All procedures and handling of mice were conducted under an approved protocol by the Institutional Animal Care and Use Committee (IACUC) at the College of Medicine, Catholic University of Korea (CMMC-2015-0042-02, 2015-2017), which adhered to the regulations set under the Korean National Animal Protection Act. The RH strain of T. gondii has been provided by the National Veterinary Research and Quarantine Service.

ARPE-19 cells (ATCC^® CRL-2302TM, Manassas, Virginia, USA) were maintained in Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F12, Invitrogen, Carlsbad, California, USA) containing 2 μM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone and 10% fetal bovine serum (FBS, Gibco Life Technologies, Grand Island, New York, USA) (complete DMEM/F12). Tachyzoites of the RH strain of T. gondii were intraperitoneally injected into BALB/c mice, and peritoneal exudates were collected at the 4th day with Dulbecco’s PBS (DPBS).

Afatinib (BIBW2992) was purchased from Selleck Chemicals (Houston, Texas, USA) as a TKI. DMSO and pyrimethamine were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Afatinib was treated with concentration of 5 mM which obtained over 98% of inhibition of T. gondii growth within the host cells [18]. Bovine serum albumin was purchased from Bovogen Biologicals (Melbourne, Australia). FITC-conjugated anti-mouse IgG antibody, TRTIC-conjugated anti-rabbit IgG antibody, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were purchased from Sigma Aldrich. Antibody against β-actin was purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Mouse Tg563 monoclonal antibody was cloned in our laboratory. PDCD4 (D29C6) XP^® anti-rabbit monoclonal antibody was purchased from Cell Signaling (Boston, Massachusetts, USA) Chemicals for the analytical uses as iodoacetamide was purchased from Sigma-Aldrich. Tris-HCl and urea were obtained from Merck (Branchburg, New Jersey, USA). All other chemicals were acquired from standard sources and were of molecular biology grade or higher.

The ARPE-19 cells were cultured in the complete DMEM/F12 (Invitrogen) at 37°C in a humidified 95% air, 5% CO₂ incubator. Cells were grown to −70% confluency (−1.6×10⁷ cells) in 150 mm culture dishes (Nunc, Naperville, Illinois, USA). The cell monolayer was rinsed carefully with serum-free medium (SFM) 3 times at RT. Then, the cells were incubated in the SFM at 37°C for 12 hr. After incubation, the SFM from 20 plates was carefully collected with 2 mM PMSF and 1 mM EDTA as protease inhibitors. Floating cells and cellular debris were removed by centrifugation (400 g, 10 min, 4°C), followed by sterile filtration (pore size: 0.22 μm, Millipore, Massachusetts, USA). The conditioned medium was concentrated through ultrafiltration using “Amicon Ultra-15” centrifugal filter devices (Millipore). Secreted proteins were precipitated by acetone at −20°C for 1 hr and then dissolved in buffer consisting of 8 M urea, 75 mM NaCl, 50 mM Tris (pH 8.2). Protein concentration was determined by a standard Bradford protein assay (Bio-Rad, Richmond, California, USA). All protein samples were stored at −80°C until use.

The protein sample (100 μg) was reduced with 10 mM DTT (Sigma) at 36°C for 25 min and alkylated with 14 mM iodoacetamide (Sigma) at 25°C for 30 min. The sample was diluted 5 times to decrease urea concentration to less than 1.6 M in solution and added 1 mM CaCl₂. The protein mixture was digested by sequencing-grade modified trypsin (Promega, Madison, Wisconsin, USA) at 37°C for 16 hr. The ratio of enzyme to protein was 1:100. Tryptic digests were directly used or further separated based on hydrophobicity by using a Micro bRPLC. Micro bRPLC columns were prepared by adding slurry of 2 mg (10 mg/1 ml acetonitrile) of Jupiter C18 material (5 μm particle diameter, Phenomenex, Torrance, California, USA) to commercially produced microcolumns (C18 Stage Tip^™; SP301, ThermoFisher Scientific, West Palm Beach, Florida, USA). All elution steps for column packing, washing, and elution were carried out with benchtop centrifugation (3,000×g for 3 min) unless otherwise stated. Prior to addition of peptide mixtures, the column was washed with 100% acetonitrile (100 μl), then with 100 μl equilibration buffer (100 mM NH₄HCO₃, pH 8.0). The sample mixture was fractionated with 100 μl portions of 7 different elution buffers (5%, 10%, 15%, 20%, 25%, 30%, and 90% acetonitrile in 100 mM NH₄HCO₃, pH 8.0).

Nano-high-performance liquid chromatography (nano-HPLC) analyses were performed using an Easy n-LC 1000 system (Thermo Fisher Scientific, San Jose, California, USA). A Q-Exactive mass spectrometer (Thermo Fisher) was used for MS analyses and was operated with Xcalibur (version 2.1) to generate peak lists. The column (30 cm×75 μm) was packed in-house with Jupiter 3 μm, 100 Å pore size C18 beads (Phenomenex). The tryptic peptides were separated using a linear gradient of acetonitrile from 5 to 60% in water in the presence of 0.1% formic acid over a period of 60 min. The mass spectrometer was operated in data-dependent mode with a full scan (m/z 350–2,000) followed by MS/MS for the top 20 precursor ions in each cycle. The acquired MS/MS spectra were subjected to searches against the Uniprot-Human database (June 2018; 73099 sequences) using SEQUEST software in Proteome Discoverer 1.4 (Thermo Fisher Scientific, version 1.4.0.288). Two missed trypsin cleavages were allowed, and the peptide mass tolerances for MS/MS and MS were set to 0.5 Da and 10 ppm, respectively. Other parameters used for the SEQUEST searches included the fixed modification of carbamidomethylation at cysteine (+57.021 Da), the variable modification of oxidation at methionine (+15.995 Da).

Proteomic data were uploaded into the Ingenuity Pathways Analysis program (IPA; Ingenuity Systems, Redwood City, California, USA) as a tab-delimited text file of UniGene numbers (transcriptomic data) or IPI accession numbers (proteomic data). Proteins in the proteomic data were mapped to corresponding gene objects in the Ingenuity Pathways Knowledge Base (IPKB). Then, biological networks were generated using the knowledge base for interactions between the uploaded gene list and all other stored gene objects. Functional analysis of the networks was performed to identify the biological functions and/or diseases that were most significant to the genes in the network. For each network, a score was computed according to the fit of the data set of significant genes by Fisher’s exact test. The score was derived from the P-value of the test and indicates the likelihood of the mapped genes in a network being found together due to random chance (score=-log10p).

Secretome (10 μg of protein) were size-fractionated by SDS-PAGE and then transferred to a PVDF membrane (Bio-Rad) that was blocked with 5% skim milk in TBS-Tween (25 mM Tris, 190 mM NaCl, 0.05% Tween 20, pH 7.5). The membranes were incubated at 4°C overnight with primary antibodies. Antibodies against RPS12, HNRNPK, PTPRK, SEMA7A, PPP2R2A, RUS1 and CAPNS1 were purchased from Cell signaling. After washing, the membranes were incubated at 25°C for 1 hr with appropriate HRP-conjugated secondary antibodies, washed and then developed with an enhanced chemiluminescence reagent (ECL, GE Healthcare).

To identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. gondii using mass spectrometric analyses of conditioned culture media devoid of serum collected from infected ARPE-19 cells. In addition, we performed the secretome analysis of treated-afatinib, which is small molecule of tyrosine kinase inhibitors (TKIs). Overall, 1,867 proteins were identified, and 230 proteins were reported as extracellular space (Fig. 1), wich showed significant in their fold changes, corresponding p-values, and relevant biological processes.

Ingenuity Pathways Analysis programs were used to predict the subcellular localization of the identified proteins and their biological functions. The searched proteins were linked to at least one annotation terms. The distribution of subcellular localizations was cytoplasm (46%), Cytosol (11%), extracellular space (10%), unknown (8%), cell surface (7%) and membrane (5%). Gene Ontology analysis showed that these proteins were mainly involved in protein synthesis and cell death and survival.

To put our proteomics data in a biological context, the expression data were analyzed using IPA software. All proteins were mapped to gene objects in the Ingenuity Pathways Knowledge Base. To further understand the changes observed in the secretome upon T. gondii infection, 269 DEPs were analyzed using IPA software. Eighteen networks were generated from the 269 DEPs. The 3 networks were merged to obtain a global view of the proteins that were differentially regulated between the T. gondii-infected and–uninfected secretome. To further understand the changes observed in the secretome upon treated-Afatinib, 168 DEPs were analylyzed using IPA software. Eleven networks were generated from the 168 DEPs. The 3 networks were merged to obtain a global view of the proteins that were differentially regulated between the treated-Afatinib and–untreated secretome. Interestingly, there were some major hubs in the network. AKT, which has been shown to play key roles at tumor progression and apoptosis, is connected to several DEPs along with the ERK/MAP kinase protein family. Further, there are 15 known drug targets in cancer ERK, p38 mitogen-activated protein kinase (MAPK), Cytokeratin, keratin, HDL-cholesterol, SAA, DNA-pK, MTORCs, CDK4/6, AKT, MTR, Cadherin, 60S ribosomal subunit, PRKAA, Cyclin D) that are a part of the network. The list of common protein in global network is shown in Table 1. In the next step, the 230 extracellular proteins were mapped onto known canonical signaling pathways in order to obtain useful information on molecular interaction networks. The top canonical signaling pathway was acute phase response signaling, which was populated with 18 proteins from the network. The list of acute phase response signaling for extracellular protein is shown in Table 2.

The initial 7 candidate proteins were narrowed down with regard to their biological functions drawn from IPA and the availability of commercial antibodies. Out of 7 proteins, only 4 showed significantly different concentrations between the groups: HNRNPK (Heterogeneous nuclear ribonucleoprotein K, hnRNP K), SEMA7A (Semaphorin-7A), PPP2R2A (Serine/threonine-protein phosphatase 2A) and CAPNS1 (Calpain small subunit 1) (Fig. 2).