mAb 4B6 22 , and MAb K8/15-15 23 . Its specificity is under scrutiny. Recently, during a survey in order to detect and quantify the presence of T. gondii oocysts in fresh vegetables and fruits, usually raw eaten, the antibodies against rTgOWP1-f proved to be helpfully (Marques et al., ongoing work). Nevertheless, its specificity is under scrutiny, and deeper investigation are warranted in order to decipher the grade of environmental Toxoplasma oocysts contamination.   www.nature.com/scientificreports www.nature.com/scientificreports/ gene possesses three introns of 206, 85 and 393 nucleotides length, and includes an amino-terminal intron that delineates a putative signal peptide sequence present on a short exon. Primers were designed in order to amplify the entirely second exon and introducing restriction enzyme recognition sites. Primers sequence were: TgOWP_SacI 5′-TGTGCCTGTGTGAGCTCCCTCCTGTG-3′ and TgOWP_KpnI: 5′-TGATGCGCGGTACCCTAGGGAACGAC-3′. TgOWP fragment was amplified by PCR using 10 µl of T. gondii DNA sample, 2 µl of 25 mM MgCl 2 , 1 µl of dNTPs mix (1 mM), 10 pmol of each primer, 5 µl of Taq buffer (Thermo Scientific), and ultra-pure water to complete a 50 µl volume. PCR amplification was performed with an initial denaturation step (4 min at 95 °C), followed by 30 cycles of denaturation (30 s at 95 °C), annealing (30 s at 50 °C), and extension (1 min at 72 °C). The program included a final extension step of 7 min at 72 °C, and it was performed in a C1000 Touch Thermal Cycler (Bio-Rad). PCR fragment was separated in LMAg and isolated with illustra GFX PCR DNA & Gel Band Purification Kit (GE Healthcare) according to the manufacturer's instructions.

Methods
Subcloning of TgOWP1-f. The TgOWP1 fragment was subcloned in pGEMT easy vector (Promega) and transformed into E. coli XL1 Blue cells. The pGEMT -TgOWP plasmid was isolated with Wizard Plus SV Miniprep DNA Purification System Kit (Promega) and sequenced at Eurofins (Germany) sequencing services. The fragment was isolated from pGEM-TgOWP plasmid upon digestion with SacI (Promega) and KpnI (Promega) restriction enzymes and subcloned in the vector pQE30 (Qiagen), the vector pQE30 with the H-tag, and the vector pQE30 with the F-tag 13,16,17 . Expression and purification of TgOWP1 proteins in E. coli. E. coli were cultivated in LB medium supplemented with ampicillin (100 µg/ml) and kanamycin (50 µg/ml) at 37 °C. The culture was induced with 1 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 5 h at 37 °C 17 . Cells were harvested by centrifugation at 4000 g during 15 minutes at 4 °C, and the pellet was lysed with 8 M urea, pH 8.0, overnight at room temperature and constant stirring of 150 rpm. Cell extracts were centrifuged at 10.000 g for 20 minutes and resulting supernatant and pellet were collected separately for further analyses. The supernatant was applied into a Ni-NTA column (Qiagen) pre-equilibrated with 8 M urea, pH 8.0. Ni-NTA purification was conducted according to the manufacturer's instructions 24 , and the protein elution performed by a pH decrease from 8.0 to 4.5. SDS-PAGE gels stained with Coomassie-blue dye was used to analyze the purity of collected fractions from Ni-NTA chromatography. Protein concentration of each collected fraction was determined by Bradford assay.
Immunization of mice. In order to remove endotoxins, rTgOWP1-f and rHTgOWP1-f were treated  T. gondii oocyst and tachyzoites extract were probed with specific anti-TgOWP1-f sera (P) or pre-immune (N) sera to evaluate the presence of the antigen in the extract. MW -molecular weights; TgO -membrane with recombinant antigen rTgOWP1-f stained with Amido Schwartz; rTgOWP1-f -Immunobloting results using rTgOWP1-f antigen; Oocyst extract -Immunobloting results using oocyst extract; Tachyzoites extract -Immunobloting results using Tachyzoites extract. rTgOWP1-f, oocysts extract and tachyzoite extract were not run on the same blotting and were not probed together with the anti-TgOWP1-f sera because we believe that specific antibodies could bind mainly to the recombinant antigen, hence decreasing the signal in the oocyst extract (competition effect).
Western blot. SDS-PAGE was carried out using SE 250 Mini-Vertical unit (GE Biosciences). Tris-tricine gel 25 was loaded with 100 µg of rTgOWP1-f or 1 mg of T. gondii tachyzoites extract (single well comb). Oocysts lysate antigen (~2 ×10 6 oocysts) was loaded in a single well (10-well). After electrophoresis in 15% SDS-PAGE Tris-tricine gel and transferred to nitrocellulose membranes using a sandwich system using a T22 mini tank transfer unit (GE Biosciences). For rTgOWP1-f and T. gondii tachyzoites antigens a strip containing the protein marker and part of the antigen was cut from both nitrocellulose membranes and stained with Amido Schwartz reagent. The rest of the membranes were saturated with 5% PBS-milk solution for 1 h at room temperature. In the case of T. gondii oocysts antigens each single lane was cut and saturated (lane containing molecular weights was attained with Amido Schwatz). Membrane strips were incubated with rabbit anti-rHTgOWP1-f, mice anti-rTgOWP1-f, or pre-immune sera overnight at 4 °C. Protein G-peroxidase and anti-mouse IgG (H + L)-HRP (Bio-Rad) were used as conjugate for rabbit and mice sera respectively, and 4-chloro-1-naphthol in cold methanol, PBS and hydrogen peroxide for protein detection 17 .
Data analysis. Data analysis was performed using the statistical analysis software SPSS v. 15.0 (SPSS Inc).
Differences between arithmetic means were evaluated by Student´s t-test. Differences with a confidence interval of 95% or higher were considered statistically significant (P ≤ 0.05).

Data availability
The datasets used and analysed in the current study are available from the corresponding author in response to reasonable requests.