Helicobacter pylorimodulates heptose metabolite biosynthesis and heptose-dependent innate immune host cell activation by multiple mechanisms

Abstract

AbstractHeptose metabolites including ADP-heptose are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent pro-inflammatory 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, how pro-inflammatory ADP-heptose biosynthesis can be regulated by the bacteria. We have characterized the inter-strain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production byH. pylori, which impact cell-autonomous pro-inflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains, and was also strain-variably expressed. Transcript amounts of genes in thehldEgene cluster displayed intra-strain and inter-strain 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. pyloriLPS heptose biosynthetic pathwayin vitrousing recombinant purified GmhA, HldE and GmhB proteins. On the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry, the structures of major reaction products were identified as β-D-ADP-heptose and β-heptose-1-monophosphate. A pro-inflammatory 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 to uncover additional strain variation in generating heptose metabolites.