Helicobacter pylori Modulates Heptose Metabolite Biosynthesis and Heptose-Dependent Innate Immune Host Cell Activation by Multiple Mechanisms.

Heptose metabolites including ADP-d-glycero-ß-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 bacterium Helicobacter pylori produces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as 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 by H. 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 the hldE gene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori lipopolysaccharide (LPS) heptose biosynthetic pathway in vitro using 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 ß-d-ADP-heptose and ß-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites. IMPORTANCE Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about how H. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production by H. pylori is strain specific. Heptose gene cluster activity is modulated by the presence of an active cag pathogenicity 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.

Central Role of Sibling Small RNAs NgncR_162 and NgncR_163 in Main Metabolic Pathways of Neisseria gonorrhoeae.

Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C1) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus. IMPORTANCE Neisseria gonorrhoeae is a major human pathogen which infects more than 100 million people every year. An alarming development is the emergence of gonococcal strains that are resistant against virtually all antibiotics used for their treatment. Despite the medical importance and the vanishing treatment options of gonococcal infections, the bacterial metabolism and its regulation have been only weakly defined until today. Using RNA-seq, metabolomics, and 13C-guided metabolic flux analysis, we here investigated the gonococcal metabolism and its regulation by the previously studied sibling sRNAs NgncR_162/163. The results demonstrate the regulation of transport processes and metabolic pathways involved in the biosynthesis of nucleotides, vitamins, and amino acids by NgncR_162/163. In particular, the combination of transcriptome and metabolic flux analyses provides a heretofore unreached depth of understanding the core metabolic pathways and their regulation by the neisserial sibling sRNAs. This integrative approach may therefore also be suitable for the functional analysis of a growing number of other bacterial metabolic sRNA regulators.

Fast Identification of Food Thickeners by Nontargeted NMR-Spectroscopy.

Food thickeners are carbohydrate additives that can only be determined by long-term, multistep analysis. Fast methods to directly determine thickeners in food matrixes are therefore welcome. In this study, a rapid procedure based on the direct 1H NMR analysis of food samples dissolved in deuterated water was developed. Individual thickeners were assigned due to specific marker signals gleaned from two-dimensional NMR analyses. The combination of one-dimensional 1H NMR and DOSY experiments enabled unequivocal assignments of thickeners even in complex matrixes. Using this approach, gum arabic, carrageenan, agar-agar, galactomannans, and pectin could be identified in pastille, glaze, and fruit spread. Because of low concentrations (<0.5%-1%, w/w), the same thickeners and others such as xanthan gum and alginate could not be determined directly by NMR in curry sauce, rice pudding, choco milk drink, and lemon peel flavor. Moreover, NMR analyses of the hydrolysate did not reveal the specific monomeric units of the thickeners under study, as shown for the hydrolysate of lemon peel flavor. Nevertheless, the NMR approach could provide welcome means in the future to directly determine intact thickeners in food.