Figure 8. Salmonella typhimurium disrupts host cell glycolysis.
(A) BMDMs were infected with S. typhimurium strain SL1344 grown to stationary phase and infected at the indicated multiplicity of infection (MOI; 25:1 and 100:1). At the indicated timepoints, cells were fixed and stained for ASC and DAPI. Inflammasome foci/nuclei were quantified. (B) BMDM were infected with △orgA (SPI-1 deficient) S. typhimurium grown to stationary phase. Cells were fixed at the indicated timepoints, stained for ASC and DAPI, and foci/nuclei quantified. (C) Cells were treated with the indicated compounds or infected with S. typhimurium grown to stationary phase (100:1 MOI) or log phase (10:1 MOI). Cells were fixed, stained for ASC, and ASC foci/nuclei were quantified. (D) BMDM were infected with 100:1 MOI stationary phase S. typhimurium for 5 hr. 2-NBDG (10 μM) or vehicle was added to media 2 hr post-infection. Cells were washed, lysed, intracellular bacteria sedimented from whole cell lysate via centrifugation, resuspended, and bacterial fluorescence (abs/em 465/540) analyzed by plate reader. (E) BMDM were treated as in (D), fixed, stained for DAPI, and visualized by confocal microscopy. Left: Representative image. White arrowheads indicate cytosolic S. typhiurium positive for 2-NBDG and DAPI. Scale bar 10 μM. right: 2-NBDG signal in areas of cytosol negative for DAPI (S. typhimurium negative) was measured. Cytosolic regions from ~200 discrete cells from 4 fields of view were measured per condition. Error bars represent mean +/- sd of different fields of view. (F) BMDMs were infected with stationary phase S. typhimurium and analyzed for cytosolic NAD+/NADH. △NAD+/NADH indicates the difference between the ratio measured at 11 hr and 5 hr post-infection. (G) BMDMs were infected with stationary phase S. typhimurium and levels of lactate in the supernatant analyzed at the indicated timepoints. (H) Quantification of the difference between lactate secretion measured at 11 hr and 5 hr post-infection.