ABSTRACTHeptose metabolites including ADP-d-glycero-β-d-manno-heptose (ADP-heptose) are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent proinflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacteriumHelicobacter pyloriproduces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. UsingH. pylorias a model, we have addressed the question of how proinflammatory ADP-heptose biosynthesis can be regulated by bacteria. We have characterized the interstrain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production byH. pylori, which impact cell-autonomous proinflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains and was also variably expressed between strains. Amounts of gene transcripts in thehldEgene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of thecagpathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of theH. pylorilipopolysaccharide (LPS) heptose biosynthetic pathwayin vitrousing recombinant purified GmhA, HldE, and GmhB proteins. On the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of major reaction products were identified as β-d-ADP-heptose and β-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product inH. pyloribacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites.IMPORTANCEBacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogenHelicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about howH. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production byH. pyloriis strain specific. Heptose gene cluster activity is modulated by the presence of an activecagpathogenicity island (cagPAI), contact with human cells, and the carbon starvation regulator A. Reconstitution with purified biosynthesis enzymes and purified bacterial lysates allowed us to biochemically characterize heptose pathway products, identifying a heptose-monophosphate variant as a novel proinflammatory metabolite. These findings emphasize that the bacteria use heptose biosynthesis to fine-tune inflammation and also highlight opportunities to mine the heptose biosynthesis pathway as a potential therapeutic target against infection, inflammation, and cancer.