SummaryImmune cells are somewhat unique in that activation responses can alter quantitative phenotypes upwards of 100,000-fold. To date little is known about the metabolic adaptations necessary to mount such dramatic phenotypic shifts. Screening for novel regulators of macrophage activation, we found nonprotein kinases of glucose metabolism among the most enriched classes of candidate immune modulators. We find that one of these, the carbohydrate kinase-like protein CARKL, is rapidly downregulated in vitro and in vivo upon LPS stimulation in both mice and humans. Interestingly, CARKL catalyzes an orphan reaction in the pentose phosphate pathway, refocusing cellular metabolism to a high-redox state upon physiological or artificial downregulation. We find that CARKL-dependent metabolic reprogramming is required for proper M1- and M2-like macrophage polarization and uncover a rate-limiting requirement for appropriate glucose flux in macrophage polarization.

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Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1β- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria (28). It has a tripartite structural organization consisting of lipid A, a conserved core oligosaccharide region, and an O-specific polysaccharide chain or O antigen. In the majority of gram-negative bacteria, the core oligosaccharide can be subdivided into an outer core, generally composed of hexoses and hexosamines, and an inner core made of 3-deoxy-D-manno-oct-2-ulosonic acid and L,Dheptose units. LPS plays an important role in maintaining the structural integrity of the bacterial outer membrane by interacting with outer membrane proteins and divalent cations (15), thereby providing a barrier against the entry of toxic hydrophobic compounds into the bacterial cell (27). Escherichia coli mutants defective in the biosynthesis of 3-deoxy-D-manno-oct-2-ulosonic acid are nonviable, whereas those impaired in L,Dheptose synthesis survive in vitro, although they display a pleiotropic phenotype referred to as "deep rough" (17). This phenotype is characterized by an extreme sensitivity to very low concentrations of novobiocin, detergents, and bile salts (32). Deep rough mutants also have defects in F plasmid conjugation and generalized transduction by the bacteriophage P1 (6, 16). Haemophilus influenzae heptose-deficient mutants were found to be serum sensitive and displayed a reduced virulence in vivo (18,36).The complete biosynthesis pathway of the L,D-heptose precursor has not been elucidated. Eidels and Osborn (11) In gram-negative bacteria, functional studies have only been performed for the isomerization reaction and the epimerization step (3,9,26), while the conversion of D,D-heptose 7-phosphate to D,D-heptose 1-phosphate and a functional proof of the activating step have not been demonstrated. The D-sedoheptulose 7-phosphate isomerase activity was described in S. enterica serovar Typhimurium (12), and the corresponding gene, gmhA, has been cloned both from E. coli and from H. influenzae (3, 4). The amino acid sequence of the GmhA polypeptide is highly conserved in different gram-negative bacteria (33). The epimerization step is catalyzed by the WaaD (formerly RfaD) protein (5), which has also been crystallized (8). We * Corresponding author. Mailing address for Paul Messner: Zentrum für Ultrastrukturforschung,

The gastric pathogen Helicobacter pylori activates the NF‐κB pathway in human epithelial cells via the recently discovered α‐kinase 1 TRAF‐interacting protein with forkhead‐associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7‐bisphosphate (HBP), an LPS intermediate produced in gram‐negative bacteria that represents a new pathogen‐associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF‐κB activation. Instead, we identified ADP‐glycero‐β‐D‐manno‐heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram‐negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β‐form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori–induced NF‐κB activation in epithelial cells, but it acts as a general gram‐negative bacterial PAMP.—Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen‐associated molecular pattern identified in Helicobacter pylori. FASEB J. 33, 9087–9099 (2019). http://www.fasebj.org