Parasites and cell cultures
All Neospora tachyzoites used in this study were propagated in a human foreskin fibroblast (HFF) line cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), as described previously [14, 16]. Both cells and parasites were incubated at 37°C with 5% CO2 in a humidified incubator.
Plasmid construction
All primer sequences are shown in Additional file 3: Table S1. The NcGRA17-targeting CRISPR plasmid (pNc_Cas9CRISPR::sgNcGRA17) was constructed as described previously [14]. All other CRISPR/Cas9 plasmids were obtained through FastPfu (TansGen Biotech Co., Ltd., China) mutagenesis of the above plasmid to change the NcGRA17 targeting gRNA to other specific gRNAs. To generate clean knockouts of NcGRA11 (a-e) genes (62240 bp), a double-gRNA CRISPR/Cas9 system was designed in which the first gRNA sequence (gRNA1) was placed close to the start codon of the first gene (NcGRA11a), with the second gRNA (gRNA2) near the stop codon of the last gene (NcGRA11e), as described previously [17]. To express two gRNAs from a single plasmid pNc_Cas9CRISPR::sgNcGRA11(a–e), the gRNA2 expression cassette (NcU6 promoter-gRNA2-RNA scaffold TTTT) was amplified using a common set of primers, 2×gRNA NcU6 F and 2×gRNA NcU6 R, and cloned into the pNc_Cas9CRISPR::sgNcGRA11(a–e)1 plasmid using a one-step cloning kit (Vazyme Biotech Co., Ltd., China). For disruption of NcGRA11 (a–e) genes, CRISPR/Cas9 double-gRNA plasmid pNc_Cas9CRISPR::sgNcGRA11(a–e) was co-transfected with its corresponding DHFR-TS amplicon containing 60-bp homology regions matching the NcGRA11 (a–e) genes. To generate a construct (pLIC-BirA*-HA-DHFR-NcGRA17) for inserting a BirA*-HA tag into the NcGRA17 gene 3′ end, upstream (830-bp) and downstream (950-bp) regions directly adjacent to the gRNA target sequence and BirA*(963 bp) sequence were cloned into the pLIC-HA vector. pDMG plasmid [16, 18, 19] was used to express NcGRA11 (a-e) genes in the Nc1 strain. The coding sequences of NcGRA11 (a–e) were cloned into the backbone plasmid pDMG with GFP replaced by HA [18] using a one-step cloning kit (Vazyme Biotech Co., Ltd., China). The vector p6 × HA-HXGPRT used for in situ C-terminal tagging, as described previously [20], was generously provided by Prof. Shaojun Long of China Agricultural University. This plasmid was modified by replacing the 6HA tag with the spaghetti monster tag smHA [21] and replacing the selectable marker HXGPRT with DHFR. The construct (pTCR-NcGRA23-CD KO) for disrupting the NcGRA23 locus was designed by inserting the 5′ (990-bp) and 3′ (930-bp) regions flanking the NcGRA23 gene region into the backbone plasmid pTCR-CD, which has been described previously [18].
Generation of NcGRA17-BirA* fusions
C-terminal BirA*-HA-tagging of NcGRA17 was performed via homologous recombination. Nc1, as the parent strain, was co-transfected with the pNc_Cas9CRISPR::sgNcGRA17 vector and homologous repair amplicon. The amplicon was amplified using primers listed in Additional file 3: Table S1 and pLIC-BirA*-HA-DHFR-NcGRA17 plasmid as a template. The parasites were cultured to the third generation in the presence of pyrimethamine (1 µM) and then screened to confirm the purity of the selected strains. The selected NcGRA17-tagged strain, named NcGRA17-BirA*-HA, was identified by polymerase chain reaction (PCR), western blotting, and immunofluorescence assay.
Affinity capture of biotinylated proteins
BirA*-tagged and parent lines were used to infect HFF monolayers and grown in medium containing 150 µM biotin for 48 h prior to parasite egress [22]. Infected cells were collected, washed in phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) supplemented with protease inhibitor cocktail (Sigma-Aldrich, USA) for 30 min on ice. Lysates were centrifuged for 15 min at 14,000×g to pellet insoluble debris and then incubated with streptavidin magnetic beads (Beaver, China) at room temperature for 4 h under gentle agitation. Beads were collected using magnets and washed five times in RIPA buffer, followed by three washes in 8 M urea buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl). Ten percent of each sample was boiled in Laemmli sample buffer, and eluted proteins were analyzed by western blotting with streptavidin-horseradish peroxidase (HRP) before mass spectrometry (MS) analysis.
Mass spectrometry
Purified proteins bound to streptavidin beads were reduced, alkylated, and digested by the sequential addition of Lys-C and trypsin proteases [22]. The peptide mixture was desalted using C18 tips and fractionated by a 75 µm inner diameter filter fused to the silica capillary column with a 5 µm pulled electrospray tip, and packed in-house with 15 cm of a Luna C18 column with 3 µm reversed-phase particles. Delivery of the gradient was performed by the EASY-nLC 1000 ultrahigh-pressure liquid chromatography (UHPLC) system (Thermo Scientific). Tandem mass spectrometry (MS/MS) spectra were collected on a Q Exactive mass spectrometer (Thermo Scientific). Data analysis was performed using ProLuCID and DTASelect2 implemented in the Integrated Proteomics Pipeline (IP2) platform (Integrated Proteomics Applications, Inc., San Diego, CA, USA). Protein and peptide identification was filtered using DTASelect and required a minimum of two unique peptides per protein and a peptide-level false-positive rate of less than 5%, as estimated by a decoy database strategy. Normalized spectral abundance factor (NSAF) values were calculated as described previously [22].
Generation of NcGRA-HA parasites
As few data are available from Neospora, we utilized homologous gene expression data of Toxoplasma to filter hits from our BioID data set for further investigation. To epitope-tag the candidate NcGRA proteins, CRISPR/Cas9-sgNcGRA plasmid was co-transfected with their corresponding smHA tag amplicon containing 42-bp homology regions matching the gene of interest. The amplicon was amplified using primers listed in Additional file 3: Table S1 and psmHA-DHFR plasmid as a template. As we failed to endogenous HA-tag the NcGRA11b gene, the vector pDMG-NcGRA11b-HA was electroporated into Nc1. The transfected parasites were grown in medium containing 1 µM pyrimethamine, and identified by immunofluorescence assay (IFA). All confirmed NcGRAs were examined in silico using the Basic Local Alignment Search Tool (BLAST) to search for other Neospora proteins with sequence similarity.
Deletion of the genes encoding NcGRA23 and NcGRA11 (a–e)
NcGRA23-KO parasites were generated via homologous recombination. The parent Nc1 strain was co-transfected with the pNc_Cas9CRISPR::sgNcGRA23 and linearized complete knockout plasmid pTCR-NcGRA23-CD KO (primers listed in Additional file 3: Table S1). The transgenic parasites were grown under chloramphenicol (20 µM) and 5-fluorine cytosine (40 µM) selection pressure. The selected NcGRA23-deficient strain, named ∆ncgra23, was identified by PCR and western blotting. For disruption of NcGRA11 (a–e) genes, CRISPR/Cas9 double-gRNA plasmid pNc_Cas9CRISPR::sgNcGRA11(a–e)1–2 was co-transfected with its corresponding DHFR-TS amplicon containing 60-bp homology regions matching the NcGRA11 (a–e) genes. The parasites were cultured in the presence of pyrimethamine (1 µM) to the third generation. Resistant clones were isolated and confirmed by PCR and western blotting.
Western blotting
Western blotting was performed as described previously [14]. The parent and modified strains of whole-parasite lysates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (w/v) gel. Samples were transferred onto polyvinylidene fluoride membranes and probed with the mouse anti-HA antibody (Sigma-Aldrich, 1:500), the mouse anti-NcGRA23 antibody (prepared in our laboratory, 1:200), or the mouse anti-NcGRA11c antibody (prepared in our laboratory, 1:200). The N. caninum F-actin subunit beta (NcActin) was used as a loading control and was incubated with the rabbit anti-NcActin antibody (prepared in our laboratory, 1:2000). For all secondary antibody incubations, HRP-labeled goat anti-mouse IgG (H+L) antibody (Sigma, USA) was diluted 1:5000, an HRP-labeled goat anti-rabbit IgG secondary antibody (Sigma, USA) was diluted 1:10,000, or streptavidin-HRP (4A Biotech Co., Ltd., China) was used at a 1:1000 dilution. Following secondary incubation, enhanced chemiluminescence reagents (CoWin, China) were used for the detection of HRP activity.
Immunofluorescence assay
Immunofluorescence assay (IFA) to detect the biotinylated proteins of BirA*-tagged parasites was performed as described previously [22]. Parasites were used to infect HFF monolayers on coverslips in a 12-well plate and grown for 30 h with the addition of 150 μM biotin. Infected cells were fixed for 15 min in 4% formaldehyde, and then permeabilized with 0.25% Triton X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for 30 min. Subsequently, the cells were incubated with a rabbit anti-HA polyclonal antibody (CWBIOTECH, China, 1:50); then incubation was performed with Cy3-conjugated goat anti-rabbit IgG (H+L) (Sigma, USA) and streptavidin/fluorescein isothiocyanate (FITC) (Solarbio, 1:100). The images were obtained using a Leica confocal microscope system (Leica TCS SP52, Germany).
To analyze NcGRA localization, parasite-infected HFFs or freshly released parasites were processed for IFA and stained with a rabbit anti-HA polyclonal antibody, mouse anti-NcGRA6 polyclonal antibody (prepared in our laboratory, 1:50), or mouse anti-NcGRA23 polyclonal antibody (1:50). To determine the trafficking route of the protein following secretion into the PV, freshly harvested parasites (NcGRA17-HA or NcGRA11c-HA) were used to pulse-infect monolayers of HFF cells for 3 min at 37 °C. Monolayers were washed with PBS and further incubated at 37 °C for 5 min, 10 min, 15 min, or 1 h, and then fixed and permeabilized in 0.002% saponin [23]. Localization of the indicated NcGRAs was performed by double immunofluorescence labeling, using both the rabbit serum anti-HA and the mouse serum anti-NcGRA6 or anti-NcGRA23. Primary antibodies were revealed using FITC-conjugated goat anti-mouse IgG (H+L) and Cy3-conjugated goat anti-rabbit IgG (H+L). The IFA process and image collection were performed as described above.
Plaque assay
Plaque assays were performed on HFF cells cultured in 12-well plates (Corning Costar, USA) as described previously [14, 16]. Briefly, 200 freshly isolated parasites were seeded into HFF monolayers and incubated at 37 °C with 5% CO2 for 9 days undisturbed. The plaques were scanned using a Canon digital scanner (model F917500, Japan), and the plaque area was measured as described previously [18].
Parasite virulence assay
The virulence of the parasites was investigated as described previously [14]. Eight-week-old female BALB/c mice (Laboratory Animal Center of Academy of Military Medical Sciences, Beijing, China) were injected intraperitoneally with ∆ncgra23, ∆ncgra11(a–e), or parent Nc1 parasites at doses of 5 × 106 and 8 × 106 parasites (n = 5 mice/dose). Mice were carefully monitored every 8 h based on their clinical signs and mortality, and humanely euthanized via cervical dislocation when they were unable to reach food or water for more than 24 h or lost 20% of normal body weight. Survival was monitored for 60 days.
Statistical analysis
All statistical data were analyzed using GraphPad Prism 5 (version 5.01; GraphPad Software, San Diego, CA, USA) except as otherwise indicated above. The results were expressed as mean ± SD and evaluated by non-parametric tests. Values of P < 0.05 and P < 0.01 were considered statistically significant.