ADP-heptose is a pathogen-associated molecular pattern of Shigella flexneri infection

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. PAMPs include among others lipopolysaccharide (LPS), flagellin, peptidoglycan, lipoteichoic acid and DNA. β-Heptose 1,7-bisphosphate (βHBP) was recently identified as a PAMP of Neisseria meningitidis [1]. βHBP is a soluble metabolite of the LPS biosynthesis pathway. In N. meningitidis, it is produced by the D-β-D-heptose 7phosphate kinase activity of the HldA protein that phosphorylates D-glycero-β-Dmanno-heptose 7-phosphate into βHBP [2]. Gaudet et al. showed that it can be secreted by N. meningitidis and sensed in the cytosol of eukaryotic cells after being internalized by endocytosis [1]. Our laboratory reported that βHBP sensing was involved in the inflammatory response of epithelial cells to Shigella flexneri and Salmonella typhimurium infection [3]. This finding was based on the analysis of the hlde and gmhb deletion mutants of the LPS biosynthesis pathway. In these bacterial species closely related to Escherichia coli, hlde encodes a bifunctional protein that harbors both D-β-D-heptose 7-phosphate kinase and D-β-D-heptose 1-phosphate adenylyltransferase activities, responsible for the production of βHBP and ADP-Dglycero-β-D-manno-heptose (ADP-D-β-D-heptose), respectively [4]. The dephosphorylation of βHBP into D-glycero-β-D-manno heptose 1-phosphate is provided by the phosphatase activity of GmhB [5]. We showed that infection with an hlde deletion (∆hldE) mutant failed to induce the expression of the inflammatory cytokine interleukin-8 (IL-8) whereas this latter was restored after infection with a mutant deleted for gmhb (∆gmhB) [3]. S. flexneri infection leads to oligomerization of the proteins TRAF-interacting protein with FHA domain-containing protein A (TIFA) and TNF receptor-associated factor 6 (TRAF6)...