Parasite culture

All the Toxoplasma parasite strains were routinely passaged in vitro in monolayers of HFF at 37 °C in 5% CO2 as previously described [13].

Culture of cell lines

The THP-1 cell line was cultured in RPMI (complete media, CM), and the DC2.4 cell line in DMEM, 10% fetal bovine serum (FBS), 2mM L-glutamine, 10mM HEPES, 1X non-essential amino acids, 1mM sodium pyruvate, 100U/mL penicillin/streptomycin, 10 µg/mL gentamicin.

THP-1 monocyte to DC differentiation

To induce differentiation into immature DCs, the human monocytic leukemia cell line THP-1 was seeded at 2 × 105 cells/mL in a 75 mm adherent culture flask (in 20 mL of media) in the presence of human GM-CSF (100 ng/mL, Preprotech) and human IL-4 (100 ng/mL, Preprotech) for 7 days in CM. Fresh cytokine-supplemented medium was exchanged every 2 days. On day 7, the semi-adherent and cells in suspension were harvested, consisting of immature DCs (modified from Holken et al. [14]).

Primary host cell culture

Bone marrow-derived Dendritic Cells (BMDCs) were isolated from 5 to 8 weeks old female CD1 mice (Charles River Laboratories) as previously described [11]. BMDCs were obtained by culturing murine bone marrow cells in RMPI 1640 with 10% FBS, 2mM L-glutamine, 10mM HEPES, 1X non-essential amino acids, 1mM sodium pyruvate, 100U/mL penicillin/streptomycin, 10 µg/mL gentamicin, referred to as complete medium (CM), and supplemented with recombinant mouse GM-CSF (40ng/mL, Peprotech) and mouse IL-4 (40ng/mL, Peprotech). Loosely adherent cells were harvested after 8 days of maturation. The medium was changed every 2 days in culture with fresh GM-CSF and IL-4 (modified from Inaba et al. [15]).

Co-immunoprecipitation

DC2.4 cells or THP-1 derived DCs were grown in a 150 mm culture dish until 100% confluency and infected (MOI 3 to 5) for 4 h with ME49 TgWIPWT parasite expressing TgWIP mutant strains or mock infected. Cells were then scraped in PBS, centrifuged and resuspended in 1 or 3 mL of lysis buffer (HEPES 10mM ph 7.9, MgCl2 1.5mM, KCl 10mM, EDTA 0.1mM, 5 mM dithiothereitol (DTT), 0.5mM, NP40 0.65%, cocktail of protease inhibitor (Roche), phenylmethylsulphonyl fluoride (PMSF) 0.5mM) for 30 min at 4 °C. The lysate was centrifuged for 30 min at 18,000 x g, 4 °C. Each sample was incubated with 35 µl of magnetic beads coupled with HA antibodies (Thermo scientific) and placed on a rotator overnight at 4 °C. The beads were washed three times with Tris-HCl 10mM pH7.5, NaCl 150mM, Triton-100 × 0.2%, PMSF 0.5mM, a cocktail of protease inhibitors (Roche), once more with Tris-HCl 62.5mM pH6.8 and beads were resuspended in 100 µl of this buffer.

Immunoblotting

30 µl of the HA magnetic beads of each sample was used to run on a 12% SDS-PAGE. The proteins were transferred to a PVDF membrane, blocked 30 min with TBST, 5% nonfat dry milk. The membrane was blotted overnight at 4 °C with rat antibody against the HA tag (Roche, 1:500 dilution), phosphorylated Tyrosine (1:500 dilution), Shp1 (Invitrogen, 1:1000 dilution), or Shp2 (Invitrogen, 1:1000 dilution) antibodies, followed by their respective secondary antibodies. For Western blot analysis of phosphorylated Rock, 1 × 106 murine BMDCs were seeded on tissue culture treated 6-well plates and infected with TgWIPWT or TgWIPY150A/Y199AToxoplasma (MOI 7) for 4 h. Cells were then scraped in PBS, centrifuged and resuspended in 40 µl of RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 25mM Tris HCl pH 7.4, cocktail of protease inhibitor (Roche), 5 mM dithiothereitol (DTT)) for 30 min at 4 °C. The lysate was centrifuged for 30 min at 18,000 x g, 4 °C and run on 10% SDS-PAGE. The proteins were transferred to a PVDF membrane, blocked 30 min with TBST, 5% nonfat dry milk. The membrane was blotted overnight at 4 °C with rabbit polyclonal antibody against phospho-ROCK2 (Tyr722) (Invitrogen, 1:500 dilution), total ROCK2 (Cell Signaling, 1:1000), and mouse monoclonal SAG1 (clone CG52) followed by their respective secondary antibodies.

Podosome assay

To test the podosome dissolution after Toxoplasma challenge, BMDCs were seeded on collagen coated glass coverslips overnight after which freshly egressed parasites were added at MOI 3 for 4 h, and the coverslips washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min, permeabilized for 5 min with 0.1% Triton X-100, and blocked for 1 h with PBS with 3% (w/v) BSA. The coverslips were incubated with antibodies against SAG1 at room temperature for 1 h, washed and incubated with the respective fluorescent secondary antibodies, DAPI to stain the nucleus, and with Alexa Fluor 488 Phalloidin for 30 min. The coverslips were mounted with Vecta-Shield mounting oil and the microscopy was performed with NIS-Elements software (Nikon) and a digital camera (CoolSNAP EZ; Roper Scientific) connected to an inverted fluorescence microscope (eclipse Ti-S; Nikon) and either phase contrast or DIC imaging. Podosomes were identified and quantified as described in [8].

DC gelatin degradation assay and image analysis

The in vitro gelatinolytic activity of DCs was analyzed by gelatinolysis of Oregon green 488 (OG 488)-conjugated porcine gelatin (Molecular probes). DCs (2.5 × 104/well) infected with freshly egressed Toxoplasma tachyzoites (ME49-GFP, MOI as indicated) were deposited on OG-488 gelatin-coated glass coverslips and incubated for 24 h in CM. Cells were subsequently fixed (4% paraformaldehyde, Sigma), stained with DAPI (Invitrogen) or Alexa Fluor 594 Phalloidin (Thermo Fisher). Imaging and analysis were performed as indicated below. The in vitro gelatinolytic activity of DCs was analyzed by gelatinolysis of Cy-3-conjugated gelatin (molecular probes). After fixation (4% PFA, Sigma), cells were stained with DAPI (1:1000) and Alexa Fluor 594 Phalloidin (1:800). Using the software ImageJ version 1.54, the threshold of the green channel (Alexa Fluor 488 Phalloidin) was adjusted to distinguish single cells. The tool ‘‘Analyze particles’’ was used to count cells and measure the area of degradation. Gelatin degradation was defined as loss of signal (gelatin, Oregon green-488). The degradation of 100 cells was manually quantified for each condition. The “Analyze particles” tool was also used to analyze cell area and roundness of DCs using phalloidin to stain for F-actin and visualize the whole body of the cell; and used for nuclear area analysis using DAPI to visualize nucleus.

Transmigration assay

The transmigration assays were performed by culturing BMDCs in CM and the addition of freshly egressed Toxoplasma strains (MOI 3) for 4 h. DCs were then transferred to transwell filters consisting of a porous membrane (8 μm; Corning) incubated at 37 °C. After 18 h, DCs from the bottom chamber were collected and quantified by hemacytometer. For transmigration assays using Rock inhibitor (Y-27,632, 40 µM), Src inhibitor (Dasatinib, 50nM), and Shp1/2 inhibitor (NSC-87,877, 100 µg), BMDCs were treated with inhibitors for 3 h before infection during the 4 h infection. Subsequently, inhibitor-treated BMDCs were spinned down and washed in media before being transferred to transwell filters.

Flow cytometry analysis

Extracellular staining of BMDCs was performed using fluorescence activated cell sorting (FACS) buffer which was prepared by combining 7.4 pH PBS with 2% heat-inactivated fetal bovine serum (FBS). 2 × 105 BMDCs were plated and either mock infected, LPS-treated, or infected with either wild-type or ∆tgwip Toxoplasma at MOI 1 for 18 h. After incubation, BMDCs were harvested, pelleted, and resuspended with FACS buffer. Cells were washed with FACS buffer one more time and subsequently incubated with Fc block using anti-mouse CD32/CD16 for 20 min on ice. Cells were then washed and incubated with CCR7 antibody for 30 min on ice. Cells were then washed with FACS buffer and fixed using 4% paraformaldehyde for 20 min. After fixing, cells were washed and resuspended in FACS buffer for analysis on the flow cytometer.

Recombinant protein purification

To express recombinant TgWIP from E. coli, a codon-optimized sequence was ordered as GeneArt Strings from Thermo Fisher and inserted into a modified pGEX expression vector (GE Healthcare) [16] using standard molecular biology procedures. The signal peptide comprising residues 2–33 was excluded and replaced with a Trp residue to facilitate protein concentration measurement using absorbance at 280 nm. Proteins were expressed using ArcticExpress™ (DE3) RIL cells (Agilent) in terrific broth following the manufacturer’s instructions. Briefly, cells were grown at 30 °C at 220 rpm till OD600 reached 1.5, when 0.5 mM IPTG was added to induce expression at 10 °C for 18–24 h. Harvested cells were resuspended in lysis buffer [20 mM Tris-HCl pH 8, 200 mM NaCl, 20% (w/v) glycerol, and 5 mM b-mercaptoethanol (BME)] and kept in -80 °C until use.

To purify TgWIP, thawed cells were lysed on ice water by sonication and clarified by centrifugation at 19,500 rpm for 45 min at 4 °C. The supernatant was mixed with Glutathione Sepharose beads (Cytiva) for 30 min. After three washes using lysis buffer, the bound proteins were eluted in elution buffer [100 mM Tris-HCl pH 8.5, 30 mM reduced glutathione, and 20% (w/v) glycerol]. Eluted proteins were further purified by cation exchange chromatography using a 8-ml Source 15 S column [10 mM HEPES pH 7.0, 20% (w/v) glycerol, and 5 mM BME, with a gradient of 0–350 mM NaCl developed over 20 column volumes], followed by size exclusion chromatography using a 24-ml Superdex 200 Increase column equilibrated in 20 mM Tris-HCl pH 8, 100 mM NaCl, 20% (w/v) glycerol, and 1 mM DTT. To obtain untagged TgWIP, proteins eluted from Glutathione Sepharose beads were treated with HRV 3 C protease overnight at room temperature to cleave off the GST tag. Treated samples were passed through an 8-ml Source 15Q column equilibrated in 10 mM Tris-HCl pH 8.5, 20% (w/v) glycerol, and 5 mM BME to absorb cleaved GST tag. Untagged TgWIP was collected in the flow through and further purified by size exclusion chromatography using a 24-ml Superdex 75 Increase column equilibrated in 20 mM Tris-HCl pH 8, 100 mM NaCl, 20% (w/v) glycerol, and 1 mM DTT.

Human Shp1 (a.a. 1-595) was obtained as a codon-optimized GeneArt String from Thermo Fisher, and human Shp2 (a.a. 1-527) was a gift from Ben Neel (Addgene plasmid # 8322) [17]. Both Shp1 and Shp2 and their individual SH2 domains were inserted into a modified pMal expression vector (New England Biolabs) for expressing MBP-tagged proteins using ArcticExpress™ (DE3) RIL cells (Agilent) as described above for TgWIP. Thawed cells were lysed on ice water by sonication and clarified by centrifugation at 19,500 rpm for 45 min at 4 °C. The supernatant was mixed with amylose beads (New England Biolabs) for 30 min. After 3 washes with lysis buffer, the bound protein was eluted in elution buffer [20 mM Tris-HCl pH 8, 2% (w/v) maltose, 20% (w/v) glycerol, 5 mM BME, and 1 mM MgCl2]. Eluted protein was then purified through anion exchange chromatography using an 8-ml Source 15Q column [10 mM Tris-HCl pH 8, 10% (w/v) glycerol, and 5 mM BME, and 1 mM MgCl2, with a gradient of 0–500 mM NaCl developed over 20 column volumes]. These proteins were then purified through a 24-ml Superdex 75 or Superdex 200 Increase column (Cytiva) equilibrated in gel filtration buffer [20 mM Tris-HCl pH 8, 20% (w/v) glycerol, 100 mM NaCl, 1 mM DTT, and 1 mM MgCl2].

The human Src kinase domain was purified following previous methods [18], using the expression vector from Amy Andreotti, Iowa State University. Briefly, Src was co-expressed with phosphatase YopH from a pCDFDuet vector using BL21 (DE3) T1R (Sigma). Cell pellets were thawed on ice water by sonication and clarified by centrifugation at 19,500 rpm for 45 min at 4 °C. Supernatant was mixed with Ni-NTA agarose resin (Qiagen) for 30 min. After 3 washes with lysis buffer, the bound protein was eluted in elution buffer [20 mM Tris-HCl pH 8, 500 mM NaCl, 500 mM imidazole pH 8, 5% (w/v) glycerol]. After dialysis overnight at 4 °C with two buffer changes in dialysis buffer [20 mM Tris-HCl pH 8, 100 mM NaCl, 5% (w/v) glycerol, 1 mM DTT], the kinase was purified using anion exchange chromatography using 4-ml Source 15Q column [10 mM Tris-HCl pH 8, 5% (w/v) glycerol, 5 mM BME] and then a Superdex 75 Increase column equilibrated in 20 mM Tris-HCl pH 8, 100 mM NaCl, 5% (w/v) glycerol, and 1 mM DTT.

All chromatography steps were performed using Cytiva columns on an ÄKTA Pure protein purification system. Purified TgWIP, Shp1, Shp2, and Src were aliquoted in single-use volumes, flash frozen in liquid nitrogen, and stored in -80 °C for up to 1 year.

In Vitro phosphorylation

Each reaction contained 20 nM Src kinase domain, 1 µM TgWIP, 2 mM ATP, and 10 mM MgCl2 in 20 mM HEPES pH 7, 100 mM NaCl, 20% (w/v) glycerol, and 1 mM DTT. After overnight incubation at room temperature, the reactions were quenched using 20 mM EDTA. For negative controls, ATP/MgCl2 was replaced with 20 mM EDTA in the reaction. Phosphorylated samples were flash frozen in liquid nitrogen and kept in -80 °C till further analysis.

To validate and quantify the completeness of phosphorylation, a small portion of each reaction (20 µl) was analyzed by the Proteomics Core facility at University of Texas Southwestern Medical Center to determine intact protein molecular weight using electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometry. Phosphorylation typically reaches 90–100% completeness in our conditions. In parallel, phosphorylation was confirmed using Western blot. Briefly, samples in 10% SDS PAGE gel were transferred to a PVDF membrane using a Trans-Blot Turbo system (Bio-RAD). The membrane was blocked with 5% (w/v) BSA for one hour and then incubated with a 1:500 dilution of an HRP-conjugated antibody against phosphotyrosine (Cat#:sc-508 HRP, Santa Cruz) at 4 °C overnight. After three washes using Tris buffered saline (TBS)-Tween20, the membrane was developed using Amersham ECL Western Blotting Detection Reagent (Cat#: RPN2134, Cytiva) and a ChemiDoc XRS + system (Bio-RAD).

Phosphatase activity assay

The impact of phosphorylated TgWIP on both Shp1 and Shp2 activity was measured using a Shp2 activity assay kit (BPS bioscience, Cat# 79,330 [19]), following the manufacturer’s instructions. The kit measures the activity of full-length human Shp2 by monitoring the dephosphorylation of its substrate, Di-FMUP (6,8-Difluoro-4-Methylumbelliferyl Phosphate), and the spontaneous increase in fluorescence. Briefly, TgWIP was incubated with full-length Shp2 (included in the kit) or Shp1 (a.a. 1-595, purified in-house) at room temperature for 1 h for Shp2 and 30 min for Shp1. Di-FMUP was then added to initiate the reaction. The final reaction contained 4 pg/µl Shp1 or Shp2 and 0.004 to 0.1 µM TgWIP. Each reaction of 180 µl was divided into 3 separate wells in a 96-well flat-bottom black plate (Costar). Fluorescence intensity of each well was recorded every 18 s at 22 °C using a Spark plate reader (Tecan), with excitation at 360 nm and emission at 460 nm (15 nm bandwidth for both wavelengths). Typically, the reaction rate remains linear for at least 5 min. Shp1 and Shp2 activity in various conditions was normalized to the reaction rate of Shp1 and Shp2 without TgWIP incubation.

In Vitro GST pull down assay

GST pull-down experiments were performed as previously described [20]. Each reaction contained 100 pmol of GST-tagged TgWIP, 200 pmol of MBP-tagged Shp1 or Shp2 (FL or individual SH2 domains), and 20 µl of Glutathione Sepharose beads (Cytiva) in 1 mL of binding buffer (100 mM HEPES pH 7, 100 mM NaCl, 20% (w/v) glycerol, and 5 mM BME). The reaction was mixed at 4 °C for 30 min. The beads were then washed three times with 1 mL of binding buffer. Bound proteins were eluted with GST elution buffer (10 mM Tris 8.5, 50 mM NaCl, 5% (w/v) glycerol, and 30 mM reduced glutathione) and further examined by SDS-PAGE.