Extended Data Fig. 1: Analyses of the ADP-Hep biosynthesis pathway and ADP-Hep-induced NF-κB activation.
From: Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose
a, Low calcium-induced secretion of the T3SS substrates in the hldE transposon mutant. yscC is required for T3SS assembly. b, Schematic of the classical ADP-Hep biosynthesis pathway in Gram-negative bacteria. c, ADP-Hep-dependent autotransporter heptosylation in ΔgmhB mutant. A fragment of the AIDA-I autotransporter (GST–AIDA-I50–600–Flag) and its heptosyltransferase (AAH) were expressed in Y. pseudotuberculosis Δ6 deleted of a gene in the ADP-Hep biosynthesis pathway. AIDA-I heptosylation was assessed using the ECL glycoprotein detection kit. d, NF-κB activation in 293T cells electroporated with HBP or H1P (obtained by treating HBP with recombinant GmhB for the indicated times). e, Effects of ALPK1 knockout on other known NF-κB pathways. NOD1, NOD2 and MYD88 were transfected into the cells. Wild-type cells and two ALPK1−/− 293T clones (KO-1/2) were treated with ADP-LD-Hep (100 μM), TNF (20 ng ml–1), C12-iE-DAP (10 ng ml–1) or MDP (10 ng ml–1). f, g, Effects of NOD1/2 deficiency on ADP-Hep-induced NF-κB activation and TIFA foci formation. Wild-type HeLa cells and two NOD1/2 double-knockout clones (DKO-1/2) were assayed. eGFP–TIFA was transfected into the cells (g). Scale bar, 20 µm. d–f, NF-κB activation was assessed by luciferase reporter assay (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, NS, not significant). All data are representative of three independent experiments.
