ALPK1 controls TIFA/TRAF6-dependent innate immunity against heptose-1,7-bisphosphate of gram-negative bacteria

Abstract

During infection by invasive bacteria, epithelial cells contribute to innate immunity via the local secretion of inflammatory cytokines. These are directly produced by infected cells or by uninfected bystanders via connexin-dependent cell-cell communication. However, the cellular pathways underlying this process remain largely unknown. Here we perform a genome-wide RNA interference screen and identify TIFA and TRAF6 as central players of Shigella flexneri and Salmonella typhimurium-induced interleukin-8 expression. We show that threonine 9 and the forkhead-associated domain of TIFA are necessary for the oligomerization of TIFA in both infected and bystander cells. Subsequently, this process triggers TRAF6 oligomerization and NF-κB activation. We demonstrate that TIFA/TRAF6-dependent cytokine expression is induced by the bacterial metabolite heptose-1,7-bisphosphate (HBP). In addition, we identify alpha-kinase 1 (ALPK1) as the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to infection with S. flexneri and S. typhimurium but also to Neisseria meningitidis. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.

ADP‐heptose is a newly identified pathogen‐associated molecular pattern of Shigella flexneri

“…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), and activation of the transcription factor NF‐κB. In line with a previous study reporting that uninfected bystander epithelial cells constitute the main source of IL‐8 during S. flexneri infection , oligomerization of TIFA and TRAF6 as well as activation of NF‐κB were found in both infected and uninfected bystander cells . TIFA is a 20‐kDa protein that was first identified as a TRAF6‐interacting protein in a yeast two‐hybrid screen .…”

“…In contrast to bacterial lysate, we find that βHBP is unable to induce rapid oligomerization of TIFA. Oligomerization is only significant 2 h post‐treatment, indicating that cellular processing of βHBP is necessary to elicit inflammatory signaling and that another PAMP was therefore responsible for the early mechanism of TIFA oligomerization that occurs during infection . By combining gene deletion mutants and functional complementation, we identify ADP‐heptose as a new PAMP involved in S. flexneri infection.…”

“…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. Our laboratory reported that βHBP sensing is also involved in the inflammatory response of epithelial cells to Shigella flexneri and Salmonella typhimurium infection . This finding was based on the analysis of the hlde and gmhb deletion mutants of the LPS biosynthesis pathway.…”

“…The dephosphorylation of βHBP into d ‐ glycero ‐β‐ d ‐ manno heptose 1‐phosphate is provided by the phosphatase activity of GmhB . We showed that infection with an hlde deletion (Δ hldE ) mutant failed to induce the production of the inflammatory cytokine interleukin‐8 (IL‐8), whereas this latter was restored upon infection with a mutant deleted for gmhb (Δ gmhB ) . 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), and activation of the transcription factor NF‐κB.…”

ADP heptose, a novel pathogen‐associated molecular pattern identified in Helicobacter pylori

“…The discovery of ALPK1 as an NF-κB pathway–associated signaling molecule (9, 10) and a novel pattern-recognition receptor acting via the rapid formation of TIFAsomes, a previously unknown signaling platform, represent major breakthroughs in the field. However, even in our original study, we were unable to isolate HBP from H. pylori for use in experiments and instead had to rely on chemically synthesized HBP.…”