FIG 5.

Validation of positive genetic hits in the pyruvate catabolism signaling pathway and effects of the corresponding inhibitor in S. flexneri infection of human THP-1 cells. (A) Schematic of positive genetic hits in the pyruvate catabolism signaling pathway and the corresponding inhibitor. (B and C) Growth of THP-1 cells (B) and the intracellular level of S. flexneri ΔvirG (C) postinfection in the presence or absence of various concentrations of the PDHB inhibitor (oxythiamine, OT) or OT combined with IRAK1/4-Inh (10 μM). (D) Schematic of inhibitor validation in PMA-stimulated THP-1 cells infected with different S. flexneri strains. Gen, gentamicin. (E) Effects of IRAK1 and PDHB inhibitors on the survival of differentiated THP-1 cells after infection with S. flexneri M90T ΔvirG (M90T ΔvirG), the S. flexneri M90T wild type (M90T WT), and the S. flexneri 2457T wild type (2457T WT), with death measured by LDH release. (F) Effects of the IRAK1 and PDHB inhibitors on the growth of three intracellular S. flexneri strains in differentiated THP-1 cells, as measured by CFU. IRAK1/4-Inh was used at 10 μM. OT was used at 0.1 mM. (G and H) Production of acetyl-CoA (G) and succinate (H) with or without S. flexneri ΔvirG infection and in the presence or absence of OT or OT combined with the IRAK1 inhibitor (10 μM). Data represent the means ± SD (B, C, G, and H, n = 3; E and F, n = 4) (Student's two-tailed unpaired t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; N.D, not detectable).