HEK293T- and T. annulata schizont-infected cells were provided by the Vector and Vector Borne Disease team, Lanzhou Veterinary Research Institute (LVRI), China. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco™, Thermo Fisher Scientific) at 37 °C in the presence of 5% CO2. Theileria annulata-infected cells were maintained in RPMI 1640 medium (Biological Industries, Kibbutz Beit Haemek, Israel) containing 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel).
Quantitative real-time PCR
Total RNA was extracted from T. annulata at three life-cycle stages (schizont, sporozoite and merozoite) using an RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the user manual. The complementary DNA (cDNA) templates were then synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa, Bio Inc., Kusatsu, Shi, Japan) following evaluation of the concentration and quality of RNA. The specific primers for the target genes were designed according to the sequences derived from the NCBI database. The levels of the β-actin transcript in T. annulata and bovine cells were used for normalization. All primer sequences used in the present study are shown in Additional file 1: Table S1. Quantitative real-time PCR assays were performed in the Stratagene MX3005P thermocycler (Agilent Technologies, Santa Clara, CA, USA) and using the TB Green® Premix Ex Taq™ kit (Tli RNaseH Plus; TaKaRa Bio Inc.) according to the user manual. The relative transcript levels of the target genes were calculated using the comparative cycle threshold (2−ΔΔCT) method.
Western blotting analysis
Cells were lysed with RIPA lysis buffer (Boster Bio, Pleasanton, CA, USA; #AR0102) containing protease inhibitors (Roche, Basel, Switzerland; #4693132001) and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland, #4906845001) for 30 min on ice. The supernatants of the cells were then collected by centrifugation at 15,000 g for 15 min at 4 °C, and the concentration of the total proteins was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; #23225). The protein samples were separated in sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred to PVDF membranes (MilliporeSigma, Burlington, MA, USA). Membranes were first blocked with blocking buffer (Tris-buffered saline, pH 7.4, containing 0.05% Tween-20 and 5% bovine serum albumin [BSA]) for 2 h at RT and then incubated with the relevant primary antibodies at 4 °C overnight. Incubations with an horse radish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Abcam, Cambridge, UK; #ab6721 or #ab6789) were performed for 1 h at room temperature (RT). The proteins levels were determined using SuperSignal™ West Pico PLUS Chemiluminescence Substrate (Thermo Fisher Scientific; #34577).
Subcellular localization of the SVSP455 protein in T. annulata-infected cells
The protein coding sequence (CDS) of SVSP455 was determined from Theileria annulata-infected cells by PCR assay, and the sequence of SVSP455 was analyzed by informatics assays. The amino acids of SVSP455 from 21 to 421 residues were used as the target sequences to prepare the antibody against SVSP455. The recombinant expression plasmid SVSP455-pET30a was then successfully obtained after sequencing and enzyme digestion identification. Finally, the recombinant protein SVSP455 was expressed in Escherichia coli and was purified via Ni–NTA Agarose. After determining the concentration and reactivity of the purified protein, 200 mg of the protein was used to immunize rabbits, with three injections at 14-day intervals. The sera against SVSP455 protein derived from rabbit immunization were collected and purified using the NAb™ Protein A/G Spin Kit (Thermo Fisher Scientific; #89980). The antibody against TaSP served as the positive control. Subcellular distribution of SVSP455 in T. annulata-infected cells was determined by confocal microscopy. In brief, for the confocal microscopy experiment, T. annulata-infected cells were seeded onto glass slides in 12-well cell culture plates at an initial density of 3.0 × 106 cells/ml and incubated at 37 °C with 5% CO2 for 24 h. The cells were then fixed with 4% paraformaldehyde at RT for 30 min, washed with phosphate buffered saline (PBS) and permeabilized with 0.5% Triton X-100 in PBS for 15 min at RT. After three washes with PBS, the cells were blocked with 3% BSA for 1 h at RT, then incubated with an anti-SVSP455 antibody at a dilution of 1:100 in PBS containing 3% BSA at 4 °C overnight. Next, 500 μl of a donkey anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Life Technologies, Thermo Fisher Scientific; #A21206) was added and incubated for 37 °C at RT. Finally, the nucleus and cytoskeleton of the cells were stained with Hoechst 33342 and Alexa Fluro™ 594-conjugated phalloidin (Life Technologies, Thermo Fisher Scientific; #H3570 and #A13281), respectively. More than 100 random cells per slide were visualized using a confocal microscope (TCA; Leica Microsystems, Wetzlar, Germany) with a 63× oil objective, and the most representative images from each sample were used for presentation.
Construction and bioinformatic analysis of the bait plasmid
A fragment of SVSP455 was amplified from the cDNA of T. annulata with specific primers designed based on the reference sequence (Accession No: XM_950455.1 or Piroplasm DB: TA05545) using PCR. The primer sequences used to detect SVSP455 were SVSP455-F (5′-CCGATTCCGTATAAATGTGTAACATAT-3′) and SVPS455-R (5′- TGCACTGCAGTGCATGTTTTATAGGTCGCTTTAAT-3′); the underlined sequences indicate restriction enzyme sites (EcoRI and PstI). The PCR products were purified using a Cycle-Pure kit (OME Bio-Tek, Norcross, GA, USA). Both the pGBKT7 vector and purified PCR products of SVSP455 were digested with the enzymes EcoRI and PstI (Thermo Fisher Scientific; #FD0274 and #FD0614), and then the digested products of the vector and SVSP455 were ligated using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA; #M0202S) according to the manufacturer’s instructions. Finally, the recombinant bait plasmid (SVSP455-pGBKT7) was sequenced (Sangon Biotech, Shanghai, China).
Assessment of the auto-activation and toxicity of the bait plasmid
The empty pGBKT7 plasmid and the constructed bait plasmid were transformed into Y2H Gold competent cells using the Quick & Easy Yeast Transformation Mix (Clontech Laboratories, Mountain View, CA, USA). The transformants were then incubated on agarose plates containing various components, including SDO, SDO/X and SDO/X/A (see Abbreviation list for description of agar plates), at 30 °C for 3–5 days. When the colonies growing on SDO and SDO/X agar plates acquired a white or pale color, the bait plasmid was not auto-activated. No colonies grew on the SDO/X/A ar plates. Moreover, SVSP455-pGBKT7 was considered toxic if the sizes of colonies cultured on the SDO and SDO/X ar plates were significantly smaller than those transformed with the pGBKT7 plasmid. The yeast two-hybrid (Y2H) system used to screen the interacting proteins could be used only when the bait plasmid was neither auto-activated nor toxic.
The Y2H screening assay was used to determine the host cell proteins that interacted with SVSP455. First, the recombinant SVSP455-pGBKT7 plasmid and prey plasmid (the cDNA library of bovine B cells)  were co-transformed into Y2H Gold competent cells using Yeastmaker™ Yeast Transformation System 2 (Clontech Laboratories) according to the manufacturer’s recommendations. The transformed competent cells were then cultured on DDO/X/A ar plates at 30 °C for 3–5 days, and the blue colonies from the DDO/X/A ar plates were picked and cultured on QDO/X/A agar plates for a further 3–5 days at 30 °C. Finally, the blue colonies were plated once again on the QDO/X/A agar plates to reduce the number of false-positive clones (see Abbreviation list for description of ar plates). At the same time, both the negative control (pDT7-T and pGBKT7-Lam plasmids) and positive control (pDT7-T and pGBKT7-53 plasmids) were co-transformed into Y2H Gold competent cells and cultured on DDO and DDO/X/A agar plates at 30 °C for 3–5 days.
Rescue and analysis of prey plasmids
The blue colonies grown on QDO/X/A agar plates were preliminarily detected with PCR using the Matchmaker™ Insert Check PCR Mix (Clontech Laboratories). The potential prey plasmids were then isolated from the identified blue colonies using the Easy Yeast Plasmid Isolation Kit (Clontech Laboratories). A 3-μl aliquot of prey plasmids extracted from yeast cells was transformed into competent cells (E. coli DH5α) for plasmid rescue and sequencing. The gene fragments were analyzed with BLAST from the US National Center for Biotechnology Information (NCBI) to identify the host genes. The biological processes and structures of the identified genes were also analyzed using the UniProt database (http://www.uniprot.org/) and SMART server (http://smart.embl-heidelberg.de/).
Expression and subcellular colocalization analysis of SVSP455 and its prey proteins
The recombinant plasmid (pcDNA3.1-SVSP455-Myc) was constructed by cloning SVSP455-Myc into the pcDNA3.1 vector after BamHI/XhoI digestion (Thermo Fisher Scientific; #FD0054 and #FD0694, respectively). The recombinant p3×Flag-CMV-prey genes plasmids were constructed by cloning the fragments of the prey genes into the p3×Flag-CMV vector at the HindIII/XbaI sites (Thermo Fisher Scientific; #FD0504 and #FD0684, respectively). The expression and subcellular localization of SVSP455 and its interacting proteins were examined by confocal microscopy. HEK293T cells were cultured on glass slides in the 6-well culture plates at an initial density of 5 × 105 cells/ml, following which the constructed plasmids carrying SVSP455 and its prey genes were transfected or cotransfected into the cells using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific; #L3000015) when the confluence rate of the cells reached 70–90%. After 24 h of transfection, the cells on the glass slides were washed, fixed, permeabilized and then blocked using the procedures described above. The cells were then stained with a rabbit anti-Myc tag monoclonal antibody (mAb) (Cell Signaling Technology, Danvers, MA, USA; #2278S) or an anti-Flag tag mAb derived from mouse (Sigma-Aldrich, St. Louis, MO, USA; #F1804) overnight at 4 °C. After the cells were washed with PBS three times, they were incubated with Alexa Fluor 594-conjugated donkey anti-rabbit or Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (Life Technologies, Thermo Fisher Scientific; #A21207 and #A11029, respectively) at a dilution of 1:1000 in PBS containing 3% BSA at RT for 1 h. Hoechst 33342 (Life Technologies, Thermo Fisher Scientific; #H3570) was used to label the nuclei followed by five washes with PBS. A confocal microscope (TCS; Leica Microsystems) was used to visualize the fluorescence with a 63× oil objective.
A co-immunoprecipitation (Co-IP) assay was performed to identify whether SVSP455 binds to the identified prey proteins. First, 2 × 106 HEK293T cells per dish were seeded into 10-cm-diameter cell culture dishes. Both the pcDNA3.1-SVSP455-Myc recombinant plasmid (10 µg) and p3×Flag-CMV-prey plasmids (10 µg) were cotransfected into the cells when the cell confluence rate was 70–90%. After 48 h of culture, the cells were collected and washed with PB, followed by lysing with 600 μl of IP/Lysis buffer containing phosphatase inhibitors (Roche; #4906845001) and protease inhibitors (Roche; #4693132001) on ice for 30 min. Cell lysates were centrifuged at 16,000 g for 10 min at 4 °C, and the supernatant was collected and used in the Co-IP experiment, which was performed with a mouse anti-Flag tag monoclonal antibody using a Pierce™ Co-Immunoprecipitation Kit (Thermo Fisher Scientific; #26149). At the same time, the empty plasmids (pcDNA3.1 and p3×FLAG-CMV) were cotransfected into HEK293T cells and used as the negative control. The eluted samples after Co-IP were used for western blotting. The target proteins were detected using rabbit anti-Myc tag and anti-Flag tag antibodies.
Bimolecular fluorescence complementation assay
A bimolecular fluorescence complementation (BiFC) assay was conducted to investigate the interactions between SVSP455 and its prey proteins in cells . SVSP455 and its potential prey genes were cloned into the pBiFC-VN173 vector at the HindIII/SalI sites (Thermo Fisher Scientific; #FD0504 and #FD0644, respectively). At the same time, the recombinant plasmids (pBiFC-VC155-SVSP455 and pBiFC-VC155-prey genes) were constructed by cloning the target fragments into the pBiFC-VC155 vector followed by digestion with SalI/KpnI (Thermo Fisher Scientific; #FD0644 and #FD0524, respectively). The pairs of pBiFC-VC155-SVSP455/pBiFC-VN173-prey genes and pBiFC-VC155-prey genes/pBiFC-VN173-SVSP455 were cotransfected into HEK293T cells. Untransfected cells served as the control. At 24 h after transfection, the green signals of BiFC and their interaction with SVSP455 and its prey proteins were visualized using confocal microscopy. For the confocal experiments, HEK293T cells were stained with a rabbit anti-HA tag mAb (Cell Signaling Technology; #3724S) or mouse anti-Flag tag mAb (Sigma-Aldrich; #F1804) followed by fixation, permeabilization and blocking of the cells at 4 °C overnight. The cells were then labeled with 1 µg/ml donkey anti-rabbit IgG (H + L) antibody conjugated to Alexa Fluor® 594 (Life Technologies, Thermo Fisher Scientific; #A21207) or Alexa Fluor® 594-conjugated goat anti-mouse antibody (Life Technologies, Thermo Fisher Scientific; #A11005) at RT for 1 h followed by three washes with PBS. The nuclei were stained with Hoechst 33342 (Life Technologies, Thermo Fisher Scientific; #H3570).
HEK293T cells were cultured in 6-well cell culture plates at an initial density of 5 × 105 cells/ml. The constructed BiFC plasmids pBiFC-VC155-SVSP455, pBiFC-VN173-prey genes, pBiFC-VC155-prey genes and pBiFC-VN173-SVSP455 were transfected individually into the cells. Moreover, the pBiFC-VC155-SVSP455/pBiFC-VN173-prey genes and pBiFC-VC155-prey genes/pBiFC-VN173-SVSP455 pairs were cotransfected into the cells. At 48 h after transfection, the cells were harvested after digestion with trypsin–EDTA (0.25%; Gibco™, Thermo Fisher Scientific) and centrifugation at 800 rpm for 5 min. The cells were then resuspended in cold PBS, and the mean fluorescence intensity (MFI) of the transfected cells was determined using an Accuri™ C6 Plus Flow cytometer (BD, Franklin Lakes, NJ, USA).
Subcellular colocalization analysis of SVSP455 and its interacting proteins in T. annulata-infected cells
Confocal microscopy was used to further identify the interaction between the native SVSP455 and its prey proteins in T. annulata-infected cells. The mouse polyclonal antibodies against prey proteins were prepared and purified using the NAb™ Protein A/G Spin Kit (Thermo Fisher Scientific; #89980) (data not shown). For the confocal experiments, T. annulata-infected cells were cultured on glass slides in 12-well cell culture plates at an initial density of 5 × 105 cells/ml. After 24 h of culture, the cells were labeled with rabbit anti-SVSP455 polyclonal antibodies and mouse anti-prey proteins at a dilution of 1:200 at 4 °C overnight, followed by fixation, permeabilization and blocking. Cells were stained with a Alexa Fluor® 594-conjugated donkey anti-rabbit secondary antibody (Life Technologies, Thermo Fisher Scientific; #A21207) and Alexa Fluor® 488-conjugated goat anti-mouse (Life Technologies, Thermo Fisher Scientific; #A11029) secondary antibody at RT for 1 h. After five washes with PBS, Hoechst 33342 was applied at a dilution of 1:2000 (Life Technologies, Thermo Fisher Scientific; #H3570) to stain the nuclei and incubated for 15 min at RT. The images were acquired using a confocal microscope (TCS; Leica Microsystems) with a 63× oil objective.
Transfection of T. annulata-infected cells
The T. annulata-infected cells were cultured in 25 cm2-cell culture flasks at an initial density of 0.5 × 106 cells/ml. The recombinant plasmid-pcDNA3.1-SVSP455-Myc (5 µg) was transfected into T. annulata-infected cells using an Amaxa® Human T-Cell Nucleofector® Kit (Lonza Group AG, Basel, Switzerland; #VPA-1002) and Nucleofector® Program V-024 (Lonza Group AG) according to the manufacturer’s instructions. The transfected cells were then treated with the antiparasitic drug buparvaquone (Bup) (MedChemExpress, Monmouth Junction, NJ, USA; #HY-17581) at a concentration 200 ng/ml for 48 h. At the same time, the untransfected cells and the cells treated with Bup served as the controls. The cell samples were then collected to identify the target molecules using qPCR and western blotting assays. The T. annulata-infected cells were also transfected with 200 nM short interfering RNA (siRNA) against the prey genes (Additional file 2: Table S2). At 48 h after transfection, the cells were examined to detect changes in the expression of the related molecules using qPCR and western blotting.
Data and statistical analysis
The GraphPad PRISM 9 software package (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analyses. In all figures, the significance of differences between groups was determined using the unpaired two-tailed Student’s t-tests. The variance of each group was estimated by calculating the standard error of the mean. Experiments were repeated independently at least 3 times, and representative results are shown in all figures.