ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.
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ABSTRACT:
SUBMITTER: Pfannkuch L
PROVIDER: S-EPMC6662969 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the β-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.
| S-EPMC8204981 | biostudies-literature
Project description:Heptose 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 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 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 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.
| S-EPMC10269868 | biostudies-literature
Project description:Helicobacter pylori is a bacterial pathogen that colonizes the gastric niche of ??50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H.?pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-?B in human gastric epithelial cells as a measure of T4SS function. Analysis of ??3000 H.?pylori mutants revealed three non-cagPAI genes that affected NF-?B nuclear translocation. Of these, the outer membrane protein HopQ from H.?pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-?B, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded cofactor of T4SS function.
| S-EPMC3797234 | biostudies-literature
Project description:Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression.
| S-EPMC2776988 | biostudies-literature
Project description:OBJECTIVES: The Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases. METHODS: Eighty H. pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n= 58) and cholelithiasis (n= 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method. RESULTS: The vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries. CONCLUSIONS: The cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer.
| S-EPMC3482671 | biostudies-literature
Project description:Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/-) mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.
| S-EPMC2855317 | biostudies-other
Project description:Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies.
| S-EPMC8266642 | biostudies-literature
Project description:The synthesis of "typical" hexa-acylated lipid A occurs via a nine-step enzymatic pathway, which is generally well conserved throughout all gram-negative bacteria. One exception to the rule is Helicobacter pylori, which has only eight homologs to the nine lipid A biosynthetic enzymes. The discrepancy occurs toward the end of the pathway, with H. pylori containing only a single putative secondary acyltransferase encoded by jhp0265. In Escherichia coli K-12, two late acyltransferases, termed LpxL and LpxM, are required for the biosynthesis of hexa-acylated lipid A. Detailed biochemical and genetic analyses reveal that H. pylori Jhp0265 (the protein encoded by jhp0265) is in fact an LpxL homolog, capable of transferring a stearoyl group to the hydroxyl group of the 2' linked fatty acyl chain of lipid A. Despite the lack of a homolog to LpxM in the H. pylori genome, the organism synthesizes a hexa-acylated lipid A species, suggesting that an equivalent enzyme exists. Using radiolabeled lipid A substrates and acyl-acyl carrier protein as the fatty acyl donor, we were able to confirm the presence of a second H. pylori late acyl transferase by biochemical assays. After synthesis of the hexa-acylated lipid A species, several modification enzymes then function to produce the major lipid A species of H. pylori that is tetra-acylated. Jhp0634 was identified as an outer membrane deacylase that removes the 3'-linked acyl chains of H. pylori lipid A. Together, this work elucidates the biochemical machinery required for the acylation and deacylation of the lipid A domain of H. pylori lipopolysaccharide.
| S-EPMC2580709 | biostudies-literature
Project description:The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.
| S-EPMC3936762 | biostudies-literature
Project description:This study about Helicobacter pylori in Number Hp0499 and Hp1490
| PRJEB53580 | ENA