Parasites, cells, and experimental animals

The Shanghai strain of E. tenella (CAAS21111611) used in the current study has been retained in our laboratory since 1993 and was propagated by passage through 14-day-old coccidia-free chickens [15]. Sporozoites were collected and purified from cleaned sporulated oocysts using standard procedures [16, 17]. DF-1 cells (ATCC CRL-12203) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (PS) (Gibco) at 37 °C in a 5% CO2 incubator. Chinese yellow chickens were purchased from Shanghai Fuji Biotechnology Co., Ltd., and kept in an environment without coccidia.

Cloning and identification of the chicken vimentin gene

The total RNA in DF-1 cells was extracted using a MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China) and the first-strand cDNA was prepared using total RNA and a reverse transcription kit (TaKaRa). The primers were designed according to the coding region (1383-base-pair [bp]) sequence of the Gallus gallus (chicken) vimentin gene (GenBank accession number: NM_001048076.2) as follows: forward primer: 5′-CGGAATTCATGAGCTTCACCAGCAGCAA-3′, reverse primer: 5′-CCCTCGAGTTACTCCAAGTCATCGTGATGCTG-3ʹ, including EcoRI and XhoI restriction enzyme cutting site, respectively (underlined label). The chicken vimentin gene was amplified and cloned into the expression vector pGEX-4 T-1 to construct the recombinant plasmid pGEX-4 T-vimentin. Then the recombinant plasmid was transformed into Escherichia coli TOP10 competent cells (Tiangen Biotech, Beijing, China) and selected with Luria-Bertani (LB) solid medium containing ampicillin. The positive-screened colonies were sent to the Shanghai Qingke Company for sequencing. Basic Local Alignment Search Tool (BLAST) programs at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) were used for the similarity analysis of the sequencing results and vimentin gene full-length sequences.

Expression, purification, and identification of recombinant vimentin protein

The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Tiangen) and cultured in a shaker of 37 °C and 180 rpm. When the OD600 reached about 0.6, IPTG was added (final concentration 1 mmol/l; Sigma-Aldrich, St. Louis, MO, USA) to induce and express recombinant vimentin protein. The cell pellet was collected by centrifugation and lysed with sonication at 4 °C. Then the lysed product was analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the recombinant protein was present as a soluble protein or in inclusion bodies. The recombinant protein was purified according to the characteristics using a published gel purification method [18]. Purified recombinant vimentin protein (10 μg) was analyzed by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was blocked in 5% skim milk for 2 h at 37 °C and incubated with Anti-GST Tag Mouse Monoclonal Antibody (1:2000; CWBio, Beijing, China) for 2 h at 37 °C, and was then washed in phosphate-buffered saline (PBS) three times for 5 min and incubated with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (1:5000; Proteintech, Chicago, IL, USA) for 45 min at room temperature. Finally, imaging was obtained using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).

Collection of DF-1 cells infected or not infected with E. tenella sporozoites

DF-1 cells (1.5 × 106 cells/well) were plated into six-well plates and cultured at 37 °C in a 5% CO2 incubator to a cell coverage of 80–90%. Freshly isolated sporozoites were incubated with DMEM (2% FBS, 5% PBS) for 2 h at 37 °C. The DF-1 cells were infected with pretreated sporozoites with a multiplicity of infection of 3 (MOI = 3). At different time points during infection (1, 2, 6, 12, 24, 36, 48, 60, and 72 h), the medium and noninvasive sporozoites were removed by washing gently with PBS three times. The DF-1 cells without infecting sporozoites were set as the uninfected control. Every cell sample was harvested with a cell scraper and stored at −80 °C for subsequent analysis.

Assessment of vimentin mRNA transcription levels after sporozoite infection

Total RNA was extracted from cells collected from different time points using the MiniBEST Universal RNA Extraction Kit (TaKaRa) according to the manufacturer’s instructions. The design of specific primers was based on published vimentin and GAPDH gene sequences, as follows: vimentin, forward primer: 5′-GCAAAGTTGAGTCCCTGCAA-3′, reverse primer: 5′-AGGGCAGCAGTAAGATCAGG-3′; GAPDH, forward primer: 5′-GGCACTGTCAAGGCTGAGAACG-3′, reverse primer: 5′- TGAGATGATAACACGCTTAGCACCAC-3′. Vimentin mRNA transcription levels in each group of samples were determined using the One-Step TB Green PrimeScript RT-PCR Kit II (TaKaRa), according to the manufacturer’s instructions. Each reaction was carried out in triplicate, and the experiment was performed three times. The relative expression of vimentin mRNA was calculated using the 2−ΔΔCt method [19], and SPSS 22.0 (https://www.ibm.com) was used for t-test analysis.

Analysis of vimentin expression levels after sporozoite infection

Western blotting was used to detect vimentin expression levels during sporozoite infection. Proteins at different time points (6, 36, and 72 h) were extracted from infected and uninfected samples using cell lysis buffer IP (Beyotime, Haimen, China) for western blotting. Protein concentration was determined by a BCA Protein Assay Kit (Beyotime), then 10 μg of protein was taken from each group for SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was blocked in 5% skim milk for 2 h at 37 °C and then incubated with polyclonal rabbit antibody against vimentin (1:200, prepared by our laboratory) or β-GAPDH (internal reference, 1:2000; Yeasen, Shanghai, China) for 2 h at 37 °C. They were then washed in PBS three times for 5 min and incubated with goat anti-rabbit IgG antibody (1:5000; LI-COR Biosciences, Lincoln, NE, USA) for 45 min at room temperature. Finally, images were obtained with the ChemiDoc Touch Imaging System.

Determination of vimentin phosphorylation levels after sporozoite infection

Proteins were extracted from infected and uninfected cells collected from different time points (1, 2, 6, 12, 24, 36, 48, 60, and 72 h) using a phosphorylated protein extraction kit (Solarbio, Beijing, China). Protein concentration was determined using a BCA protein assay kit (Beyotime), and 10 μg of protein from each group was used for Phos-tag acrylamide-based gel (Wako, Tokyo, Japan) analysis. After electrophoresis, Phos-tag acrylamide gels were immersed three times in transfer buffer containing 1 mM EDTA for 10 min and then washed in transfer buffer without EDTA for 10 min. Then the proteins were transferred to a PVDF membrane for western blot analysis.

Assessment of vimentin distribution after sporozoite infection

Cell climbing films were placed in six-well plates. DF-1 cells (1.5 × 106 cells/well) were plated into the six-well plates and cultured to a cell coverage of 80%–90%. Sporozoites were labeled with PBS containing carboxyfluorescein diacetate succinimidyl ester (CFSE) (1:20,000; Invitrogen, Carlsbad, CA, USA) for 15 min and then incubated with DMEM (2% FBS, 5% PS) for 2 h at 37 °C. Labeled sporozoites were added to the DF-1 cells with a multiplicity of infection of 3 (MOI = 3). Samples were collected from the six-well culture plates at different time points (1, 12, and 24 h). The uninfected and infected DF-1 cells were fixed with 4% paraformaldehyde (Solarbio) for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked in 2% bovine serum albumin (BSA) (Solarbio) at 4 °C for 12 h. DF-1 cells were incubated with polyclonal rabbit antibody against vimentin (1:200) for 2 h at 37 °C, and then washed in PBS three times for 5 min and incubated with Alexa Fluor 647 Goat Anti-Rabbit IgG (1:500; Invitrogen) for 45 min at room temperature. DF-1 cells were washed again with PBS, stained with DAPI (1:500; Beyotime) for 15 min, treated with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich), and imaged under a fluorescence microscope (Olympus, Tokyo, Japan).

Analysis of anti-vimentin polyclonal antibodies on sporozoite invasion

DF-1 cells (3.0 × 105 cells/well) were plated into 24-well plates and cultured to a cell coverage of 80–90%. Polyclonal rabbit antibody against vimentin (prepared by our laboratory) was purified using protein A+G agarose (Beyotime). Then, purified IgG was added to DF-1 cells at a final concentration of 25, 50, 100, 200, 300, or 400 μg/ml in DMEM (2% FBS, 5% PS) at 37 °C for 3 h, respectively. The same quantity of purified IgG from rabbit sera was used as negative control, and DF-1 cells incubated without antibodies were used as a positive control. After 3 h, the original medium was replaced with DMEM (2% FBS, 5% PS), and the sporozoite invasion rate was determined by an invasion assay in vitro [9]. Labeled sporozoites (as described above) were added to DF-1 cells at a multiplicity of infection of 3 (MOI = 3). After invasion for 6 h at 37 °C in 5% CO2, DF-1 cells were washed with PBS to remove noninvasive sporozoites, digested with trypsin, and analyzed by flow cytometry (Cytomics FC 500; Beckman Coulter, USA). A total of three tests were carried out.

Effects of downregulation of vimentin expression by small interfering RNA (siRNA) on sporozoite invasion ability

Transfection experiments were performed when cell coverage reached 40–50% using the siRNA sequences published by Schäfer et al. (Table 1) [20]. The DF-1 cells were transfected with siRNA 1 + 2 + 3 or negative siRNA using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Briefly, DNA and the transfection reagent were mixed (10 ml lipofectamine 3000 and 4 μg DNA), incubated at room temperature for 30 min, and added to the cells. Six hours later, the DNA-transfection reagent mixture was replaced by DMEM containing 10% FBS. All plasmids were transfected in triplicates. In the mock-treated cells, only the transfection reagent was used. At 24 h post-transfection, the protein samples were collected to test vimentin expression levels by western blot analysis.

Table 1 Sequences of siRNA used to downregulate the vimentin expression

In order to determine the cell growth after transfection with siRNAs, we examined cell activity. DF-1 cells (0.8 × 105 cells) were plated into 96-well plates, then transfected using the above method. The original medium was discarded after 24 h transfection, 90 μl complete medium and 10 μl CCK-8 solution were added to each well, and then the OD450 value of the cells was measured every hour for 6 h.

DF-1 cells (3.0 × 105 cells) were plated into 24-well plates and transfected with siRNA. After 24 h transfection, the CFSE-labeled sporozoites (as described above) were added to DF-1 cells at a multiplicity of infection of 3 (MOI = 3). After invasion for 6 h at 37 °C in 5% CO2, DF-1 cells were washed with PBS to remove noninvasive sporozoites, digested with trypsin, and analyzed by flow cytometry (Cytomics FC 500; Beckman Coulter, USA). DF-1 cells that were not transfected and transfected with negative siRNA were used as control groups. A total of three tests were carried out.

Statistical analysis

The experimental data were compared using SPSS version 22.0 statistical software (SPSS IBM Corp., Armonk, NY, USA). The t-test was used for two independent samples, and one-way analysis of variance (ANOVA) and Duncan’s multiple range test were used for multiple groups. Significance was set at P < 0.05, with P < 0.01 indicating an extremely significant difference. GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for graphing the results.

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