ABSTRACT During an infection, the detection of pathogens is mediated through interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. β-Heptose 1,7-bisphosphate (βHBP), a metabolite of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a PAMP of gram-negative bacteria. It was reported that βHBP sensing leads within minutes to oligomerization of the protein TIFA, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compared the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. In contrast to lysate, we found that βHBP fails to trigger rapid oligomerization of TIFA. βHBP only induces delayed signaling, suggesting that it has to be processed intracellularly to induce inflammation. By dissecting the LPS biosynthesis pathway with deletion mutants and functional complementation experiments, we show that ADP-D- glycero -β-D- manno -heptose and ADP-L- glycero -β-D- manno -heptose are the bacterial metabolites responsible for rapid TIFA oligomerization, and that they strongly induce interleukin-8 expression during S. flexneri infection. Altogether, our results rule out a major role of βHBP in S. flexneri infection and identify ADP-heptose as a new PAMP.