Extended Data Fig. 3: The ALPK1–TIFA pathway mediates T3SS-dependent and -independent activation of NF-κB by various bacterial pathogens.
From: Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose
a, b, Requirement of ALPK1 kinase activity and TIFA for Y. pseudotuberculosis-induced activation of NF-κB. Wild-type or ALPK1−/− 293T cells expressing Flag–ALPK1 (wild type or K/M) or TIFA−/− 293T cells expressing Flag–TIFA were infected with indicated Y. pseudotuberculosis strains. NF-kB activation was assessed by luciferase reporter assay (a) or eGFP reporter expression (b). Scale bar, 50 μm (b). c–e, Requirement of ALPK1 kinase activity for Y. pseudotuberculosis (or S. flexneri 2457T)-induced phosphorylation of TIFA (c, d) or formation of eGFP–TIFA foci (e). Flag– or eGFP–TIFA was expressed in wild-type or ALPK1−/− 293T cells expressing Flag–ALPK1 (wild type or K/M). TIFA phosphorylation was assessed by anti-pT9-TIFA immunoblotting (c, d). Y. pseudotuberculosis Δ6 was labelled with mCherry (scale bar, 20 μm) (e). f, g, NF-κB luciferase reporter activation and formation of eGFP–TIFA foci induced by T3SS-negative bacteria. Wild-type, ALPK1−/− or Flag–ALPK1-complemented ALPK1−/− 293T cells were infected with DAEC 2787, ETEC H10407, B. cenocepacia J2315 or their ΔhldE mutants. Scale bar, 20 μm. a, f, Luciferase data are shown as mean ± s.d. from three technical replicates; two-tailed unpaired Student’s t-test was performed (*P < 0.05, **P < 0.01, *** P < 0.001). b, e, g, Confocal images with Hoechst-stained nuclei. All data shown are representative of three independent experiments.
