Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity

NOTE: All protocols that involve the use of animals were performed in accordance with the guidelines and regulations set forth by the New York Medical College’s Animal Care and Use Committee.
NOTE: Detailed protocols for chemical mutagenesis ³⁸, isolation of parasites by limiting dilution ³⁸, isolation of murine bone marrow derived macrophages ³⁹, growth of T. gondii in human foreskin fibroblast (HFF) cells and cyst production in macrophages and basic immunofluorescence analysis (IFA) ³² are referenced. Carry out all cell culture at 37 °C in 5% CO₂ in D10 media (Dulbecco’s Modified High Glucose Eagle Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin). Keep all reagents sterile throughout cell isolations and cell culture.

1. Dose Titration of IFN-γ and LPS to Determine the Concentrations to Use to Activate Infected Macrophages for the Forward Genetics Screen

1. Culture murine bone marrow-derived macrophages O/N in eight chamber glass slides at a concentration of 3 × 10⁵ cells/ml in 250 μl of D10 media per chamber. Use macrophages one to two-weeks after isolation from bone marrow.
   NOTE: Chamber slides are purchased that have a growth enhancing RS wash.
2. Use a hemocytometer to count wild type parasites harvested from human foreskin fibroblast (HFF) cells grown in a T25 tissue culture flask. Resuspend parasites at a concentration of 5 x 10⁵ wild type parasites per ml in D10 media. This concentration will result in a multiplicity of infection of approximately 1:1 as the macrophages will have proliferated O/N . Use polystyrene or PET tubes for all manipulations with parasites as the parasite sticks to polypropylene.
3. Remove the D10 media in the chamber slides and replace with 250 μl of the suspension of wild type parasites. Gently add the parasite suspension to each chamber of the slide by allowing it to flow down the inner face of each chamber. Do not allow macrophages to dry out at any point during the protocol.
4. Cover chamber slides infected with parasites with the chamber slide cover and place in an incubator at 37 ° C in 5% CO₂ for 4 hr to allow time for the parasite to invade and establish a PV.
5. Make dilutions of LPS and IFN-γ in 1 ml of D10 media in sterile tubes for the dose titrations. For dose titrations keep either LPS or IFN- concentration constant and vary the other stimulus ⁴⁰.
   1. For example, keep LPS constant at 10 ng LPS per ml and vary the concentration of IFN- γ (0, 1 unit/ml, 10 units/ml, 100 units/ml, 1,000 units/ml). Keep IFN- γ constant at 100 units/ml and vary the concentration of LPS (0, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml). Briefly sonicate LPS stock for 2 min without heating in a bath sonicator (not with a tip sonicator) prior to dilution.
      NOTE: Sonication is recommended to disrupt micelle aggregates formed by LPS that may not bind properly to host cells.
6. 4 hr after initiation of co-culture between the parasites and adherent macrophages in the chamber slide, discard the D10 media in each chamber and replace it with 300 μl of the appropriate dilutions of IFN- γ and LPS in D10 media. Include a control with only D10 media to evaluate parasite replication in the absence of macrophage activation.
7. Re-cover chamber slides with the chamber slide cover and place in an incubator at 37 ° C in 5% CO₂ for 24 to 30 additional hr.
   NOTE: Incubation time depends on the doubling time of the wild type parasite strain that can range from 6-12 hr depending on the parasite strain. A time point is chosen prior to parasite lysis of naïve macrophages but long enough to enable at least 3-4 doubling times.
8. Make a 2.5% formaldehyde solution in Dulbecco’s phosphate buffered saline (PBS). Discard the media in the chamber slide and replace with 300 μl of the 2.5% formaldehyde solution. Fix for 20 min at RT.
9. Rinse slide(s) twice with 300 μl of PBS per chamber. For each rinse, discard the PBS in the slide and add new PBS gently down the inner face of each slide chamber to avoid disrupting the macrophages adhered to the slide. Add 300 μl of PBS per chamber and place cover on chamber slide to prevent slides from drying out prior to staining.
   NOTE: Slides can be placed at 4 C for up to 3 days at this point prior to staining.
10. Staining protocol for T. gondii detection by immunofluorescence microscopy (IFA).
    1. Make a 0.2% Triton X-100 solution in PBS (permeabilization buffer). Place the solution in a tube in a 37 °C water bath and briefly vortex it to ensure the Triton X-100 goes into solution. Discard the PBS in the chamber slides and replace with 300 μl of the permeabilization buffer. Leave at RT for 30 min.
    2. Make a solution of 10% goat serum in PBS (blocking solution). Discard the permeabilization buffer in the slide and replace with 300 μl of blocking solution per chamber. Leave at RT for 30 min.
       NOTE: Fetal calf serum can be substituted for goat serum in all staining protocols.
    3. For a one step IFA staining protocol, make a 1:1,000 dilution of a fluorescently-conjugated antibody to T. gondii in blocking solution. Remove blocking solution from slide and add 150 μl of antibody solution per chamber. Replace cover slide on chamber slide to prevent cells from drying out. Incubate for 1 hr at RT .
       NOTE: Antibody concentration must be empirically determined depending on the source. Antibody is purchased directly conjugated to a fluorochrome or conjugated to a fluorochrome by the user.
       1. For a two-step IFA staining protocol with an unconjugated antibody to T. gondii and a fluorescently conjugated secondary antibody, stain slides with 150 μl of unconjugated primary antibody against T. gondii in blocking buffer for 1 hr at RT . Rinse 3x with 300 μl of PBS plus 1% goat serum (wash buffer).
       2. Add 150 μl of fluorescently-conjugated secondary antibody specific for the species of origin for the primary antibody. Replace cover slide on chamber slide to prevent cells from drying out. Incubate for 1 hr at RT .
    4. Rinse slides 3x with PBS plus 1% goat serum (washing buffer). For each rinse, remove the solution in the chamber slides by dumping out its contents and replace with 300 μl of washing buffer.
    5. Rinse slides 2x with 300 μl of PBS.
    6. Remove the chamber from the chamber slide as per manufacturer’s instructions.
    7. Mount slides with mounting media containing DAPI (4’6-diamidino-2-phenylindole, dilactate) and a 22 mm x 50 mm coverslip.
11. Examine slides using phase contrast and fluorescence microscopy. Determine the average number of parasites per vacuole by examining a minimum of 100 PVs per chamber well. The chosen dose of IFN- γ and LPS for the screen needs to be sufficient to suppress, but not prevent, replication of wild type parasites (see Figure 1).

2. Isolation of Parasite Mutants with a Fitness Defect Following Activation of Infected Macrophages

NOTE: A library of random T. gondii mutants is required for the forward genetics screen. Random mutagenesis of T. gondii can be performed by chemical (ENU/EMS) or insertional mutagenesis ^27,28,38. Following mutagenesis, clone parasites by limiting dilution and grow individual clones in 96-well plates containing adherent HFF cells in a volume of 200 µl of D10 media ^32,38. It is critical that 96-well plates for screening of parasites in macrophages by microscopy have optical bottoms to enable microscopic screening. Phase contrast and fluorescence microscopy for screening stained 96-well plates requires an inverted fluorescence microscope with phase contrast 4, 10, 20 or 40X objective equipped for long working distances. A 4X objective is useful for seeing the entire well but better resolution of the parasites is achieved with the 20X objective.

1. Transfer 50 µl of tachyzoite mutants grown in confluent HFF cells in 96-well tissue culture plates into two replicate 96-well plates containing murine bone marrow-derived macrophages (1-2 weeks after isolation) in 100 µl of D10 media.
   NOTE: Perform the initial screen in 96-well plates of the parasite based on volume of parasite culture to avoid having to count the number of parasites transferred per each well. Thus, the microscopy analysis in 2.6 is based qualitatively on whether parasites have generally replicated or not replicated. Step 3 describes the approach to confirm the phenotype of the mutants in chamber slides using equal numbers of wild type or mutant parasites to infect the macrophages.
   1. Directly add parasites to the D10 media already in each well. Infect two wells with wild type tachyzoites as a positive control for parasite replication.
2. Place the infected cells into an incubator (37 ° C and 5% CO₂) for 4 hr to allow parasites time to invade the cells and create their PV.
3. Remove media from one plate by dumping the contents of the plate. Use a single flip of the wrist to dump the media in the plate into a discard basin containing a detergent cidal for the parasites.
   1. Add 100 µl of “macrophage activation media” using a sterile basin for the media and a multichannel pipette. Use the optimal concentration of LPS and IFN-γ determined from Step 1 for the macrophage activation media. Similarly remove media from the duplicate control plate and replace with 100 µl of D10 media.
4. Place the infected cells into an incubator (37 ° C and 5% CO₂) for an additional 24-30 hr.
5. Staining protocol for 96-well plates for IFA.
   1. Discard the media in the plate and replace with 100 µl of 2.5% formaldehyde in PBS. Incubate for 20 min at RT . Use a sterile basin for the formaldehyde and a multichannel pipette.
   2. Discard the formaldehyde solution in the plate and add 100 µl PBS to rinse the formaldehyde from the plate.
   3. Discard the solution in the plate and add 100 µl of permeabilization buffer (PBS with 0.2% Triton X-100) to each well. Incubate for 30 min at RT .
   4. Discard the solution in the plate and add 100 µl of blocking solution (PBS with 10% goat serum) to each well. Incubate for 30 min at RT .
   5. Discard the solution in the plate. Add 50 µl/well of a fluorescently-conjugated anti-T. gondii antibody diluted 1:1,000 in PBS plus 1% goat serum. Incubate for 1 hr at RT with the plate cover on and on a rocker.
      1. Alternatively use an unconjugated primary antibody against T. gondii followed by staining with a fluorescently-conjugated secondary antibody as described in 1.10.3.1.
   6. Discard the antibody solution in the plate and replace with 100 µl/well of PBS plus 1% goat serum. Perform this rinse 3x followed by 2 additional rinses with PBS alone. Leave 200 µl of PBS in each well and replace the cover on the 96 well plate to prevent the wells from drying.
      NOTE: The plate with the cover on can be wrapped in parafilm and placed at 4 ° C for up to 3 days prior to analysis by microscopy.
6. Examine cells under an inverted fluorescent microscope using a 20-40X objective. Select mutants that replicate in the naïve macrophage plate but predominantly fail to replicate beyond one parasite/vacuole in “activated macrophages” or that appear amorphous or degraded in “activated macrophages”.
   NOTE: The number of wild type parasites per vacuole will depend on the dose of IFN-γ and LPS used but ideally is around 4 parasites per vacuole; a number that reflects growth suppression but not complete inhibition of replication by the activation stimulus.

3. Evaluate the Mutants to Determine if the Defect is at the Level of Parasite Survival or Replication Following Activation of Infected Macrophages

1. Transfer selected parasite mutants from 96-well plates (contents of entire well) to T25s containing HFF cells and allow replication.
2. Culture bone marrow derived macrophages O/N in 8 chamber glass slides at a concentration of 3 × 10⁵ cells/ml in 250 μl of D10 media. Prepare one slide to compare parasite replication in naïve macrophages and an additional slide to compare parasite replication following activation of infected macrophages.
3. Harvest tachyzoite parasites and count in a hemocytometer. Add 5 × 10⁴ T. gondii parasites to a chamber well containing macrophages in D10 media and culture for 4 hr. Include one well with wild type parental parasites as a control. Inoculate an identical replicate slide with the same parasite clone that will not receive activation media in order to monitor replication of parasites in naïve macrophages.
4. Discard D10 media from 1 of the replicate chamber slides and replace with 250 µl of “activation media”. Discard D10 media from the other replicate chamber slide and replace with 250 µl of D10 media. Incubate both the “activated” and “naïve” macrophage slide with the chamber slide cover on at 37 ° C and 5% CO₂ for an additional 24-30 hr.
5. Fix, permeabilize, and block slides for IFA staining and analysis of parasites as described in Step 1. Perform IFA staining with the chambers intact.
   1. Co-stain cells with an antibody to lysosomal membrane associated 1 (LAMP1) or Lysotracker and antibody to T. gondii to evaluate whether activation of infected macrophages is triggering fusion of the mutant PVs with lysosomes (Figure 4).
   2. Use antibodies to specific compartments/organelles within the parasite/PV for staining by IFA to identify alterations in the parasite mutant that are evident early following macrophage activation ²⁸.
      NOTE: Such alterations may provide insight into the mechanism that underlies the deficient survival/replication of the mutant following macrophage activation.
6. Examine slides using a 100X phase oil objective and fluorescence microscopy.
   1. Evaluate parasite replication by determining the number of parasites per PV in 100 random vacuoles. Perform at least 2 counts of 100 vacuoles each for statistical analysis.
   2. Evaluate general parasite morphology using both fluorescence analysis as well as phase contrast microscopy. View parasites under phase 100x objective. Healthy parasites have parasites tightly enclosed within a close-fitting parasitophorous vacuole. Parasites that have amorphous spacious vacuoles with phase dense rims or amorphous parasites suggest parasite death rather than just a defect in replication (Figures 1 and 3).

4. Evaluate whether the Susceptibility of Mutant Parasites to Activation of Infected Macrophages is Associated with Known Anti-microbial Mediators

1. Isolate bone marrow-derived macrophages from wild type C57/BL6 mice, iNOS^-/-, gp91-phox^-/- or Irgm1/Irgm3^-/- mice ³².
2. Culture respective bone marrow derived macrophages O/N in 8 chamber glass slides at a concentration of 3 × 10⁵ cells/ml in 250 μl of D10 media.
3. Proceed with parasite challenge, IFA staining, and analysis as described in Step 3.
4. Determine if the ability of the mutant parasites to survive and replicate following activation of infected macrophages is restored in the absence of reactive oxygen or nitrogen species or specific immunity related GTPases ²⁸.
5. Use pharmacological agents, such as nitric oxide donors, to evaluate whether specific anti-microbial mediators are sufficient by themselves to impair mutant parasites in HFF cells or naïve macrophages or if they only act in concert with other mediators of macrophages activation ²⁸.

5. Evaluate whether the Defect in the Mutant Parasite Compromises Chronic Infection

1. Isolate tachyzoites from wild type, mutant or targeted gene deleted parasites grown in T25s containing HFF cells. Parasites must be freshly lysed from HFF cultures.
2. Count parasites using a hemocytometer. Resuspend parasites in Hanks balanced salt solution (HBSS) at a concentration per ml appropriate for a sub-lethal dose of the parasite delivered in 200 μl by intraperitoneal (IP) injection.
3. Use a 1 ml tuberculin syringe and 25 G needle to inject parasites with a 200 μl volume intraperitoneally (IP). The dose depends on the wild type strain of the parasite and the mouse genotype.
   NOTE: Type I genotypes of the parasite are lethal to mice during the acute stage of infection regardless of parasite dose and are not appropriate for studies of chronic infection in the absence of chemotherapy for T. gondii to suppress parasite replication.
4. Thirty days after parasite challenge, sacrifice mice by inhalation of CO₂ from a pressurized tank in an uncrowded chamber (a standard size mouse cage may contain no more than 5 mice) followed by cervical dislocation.
5. Isolate the brain from the mouse using established procedures^41-43.
   1. Lay the mouse on its front side. Spray the head with 70% ethanol to sterilize the area.
   2. Use sharp scissors to cut through the brain stem. Use dissecting scissors to make a shallow cut laterally around the right and then the left side of the skull starting from the base of the brain stem. Use forceps to gently peel back the skull to expose the brain.
   3. Gently lift the brain and place scissors between the brain and base of the skull to cut the olfactory nerve to free the brain. Lift out the brain with sterile forceps or a spatula and place in a bacteriological Petri plate containing 10 ml of sterile PBS.
6. Cut the brain in half with a sterile scalpel through the center between the right and left hemispheres.
7. Add half of the brain to a small mortar containing 1 ml of PBS. Use the mortar and pestle to make a fine tissue suspension of the brain that can pass through a wide bore 20-200 μl pipette tip.
   NOTE: The other half of the brain should be fixed in 2.5% formaldehyde for 1 hr if tissue sections are needed for histopathology.
8. Place 100 μl of the brain lysate in a 1.7 ml microcentrifuge tube. Add 1 ml of 2.5% formaldehyde in PBS into the tube containing the brain lysate to fix the brain suspension for staining. Incubate at RT for 30 min.
9. Centrifuge the lysate at 8,000 x g for 5 min in a microcentrifuge and carefully discard the supernatant.
10. Add 1 ml of permeabilization/blocking buffer to the lysate (10% goat serum, 0.2% Triton X-100 in PBS). Incubate at RT for 30 min.
11. Centrifuge lysate at 8,000 x g for 5 min and remove supernatant.
12. Add 200 μl of FITC-conjugated dolichos biflorus agglutin (DBA). Use a 1:100 dilution of the lectin in PBS plus 10% goat serum. Gently resuspend the lysate by pipetting. Incubate for 1 hr at RT .
    NOTE: DBA is a ligand that binds to CST1 on the parasite cyst wall.
13. Centrifuge lysate at 8,000 x g for 5 min and discard supernatant. Add 1 ml of PBS to the pellet. Incubate at RT for 5 min. Repeat PBS wash 3x. Remove the supernatant following the final wash using a pipette.
14. Use a wide bore 20-200 μl pipette tip to draw up 5 μl of the stained brain lysate and place on a microscope slide. Mount the slide with a 25 x 25 mm cover slip to create a wet mount of the brain lysate. Make 3 replicate slides using 5 μl of brain lysate each.
15. Count the number of cysts per each 5 μl aliquot by fluorescence microscopy using a 10X objective (Figure 6).
    NOTE: Some cysts are large (8 or more parasites approximately) while some are very small (1-2 parasites per cyst). Count total number of cysts per each slide but document number of large versus number of small cysts.
16. Add the number of cysts detected in the three different 5 µl aliquots and multiply by the total dilution factor x 2 (only half of the brain was made into a lysate) to estimate the number of total cysts per brain.