Toxoplasma gondii GRA9 Regulates the Activation of NLRP3 Inflammasome to Exert Anti-Septic Effects in Mice

Dense granule proteins (GRAs) are essential components in Toxoplasma gondii, which are suggested to be promising serodiagnostic markers in toxoplasmosis. In this study, we investigated the function of GRA9 in host response and the associated regulatory mechanism, which were unknown. We found that GRA9 interacts with NLR family pyrin domain containing 3 (NLRP3) involved in inflammation by forming the NLRP3 inflammasome. The C-terminal of GRA9 (GRA9C) is essential for GRA9–NLRP3 interaction by disrupting the NLRP3 inflammasome through blocking the binding of apoptotic speck-containing (ASC)-NLRP3. Notably, Q200 of GRA9C is essential for the interaction of NLRP3 and blocking the conjugation of ASC. Recombinant GRA9C (rGRA9C) showed an anti-inflammatory effect and the elimination of bacteria by converting M1 to M2 macrophages. In vivo, rGRA9C increased the anti-inflammatory and bactericidal effects and subsequent anti-septic activity in CLP- and E. coli- or P. aeruginosa-induced sepsis model mice by increasing M2 polarization. Taken together, our findings defined a role of T. gondii GRA9 associated with NLRP3 in host macrophages, suggesting its potential as a new candidate therapeutic agent for sepsis.


GST pulldown, immunoblot, and immunoprecipitation analysis
For GST pulldown, cells were harvested and lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After centrifugation, the supernatants were precleared with protein A/G beads at 4 °C for 2 h. Pre-cleared lysates were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was incubated for 4 h at 4 °C. Precipitates were washed extensively with lysis buffer. Proteins bound to glutathione beads were eluted with SDS loading buffer by boiling for 5 min.
For immunoprecipitation, cells were harvested and then lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After pre-clearing with protein A/G agarose beads for 1 h at 4 ˚C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1-4 μg of commercial antibody was added to 1 ml of cell lysates and incubated at 4°C for 8 to 12 h. After the addition of proteins A/G agarose beads for 6 h, immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min.
For immunoblotting, polypeptides were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane (Bio-Rad). Immuno detection was achieved with specific antibodies. Antibody binding was visualized by chemiluminescence (ECL; Millipore) and detected by a Vilber chemiluminescence analyzer (Fusion SL 3; Vilber Lourmat).

Protein purification and Mass spectrometry
To identify GRA9-binding proteins, THP-1 cells expressing Flag-GRA9 or vector were harvested and lysed with NP-40 buffer (50 mM HEPES, pH 7.4,150 mM NaCl, 1 mM EDTA, 1% (v/v) NP40) supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland). Postcentrifuged supernatants were precleared with protein A/G beads at 4 °C for 2 h. Precleared lysates were mixed with αFlag antibody-conjugated with agarose beads for 4 h at 4 °C. Precipitates were washed extensively with lysis buffer. Proteins bound to beads were eluted and separated on a Nupage 4-12% Bis-Tris gradient gel (Invitrogen). After silver staining (Invitrogen), specific protein bands were excised and analyzed by ion-trap mass spectrometry at the Korea Basic Science Institute (Seoul, Korea) Mass Spectrometry facility, and amino acid sequences were determined by tandem mass spectrometry and database searches.

Confocal fluorescence microscopy
Immunofluorescence analysis was performed as described previously [32]. The cells were fixed on coverslips with 4% (w/v) paraformaldehyde in PBS and then permeabilized for 10 min using 0.25% (v/v) Triton X-100 in PBS at 25 ˚C. TRAF6 or His was detected using a 1/100 dilution of the primary Ab for 1 h at 25 ˚C. After washing, the appropriate fluorescently labeled secondary Abs were incubated for 1 h at 25 ˚C. Slides were examined using laser-scanning confocal microscopy (model LSM 800; Zeiss).

Cellular fractionation
Cytosol and mitochondria were isolated from cells using a Mitochondria Fractionation Kit (Active Motif, 40015) or as described previously [34]. Subcellular fractionated proteins were lysed in buffer containing 2% SDS and boiled with 2x reducing sample buffer for SDS-PAGE.

MTT assay
Cell viability relative to non-treated group was measured by MTT assay, as described previously [2]. After incubating for the indicated time points, 5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) solution was added in the place of media, and cells were incubated for further 4 h. Then, all the media was removed and the same volume of dimethyl sulfoxide (DMSO) solution was added for 15 min to dissolve the formazan. Using UV/VIS spectrophotometer, each well of the plate was measured at 540 nm to measure relative cell viability.