FIG 1.

MTA treatment of S. Typhimurium reduces pyroptosis and invasion in vitro. (A) A modified gentamicin protection assay using an inducible GFP plasmid was used to detect pyroptosis (7-AAD⁺ red nuclear staining) and host cell invasion (intracellular GFP⁺ bacteria). This is observable by fluorescence microscopy and quantifiable by flow cytometry. Bar, 100 μm. (B) Exogenous MTA has no effect on the growth of S. Typhimurium in rich medium. The optical density at 600 nm (OD₆₀₀) for S. Typhimurium treated with 300 μM MTA or 0.5% DMSO was measured every 30 min and showed that it exhibited equivalent growth with both treatments (n = 3). (C, D) Treatment of bacteria with 300 μM MTA during growth to late log phase (2 h 40 min) reduced pyroptosis (MOI, 30) and host cell invasion (MOI, 10) in LCLs measured at 3 h postinfection in 18592 LCLs (C) and THP-1 cells (D). Percent cell death represents all 7-AAD-positive cells under each infected condition, with the baseline uninfected cell death being subtracted. See the gating in panel A. Data were normalized to the global mean across five experiments, and P values, indicated at the top, were generated by a Student's t test. (E, F) Treating bacteria with other methionine-related metabolites (methionine, SAM, MTOB, and phenylalanine) or adenosine does not suppress host cell pyroptosis (MOI, 30), based on 3 to 5 independent experiments in 18592 LCLs (E) or THP-1 cells (F). Data were normalized to the global mean, and P values were generated by a one-way analysis of variance with Dunnett's multiple-comparison test. All error bars represent the standard error of the mean. ns, not significant.