在革兰氏阴性菌假结核耶尔森菌（Y.假）诱导的突变体21000转座子诱变研究表明，细菌转座子携带hldE突变已经受损的诱导核因子κB的活化能力（NF-κB）途径在人胚胎肾293T细胞系（HEK293T）（周P等。2018）。基因hldE为ADP -L-甘油基-β-d甘露 - 庚糖（ADP-庚糖），细菌代谢中间体在脂多糖（LPS）的生物合成，这是存在于所有的革兰氏阴性菌和革兰氏一些的生物合成必需阳性菌（唐W等一。2018）。此外，所需的ADP-庚糖相关的代谢物的生物合成相关基因的缺失，d甘油基-β-d甘露 - 庚糖-1,7-二磷酸（HBP），NF-κB的活化防止由假结核菌（周鹏等人。2018）。此外，合成的ADP-庚糖但不HBP，当加入到细胞外空间，诱导的NF-κB的活化和白细胞介素8（IL8或CXCL8）的分泌在HEK293T细胞（周P等。2018）。的荧光激活细胞分选（FACS）为主，全基因组CRISPR-Cas9屏幕上识别的α蛋白激酶1（ALPK1），肿瘤坏死因子（TNF-α）受体相关因子一个HEK293T NF-κB报道细胞系（TRAF）-interacting蛋白与所述叉头相关联的域（TIFA）和TRAF6作为NF-κB活化通过ADP-庚糖或假结核菌（周P等2018）诱导的介导物。ALPK1 - / - 或TIFA - / - HEK293T细胞显示废除NF-κB激活和细胞因子的表达。有缺陷的NF-κB活化中ADP-庚糖处理ALPK1 - / - 是由野生型ALPK1恢复HEK293T细胞而不是通过它的激酶失活突变体K1067M（周P等2018）。此外，ADP-庚糖刺激TIFA的共免疫沉淀与ALPK1（周P等。2018）。此外，ALPK1激酶需要在HEK293T细胞中的TIFA ADP-庚糖诱导的磷酸化活性，因此表明ALPK1作用TIFA的上游（周P等。2018）。 Similarly, ADP‐heptose sensing was ALPK1‐dependent during S. flexneri infection (Garcia-Weber D et al. 2018). These findings are supported by studies showing that cytosolic HBP, found in the bacteria Neisseria meningitidis, Shigella flexneri, Salmonella enterica serovar Typhimurium and Helicobacter pylori (H. pylori), induced activation of ALPK1-TIFA-dependent NF-κB signaling in host cells (Zimmermann S et al. 2017; Milivojevic M et al. 2017; Gaudet RG et al. 2017). In addition, HBP failed to directly activate ALPK1 (Garcia-Weber D et al. 2018; Zhou P et al. 2018); instead, HBP is converted by host-derived adenylyltransferases, such as nicotinamide nucleotide adenylyltransferases, to ADP-heptose 7-P, a substrate which activates ALPK1 and the downstream NF-κB response (Zhou P et al. 2018). ALPK1 contains a kinase domain (KD) and an α-helical domain linked by an unstructured region (Zhou P et al. 2018). Co-expression of the N-terminal domain (NTD) and KD of ALPK1 (ALPK1-NTD (1–473) and ALPK1-KD (959–1244), respectively) in HEK293T cells was sufficient to allow ADP-heptose or Y. pseudotuberculosis to induce activation of NF-κB and phosphorylation of TIFA (Zhou P et al. 2018). High-performance liquid chromatography (HPLC)- mass spectrometry (MS) fractionation of small-molecule extracts from His6–ALPK1-NTD purified from wild-type E. coli coupled with anti-pT9-TIFA immunoblotting and NF-κB luciferase reporter assays in HEK293T identified one active fraction that contained the presumed ADP-heptose ion. Direct binding of E. coli ΔhldE-derived apo-ALPK1-(NTD+KD) complex to ADP-heptose was detected by BioLayer Interferometry (BLI) assay in vitro (Zhou P et al. 2018). The structural studies revealed that a narrow pocket in ALPK1-NTD directly binds ADP-heptose (Zhou P et al. 2018). The ADP-heptose-binding residues are conserved in ALPK1 of other vertebrates. Introducing several mutations in respective residues at the NTD of ALPK1 impaired the ability of ALPK1 to activate NF-κB in response to ADP-heptose (Zhou P et al. 2018). The ADP-heptose binding to ALPK1 is thought to trigger conformational changes and stimulate the kinase domain of ALPK1 to phosphorylate and further activate TIFA, which eventually trigger activation of the downstream NF-κB pathway (Zhou P et al. 2018). Many Gram-negative bacteria induce host cytokine expression in a type III secretion system (T3SS)-dependent manner. T3SS of Y. pseudotuberculosis was required for ADP-heptose induced activation of NF-κB signaling, indicating that a bacterial injection system mediated ADP-heptose translocation to induce activation of ALPK1 upon Y. pseudotuberculosis infection (Zhou P et al. 2018). However, sensing of ADP-heptose by ALPK1 is not just limited to bacteria with T3SS. Burkholderia cenocepacia, enterotoxigenic E. coli, and diffuse-adhering E. coli, also stimulated NF-κB activation through the ALPK1-TIFA axis in an hldE-dependent manner in HEK293T cells (Zhou P et al. 2018). Further, TIFAsome formation and NF-kB activation could be triggered by H. pylori’s component secreted in a type IV secretion system (T4SS)-dependent fashion (Zimmermann S et al. 2017; Stein SC et al. 2017). Animal studies indicate that injection of ADP-heptose induced massive neutrophil recruitment with increased production of several NF-κB-dependent cytokines and chemokines in wild type (WT), but not in Alpk1-/- mice. On infection with B. cenocepacia, which triggers lung inflammation in WT, but not Alpk1-/- mice showed increased expression of NF-κB-dependent cytokines and chemokines in the lungs, which led to higher bacterial load in the lungs of Alpk-/-, but not WT, mice (Zhou P et al. 2018). Thus, in vitro and in vivo studies identified ADP-heptose as a bona fide bacterial immunomodulator which is sensed by cytosolic innate immune receptor ALPK1.