A fluorescence-activated cell sorting (FACS)-based, genome-wide CRISPR-Cas9 screen on a HEK293T NF-κB reporter cell line identified alpha protein kinase 1 (ALPK1), tumor necrosis factor (TNF-α) receptor-associated factor (TRAF)-interacting protein with the forkhead-associated domain (TIFA) and TRAF6 as mediators of NF-kB activation induced by bacterial ADP L-glycero-β-d-manno-heptose (ADP-heptose) or by Yersinia pseudotuberculosis (Y. pseudotuberculosis) (Zhou P et al. 2018). ADP-heptose is metabolic intermediate in the lipopolysaccharide (LPS) biosynthesis, which is present in all Gram-negative and some Gram-positive bacteria (Tang W et a. 2018). ADP-heptose stimulated coimmunoprecipitation of TIFA with ALPK1 and TRAF6 in HEK293T cells (Zhou P et al. 2018). The co-localization of both proteins was visualized in Shigella flexneri (S. flexneri)-infected HeLa cells co-transfected with TIFA-myc and TRAF6-Flag cDNA constructs (Milivojevic M et al. 2017). The same result was obtained upon infection of human epithelial colorectal adenocarcinoma Caco-2 cells. The interaction between TIFA and TRAF6 was further confirmed by co-immunoprecipitation assay in HeLa cells co-transfected with TIFA-myc and TRAF6-Flag cDNA constructs (Milivojevic M et al. 2017). The E178A TIFA mutant was unable to bind TRAF6 suggesting that TRAF6 activation was dependent on the TRAF6 binding motif of TIFA (Milivojevic M et al. 2017). Finally, structural studies further support the interaction between TIFA and TRAF6 (Huang WC et al. 2019). Small interfering RNA (siRNA) oligonucleotides targeting Ubc13, TRAF6, or TRAF2 strongly inhibited TIFA-mediated NF-κB activation upon the expression of these genes in HEK293 cells transfected with TIFA expression vector and a luciferase reporter gene thus suggesting that Ubc13, TRAF2, and TRAF6 are required for TIFA-mediated NF-κB activation in living cells (Ea CK et al. 2004). Further, analysis of the molecular sizes by glycerol-gradient ultracentrifugation showed that only the high-molecular-weight forms of TIFA co-sedimented with TRAF6, suggesting that oligomerization of TIFA greatly enhances its ability to bind to TRAF6 (Ea CK et al. 2004). The TIFA mutant that did not bind to TRAF6 was also unable to induce TRAF6 oligomerization (Ea CK et al. 2004). In vitro ubiquitination assay in the presence of E1, Ubc13-Uev1A, purified endogenous TRAF6, Ub, and ATP showed that TIFA enhanced the Ub ligase activity of TRAF6 (Ea CK et al. 2004). ALPK1 kinase activity was found to control TIFA oligomerization and TRAF6 activation in response to the invasive bacteria Y. pseudotuberculosis, S. flexneri and Salmonella typhimurium as well as to the extracellular pathogen Neisseria meningitidis (Milivojevic M et al. 2017; Zhou P et al. 2018). Thus, ALPK1 induces TIFA oligomerization upon bacterial infection (Zhou P et al. 2018; Milivojevic M et al. 2017). The oligomerized forms of TIFA bind to TRAF6 and promote TRAF6 oligomerization (Ea CK et al. 2004). As a result, the TRAF6 Ub ligase is activated to catalyze K63-linked polyubiquitination in conjunction with the Ubc13-Uev1A E2 complex (Ea CK et al. 2004). Activated TRAF6 promotes polyubiquitin-mediated activation of the protein kinase TAK1 (MAP3K7) complex (Ea CK et al. 2004). TAK1 is then phosphorylates IkB kinase (IKK) at key serine residues within the activation loop, thereby activating IKK complex (Israël A 2010).