Specific EGFR and phospho-EGFR (Tyr1068) rabbit monoclonal antibodies (mAb) were from Cell Signaling Technology, Inc. (Beverly, MA, United States). AG1478 were from Selleck Chemical., Co. (Houston, TX, United States). Beta-actin mouse mAb was from Proteintech (West Grove, PA, United States). Cy3- or FITC-conjugated goat anti-rabbit or anti-mouse IgG, peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG were all from Proteintech. Recombinant hEGF was from Sigma Chemical., Co. (St. Louis, MO, United States). Prokaryotic recombinant protein NcMIC3 (rNcMIC3) and mouse antiserum specific for NcSAG1 was produced in our Laboratory. Full-length gene of N. caninum MIC3 was subcloned into the pET-28a (+) vector (TaKaRa, Japan) containing the His-tag, and the recombination pET-28a-NcMIC3 contains four EGF sequence repeats.

HEK-293 cells (ATCCCRL-1573) and Vero cells (ATCCCCL-81) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic–antimycotic reagents (all from Gibco BRL, Rockville, MD, United States). One day before treatment, the culture medium was changed to DMEM with 2% FBS.

Tachyzoites of N. caninum-1 strain (ATCC 50843) were restored at our laboratory. Vero cells were infected with tachyzoites of Nc-1 and cultured at 37°C and 5% CO₂ for 3–5 days in DMEM/F12 supplemented with 2% heat-inactivated FBS and antibiotic–antimycotic reagents. After spontaneous host cell rupture, parasites and cell debris were washed in cold DMEM/F12 without FBS and harvested by centrifugation at 850 × g at 4°C for 10 min. After centrifugation, the pellet was resuspended in cold DMEM/F12 and passed through a 26-gauge needle (Millipore, Billerica, MA, United States) to further break the cells. The obtained mixture was slowly added onto 40% percoll solution (GE Healthcare, United States) in DMEM/F12 without FBS and separated by centrifugation at 850 × g in a horizontal centrifuge for 30 min. The fraction containing tachyzoites at the bottom of the tube was collected, resuspended in DMEM/F12 without FBS and centrifuged at 850 × g at 4°C for 10 min. The final pellet was purified tachyzoites.

HEK-293 cells were cultured in 24-well plates containing glass slides and serum-starved overnight. Serum-starved Nc-1 tachyzoites were inoculated into the cells or not at a MOI of 10 for 10 min. For parasite immunofluorescence, purified Nc-1 tachyzoites were fixed on coverslips coated with polylysine. Coverslips were rinsed in 1× PBS with 0.05% Tween-20. Monolayers were fixed with 4% paraformaldehyde for 15 min, washed three times in PBST and permeabilized in cold 0.25% Triton X-100 (Life Technologies Corporation, CA, United States) for 10 min. After PBST washing, coverslips were blocked in 5% w/v bovine serum albumin (BSA) for 1 h at 37°C. Slides were incubated in the mAb against phospho-tyrosine 1068 EGFR and EGFR (1/1,000) or antiserum against NcSAG1 (1/100) in PBST containing 3% BSA for 1 h at 37°C. After washing three times in PBST, slides were incubated with the corresponding secondary antibody conjugated to Cy3 or FITC (1/500) in PBST containing 3% BSA for 45 min in the dark. Slides were washed three times in PBST and counter stained with DAPI (1/1,000; GeneCopoeia, United States) for 5 min. After washing, monolayers were then observed using a FV1000 confocal microscopy (Olympus Co., Japan). Specificity of staining was determined by incubating monolayers with secondary antibody only.

Nc-1 tachyzoites were prepared as above. For treatment with EGFs, 2 × 10⁶ tachyzoites were serum-starved overnight and subjected to treatments with 25–400 ng/ml hEGF or vehicles for 10 min, and with 0 or 100 ng/ml rNcMIC3 for 5–120 min, respectively. Some sets of tachyzoites (2 × 10⁶) were incubated with 100 ng/ml rNcMIC3 or 50 ng/ml hEGF for 0, 10, 30, and 60 min in the presence of AG1478 (20 μM) or DMSO. Before and after treatments, parasite pellets were collected, rinsed with cold PBS for three times, and gently sonicated on ice for 15 s (amplitude 30, 5 s for 10 s; Sonics and Materials Inc., United States) in RIPA lysis buffer supplemented with protease and phosphatase inhibitors and PMSF (all from Sangon Biotech Co., Shanghai, China) and then lysed on ice. After mixing with 5× loading buffer, protein extracts were boiled for 5 min and centrifuged at 10,000 × g for 5 min at 4°C. The supernatants were then subjected to electrophoresis on 10% SDS-PAGE gels (Bio-Rad Laboratories, Inc., United States) in the same amount and transferred to PVDF membrane (Pall Life Sciences, United States) by electroblotting (Bio-Rad). Membranes were blocked for 1 h in 5% (w/v) skim milk in TBST. The expression of N. caninum EGFR was detected using antibody to total EGFR (170 kDa, 1/1,000) or phospho-tyrosine 1068 EGFR (170 kDa, 1/1,000) in TBST overnight at 4°C, followed by four times washing and incubation with horseradish peroxidase-conjugated affinipure goat anti-rabbit IgG (1/2,500) in TBST for 1 h at room temperature. Protein bands were visualized by using an enhanced chemiluminescence kit (Proteintech Group Inc., United States) and detected using ChemiScope series 5300 (Clinx Science Instruments Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Beta-actin and NcSAG1 was used as the loading control. Intensities of phospho-EGFR were calculated using ImageJ (NIH) and normalized against total EGFR.

To determine whether rNcMIC3 or hEGF could induce PKC activity in tachyzoites, a non-radioactive PKC assay kit (Calbiochem, La Jolla, CA, United States) was used. Serum-starved tachyzoites were prepared and subjected to different treatments. One set of tachyzoites (1 × 10⁷) were incubated with rNcMIC3 at concentrations of 0.01–1 μg/ml or with vehicles at 37°C for 10 min. One set of tachyzoites (1 × 10⁷) were incubated with 0 or 0.05 μg/ml hEGF for 10–60 min. To test for activity inhibition, tachyzoites (1 × 10⁷) were incubated with or without the EGFR inhibitor AG1478 (20 μM) for 2 h, and then stimulated with 0.1 μg/ml rNcMIC3 or 0.05 μg/ml hEGF for 10 min. After treatments, tachyzoites pellets were collected, rinsed with HEPES buffer, and lysates were obtained as described above. The lysates were centrifuged at 10,000 × g for 15 min at 4°C and supernatants were used for protein-kinase activity analysis. PKC activity was measured with ELISA according to the manufacturer’s instructions. Samples were applied in triplicates in 96-well plates and read at 450 nm on a Powerwave 200 spectrophotometer (Bio-Tek, Winooski, VT, United States). Relative PKC activity was calculated by dividing PKC activity induced by EGF by the PKC activity of its respective mock-treated control.

HEK-293 cells were cultured in 24-well plates containing glass coverslips prior to inoculation with freshly purified Nc-1 tachyzoites at a MOI of 10 in the presence of AG1478 or DMSO with the concentration of 20 μM. For pretreatment on parasites only, tachyzoites were pretreated with DMSO or AG1478 (20 μM), then inoculated to HEK-293 cells after rinse with PBS. At 2 h, the coverslips were rinsed with PBS to remove free tachyzoites. After 24 h of incubation, the coverslips were rinsed three times with PBS and cells were collected and genome were obtained using a Genome DNA Extraction Kit (Sangon Biotech Co., China). Then the DNA was adjusted to 20 ng/μl and the parasite burden was determined by real-time fluorescent quantitative PCR. The Nc-5 sequence of N. caninum was detected using a BioEasy SYBR Green I Real Time PCR Kit (Roche, United States) following the manufacturer’s instructions with the primer pairs (forward primer, 5′-TCCCTC GGTTCACCCGTTCACACAC-3′, reverse primer, 5′-CACGTATCCCACCTC TCACCGCTACCA-3′). Cell GAPDH were used as the control and the relative numbers of N. caninum in cells were calculated by SPSS. In addition, some sets of monolayers were fixed with cold methyl alcohol and stained with acridine orange (Life Technologies, United States), which were observed using a FV1000 confocal microscopy (Olympus Co., Japan). The number of tachyzoites per 100 cells and the numbers of tachyzoites in vacuoles were determined by counting at ×1,000 magnification. Experimental groups had triplicate samples and at least 100-200 cells or 100 vacuoles per sample were counted.

Results were obtained from triplicate values in each independent experiment and analyzed for statistical significance using Student’s t-test and one-way ANOVA by SPSS 19.0 software. Results are expressed as the mean ± standard errors. Differences were considered statistically significant when p-values were <0.05 (^∗p < 0.05 and ^∗∗p < 0.01).

To examine whether N. caninum could induce phosphorylation of EGFR on the cell surface, purified tachyzoites were inoculated to HEK-293 cells. Immunofluorescence staining with tyrosine residue 1068 specific phosphor antibody suggested that not only cell EGFR was phosphorylated; N. caninum infection also induced activation of EGFR-like on tachyzoites themselves (Figure 1). Phospho-EGFR-specific fluorescence could be observed as early as 10 min after infection, while phospho-EGFR (Tyr1068) expression could not be observed in tachyzoites which were not inoculated into cells, as shown in Figure 1. Thus, N. caninum infection caused rapid EGFR-like activation in tachyzoites.

In order to determine the expression of parasite EGFR, protein extracts from N. caninum tachyzoites and HEK-293 cells were subject to immunoblot using mAb against EGFR and β-actin, respectively. An EGFR-specific protein band with a molecular weight of 170 kDa was shown in both tachyzoite and HEK-293 cell lysates as shown in the left panel of Figure 2A. Moreover, the β-actin protein band (45 kDa) was present in HEK-293 cell lysates while not in tachyzoite lysates, suggesting that tachyzoite extracts were not contaminated with cell proteins as shown in the right panel of Figure 2A. Meanwhile, the results from immunofluorescence staining using the antibody specifically against EGFR also suggested the expression of EGFR-like in N. caninum tachyzoites (Figure 2B). In addition, co-localization of the EGFR-like with SAG1 in N. caninum by IFA indicated that N. caninum EGFR-like located on the tachyzoite surfaces (Figure 2C).

Next, the ability of EGFs to induce PKC activities in tachyzoites was investigated. PKC activities in tachyzoites before or after stimulation with rNcMIC3 or hEGF were measured. The results showed that after treatment with the recombinant proteins, the PKC activities in tachyzoites were significantly increased in a concentration and time dependent manner. When incubated with rNcMIC3 (Figure 3A, p < 0.01) at 100 ng/ml or hEGF (Figure 3B, p < 0.01) at 50 ng/ml for 10 min, the PKC activity peaked and increased by 50.7 and 48.8% compared to control, respectively, while these increases were both effectively inhibited when treatment with 20 μM EGFR-inhibitor AG1478 (Figure 3C, p < 0.01). The results suggested that the EGF-dependent PKC activity was present in N. caninum tachyzoites.

To examine whether N. caninum MIC3 which contains four EGF repeats and hEGF were involved in the activation of parasite EGFR-like, purified tachyzoites were treated with or without rNcMIC3 or hEGF with or without the presence of AG1478. Immunoblot results suggested that phosphorylation of tachyzoites EGFR-like (170 kDa) increased with recombinant hEGF and NcMIC3 in a dose- and time-dependent manner, respectively, compared to control. EGFs-induced phosphorylation of parasite EGFR-like peaked at 50 ng/ml of hEGF (Figure 4A, p < 0.01) or at 100 ng/ml of rNcMIC3 (Figure 4B, p < 0.01) both for 10 min. Furthermore, results from co-incubation of 50 ng/ml hEGF (Figure 4C, p < 0.05) or 100 ng/ml rNcMIC3 (Figure 4D, p < 0.05) with 20 μM AG1478 for 10–60 min showed that the phosphorylation levels of tachyzoites EGFR-like induced by the recombinant proteins were significantly decreased at every time point. These results showed that rNcMIC3 and hEGF promoted the activation of tachyzoite EGFR-like.

Finally, we explored the role of tachyzoite EGFR-like activation in parasite proliferation in host cells. The results from acridine orange staining indicated that treatment or pretreatment with specific EGFR-inhibitor AG1478 (20 μM, which was close to the highest IC50 and exhibited no toxicity to cells) obviously decreased the ability of the tachyzoites to grow within vacuoles in HEK-293 cells. AG1478 treatment decreased not only the number of tachyzoites per 100 cells by 52.9% (Figures 5A,C, p < 0.01), but also the number of tachyzoites per vacuole (Figures 5B,C, p < 0.01) at 24 h. AG1478 pretreatment on tachyzoites only induced a small but significant reduction in the number of tachyzoites per 100 cells of 21.2% and per vacuole at 24 h (Figures 5A–C, p < 0.05). In addition, the results from real-time fluorescent quantitative PCR showed that treatment or pretreatment with AG1478 significantly reduced the parasite burden in HEK-293 cells by 42.3% (p < 0.01) and 23.4% (p < 0.05) at 24 h, respectively (Figure 5D). These data suggested that inhibition of EGFR kinase activities both on parasites and hosts by AG1478 hindered the intracellular development of parasites, suggesting that tachyzoite EGFR-like might be involved in N. caninum proliferation within host cells.