Xanthine

Prugniaud (Pru) T. gondii-expressing luciferase were filtered, washed and resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 0.15 mM CaCl₂, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA). Parasites were mixed with 50 μg of sgTgIST-1 and sgTgIST-2 CRISPR plasmid along with 40 μg of the targeting vector linearized by Kpnl and SacI, and supplemented with 2 mM ATP, 5 mM GSH. Parasites were electroporated by GENE PULSER II (Bio-Rad Laboratories). Selection by growth for 14 days in 25 μg/ml mycophenolic acid (Sigma) and 25 μg/ml xanthine (Wako) were used to obtain stably resistant clone. And then parasites were plated in limiting dilution in 96-well plates to isolate single clones. To confirm the disruption of the gene encoding TgIST, we analyzed messenger RNA of TgIST from WT and TgIST-KO parasites by quantitative RT-PCR. In addition, we observed comparable in vitro growth and in vivo virulence to each other and to the parental line.

The IWS1-deficient parasites lacking HXGPRT were filtered, washed, and resuspended in Cytomix. Parasites were mixed with 50 μg of the ROP18 expression vector containing ROP18 cDNA (18 (link)), in which 1 kb promoter of the SAG1 gene was fused to the coding sequence of ROP18 cDNA, followed by the poly A sequence and hxgprt expression cassette in pBlueScript plasmid vector (Fig. S2B), and supplemented with 2 mM ATP and 5 mM GSH. Parasites were electroporated using a Gene Pulser II. Selection by growth for 14 days in 25 μg/mL mycophenolic acid (Sigma) and 50 μg/mL xanthine (Wako) was used to obtain stably resistant clones. Then, parasites were plated in limiting dilution in 96-well plates to isolate single clones. To confirm expression of rop18, we analyzed ROP18 mRNA from the rescued parasites by quantitative RT-PCR using the primers listed in Table S3.

Prugniaud (Pru) T. gondii parasites expressing luciferase were filtered, washed, and resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 0.15 mM CaCl₂, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA). Parasites were mixed with 50 µg of sgGRA15-1 and sgGRA15-2 CRISPR plasmid along with 40 µg of the targeting vector linearized by KpnI and SacI and were supplemented with 2 mM ATP and 5 mM glutathione (GSH). Parasites were electroporated by the use of a Gene Pulser II instrument (Bio-Rad Laboratories). Selection by growth for 14 days in 25 µg/ml mycophenolic acid (Sigma)–25 µg/ml xanthine (Wako) was used to obtain a stably resistant clone. And then parasites were plated in limiting dilution in 96-well plates to isolate single clones. To confirm the disruption of the gene encoding GRA15, we analyzed mRNA of GRA15 from WT and GRA15-KO parasites by quantitative reverse transcription-PCR (RT-PCR). In addition, we observed comparable levels of in vitro growth and in vivo virulence with respect to each of the mutants and to the parental line.

The direct inhibitory effect of BLEx and its fractions on XOD activity was evaluated as the conversion of xanthine to Uric acid under XOD (from bovine milk, Sigma). Uric acid, xanthine, and allopurinol (Fujifilm Wako) were dissolved in 0.1 M phosphate buffer (PB; pH 8.0). Next, 50 μL PB, 50 μL samples (appropriate concentrations of BLEx, fractions, or 1 mM allopurinol), and 100 μL of 1 mM xanthine were added to a 96-well plate (UV-star, Greiner Bio-One, Kremsmünster, Austria). Finally, XOD was added to a final concentration at 0.1 mU and reacted for 3 min at 37 °C. The production of Uric acid was measured as the absorbance at 293 nm using SpectraMax (Molecular Devices, San Jose, CA, USA).

B. breve MCC1274 was cultured in YC medium supplemented with 200 μM each of adenosine, inosine, hypoxanthine, and xanthine (FUJIFILM Wako Pure Chemical) for 24 h under anaerobic conditions. Then, the culture medium was centrifuged (10,000 × g, 5 min, 4°C), and the supernatant was stored at −20°C until analysis.

RHΔhxgprtΔku80 strain parasites expressing firefly luciferase were filtered, washed, and resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 0.15 mM CaCl₂, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA). Parasites were mixed with 50 μg of each gRNA1 and gRNA2 CRISPR plasmid for each gene, along with the PCR-amplified targeting fragment for each gene, and supplemented with 2 mM ATP and 5 mM glutathione (GSH). Parasites were electroporated by Gene Pulser II (Bio-Rad Laboratories). Selection by growth for 14 days in 25 μg/mL mycophenolic acid (Sigma) and 50 μg/mL xanthine (Wako) were used to obtain stably resistant clones. Next, parasites were plated in limiting dilution in 96-well plates to isolate single clones. To confirm the disruption of the gene, we analyzed mRNA of IWS1, SUB2, RAMP4 or DRL1 from WT and each KO (knockout) parasite by quantitative RT-PCR using the primers listed in Table S3.

NC-6300 was obtained from NanoCarrier Co., Ltd. (with material transfer agreement) and diluted in Dulbecco’s phosphate-buffered saline (DPBS) to prepare a stock solution (EPI concentration of 2 mM). EPI, distilled water, superoxide dismutase, RPMI 1640 medium, and accutase were from Nacalai Tesque (Kyoto, Japan). DPBS, iron(II) FeSO₄, H₂O₂, xanthine, and xanthine oxidase were from Fujifilm Wako Pure Chemical (Osaka, Japan). Fetal bovine serum was from Gibco (Grand Island, NY, United States). Penicillin-streptomycin solution was from Sigma (St. Louis, MO, United States). Hydroxy phenyl fluorescein (HPF) was from Goryo Chemical (Sapporo, Japan), and Cell Counting Kit-8 (CCK-8) and WST-1 were from Dojindo Laboratories (Kumamoto, Japan). The Annexin V fluorescein isothiocyanate (FITC)-propidium iodide (PI) kit was from Medical and Biological Laboratories (Nagoya, Japan).