@phdthesis{Ye2004,
  author    = {Ye, Fang},
  title     = {The role of DNA supercoiling in the coordinated regulation of gene expression in Helicobacter pylori},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-9878},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2004},
  abstract  = {Summary Mechanisms of global gene regulation in bacteria are not well characterized yet. Changes in global or local supercoiling of chromosomal DNA are thought to play a role in global gene silencing and gene activation. In Helicobacter pylori, a bacterium with few dedicated transcriptional regulators, the structure of some promoters indicates a dependency on DNA topology. For example, the promoter of the major flagellar subunit gene flaA ({\´o}28-dependent) has a shorter spacing of 13 nucleotides (nt) in comparison to the consensus promoter (15 nt). Supercoiling changes might be a mechanism of gene-specific and global transcriptional regulation in this bacterium. The aim of this study was to elucidate, if changes in global supercoiling have an influence on global gene regulation in H. pylori, and on the temporal regulation of the flagellar biosynthesis pathway in this organism. In the present work, global DNA supercoiling in H. pylori was visualized for the first time, by determining the supercoiling state of plasmids under different growth conditions. Using this method, we showed that cellular supercoiling was clearly growth phase-dependent in H. pylori. Coinciding with increased supercoiling during the growth phases, transcription of the flaA gene was increased, while the transcription of a second {\´o}28-dependent gene with regular promoter spacing (HP0472) was reduced, supporting the hypothesis that growth phase-dependency of promoters might be mediated by changes of DNA topology. Supercoiling in H. pylori could be influenced in a reproducible fashion by inhibition of gyrase using novobiocin, which led to DNA relaxation and to a concomitant decrease of flaA transcript levels. Promoter spacer mutagenesis of the flaA promoter was performed. With flaA promoters of increased or reduced length, transcription of flaA was reduced, less susceptible to supercoiling changes, and, under specific conditions, inverted as compared to the wild type promoter. Transcriptional interdependence between the coupled topA-flaB genes and flaA was found by analysis of the flaA promoter mutants. Chromosomally linked gyrA-flgR, and topA-flaB genes were all dependent on supercoiling and coregulated with each other. Comprehensive transcript profiling (DNA microarrays) of wildtype H. pylori with and without novobiocin treatment identified a number of genes (10\% of total genes), including flagellin, virulence and housekeeping genes, which were strongly dependent on and appeared to be synchronized by supercoiling changes (transcriptional up- or downregulation). These findings indicate a tightly coupled temporal regulation of flagellar biogenesis and metabolism in H. pylori, dependent on global supercoiling. A specific group of genes was also regulated in H. pylori by overexpression of Topoisomerase I, as detected by genome-wide analysis (DNA microarray). The DNA-bending protein HU is thought to be responsible for influencing the negative supercoiling of DNA, through its ability to wrap DNA. HU is encoded by the hup single gene in H. pylori, and constitutively expressed during the whole growth curve. An H. pylori hup mutant was constructed. H. pylori cells lacking HU protein were viable, but exhibited a severe growth defect. Our data indicate that the lack of HU dramatically changes global DNA supercoiling, indicating an important function of HU in chromosome structuring in H. pylori. Transcriptome analyses were performed and demonstrated that a total of 66 genes were differentially transcribed upon hup deletion, which include virulence genes and many other cell functions. The data indicate that HU might act as further important global regulator in H. pylori. Increased gene expression of heat shock proteins and a decreased transcription of the urease gene cluster may indicate a co-ordinated response of H. pylori to changes of environmental conditions in its specific ecological niche, mediated by HU. After the whole genomic sequences of H. pylori strains 26695 and J99 were published, two ORFs (HP0116 and HP0440) were presumptively annotated as topoisomerase I orthologs. HP0116 is the functional H. pylori topoisomerase I (TopA). HP0440 (topA2) was found in only few (5 of 43) strains. Western blot analysis indicated that TopA2 is antigenically different from TopA. TopA2 is transcribed in H. pylori, but the protein must be functionally different from TopA, since it is lacking one functionally essential zinc finger motif, and was not able to functionally complement a TopA-deficient E. coli. Like topA, topA2 was also transcribed in a growth phase-dependent manner. We did not find a function of TopA2 in DNA structuring or topology, but, in the present study, we were able for the first time to establish a unique function for TopA2 in global gene regulation, by comprehensive transcriptome analysis (DNA microarray). Transcriptome analysis showed that a total of 46 genes were differentially regulated upon topA2 deletion, which included flagellar genes and urease genes. These results suggest that TopA2 might act as a novel important regulator of both flagellar biosynthesis and urease in H. pylori.},
  subject      = {Helicobacter pylori},
  language  = {en}
}
@phdthesis{Lee2006,
  author    = {Lee, Sae Kyung},
  title     = {Interaction of Helicobacter pylori flagellins with the host innate immune system},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19917},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium. It resides in the gastric mucous layer and epithelial lining of the stomach, often clustering at the junction of epithelial cells. H. pylori colonization usually occurs during childhood, and, when left untreated, generally persists for the host's lifetime. Persistent H. pylori infection can cause chronic superficial gastritis and gastric duodenal ulcers, which is possibly linked to the development of gastric carcinoma and primary gastric lymphoma, especially of the mucosa-associated lymphoid tissue (MALT) type. It was recently defined as a class 1 carcinogen. The gastric inflammatory response to H. pylori infection is characterized by infiltration of the mucosa by neutrophils, T and B cells, plasma cells and macrophages. This reaction is initially induced by H. pylori attachment, followed by cytokine release by gastric epithelial cells. Epidemiological studies revealed that more than 50\% of adults are infected with H. pylori all over the world. However, interestingly, only a subset of individuals develops serious H. pylori-related disease, while most infected individuals show no clinical symptoms. Gastric epithelial cells, like intestinal epithelial cells, express a subset of Toll-like receptors (TLRs) and similar pattern recognition receptors, which are important for the activation of the innate immune system. Bacterial components such as lipopeptides, peptidoglycan, LPS, flagellin, and CpG DNA are the ligands of TLRs. Thus, TLRs in gastric epithelial cells might be able to contribute to innate immune responses to H. pylori infection. However, there is scant knowledge about the mechanisms of innate immune response to acute and chronic H. pylori infection. This study is focused on host cell interaction with H. pylori flagellins, which are major components of the flagellar apparatus, and innate immune responses against them. The flagellins, which are essential for bacterial motility, are important for H. pylori to survive in the stomach mucus during the whole infectious cycle. Flagellins are known to act as the main determinant of many mucosal pathogenic bacteria that mediates proinflammatory signaling, including transcriptional factor NF-\&\#61547;B activation via TLR5. In the first part of the study, we investigated the effects of H. pylori flagellins on TLR5 expression, NF-\&\#61547;B activation and IL-8 production in various human intestinal and gastric epithelial cell lines by using Western blotting, semi-quantitative RT-PCR and ELISA. IL-8 is a potent neutrophil-activating chemokine expressed by gastric epithelial cells. When we stimulated the cells with the native form of or E. coli-expressed recombinant H. pylori flagellins, FlaA and FlaB, IL-8 was not induced in any case, while S. typhimurium flagellin (FliC) induced it significantly. H. pylori was able to modulate TLR5 protein expression and NF-\&\#61547;B activation in epithelial cells regardless of the presence of flagellins. Having established the finding that H. pylori flagellins have unusually low immune-stimulatory properties, we further investigated to find out possible reasons why H. pylori flagellins are distinct from other flagellins of pathogenic bacteria in terms of immune-stimulatory activity. From amino acid sequence comparisons, we found that some regions in the terminal D0D1 protein domains of H. pylori flagellins are different from flagellins of other pathogenic bacteria. D0D1 is the domain which is known to interact with TLR5 in Salmonella FliC. To examine whether the differences endow H. pylori flagellins with low immune-stimulatory properties, we created several mutated H. pylori flagellins (FlaA and FlaB) by site-directed mutagenesis that contain one to four epitopes of Salmonella flagellin D0D1 domain amino acid sequences. The mutant flagellins expressed both in H. pylori and E. coli were used to determine their influence on TLR5-signaling mediators and cytokines, such as MAPkinases, (ERK, p38), NF-\&\#61547;B, IL-8, and MIP-3\&\#61537;. Salmonella FliC expressed in E. coli induced activation of p38, I\&\#61547;B\&\#61537; and NF-\&\#61547;B leading to IL-8 and MIP-3\&\#61537; production in gastric epithelial cells. However, none of the H. pylori flagellin mutants activated MAP kinases or induced those cytokines. In a co-immunoprecipitation assay none of the recombinant wild type or mutated H. pylori flagellins showed any direct physical interaction with TLR5, while Salmonella FliC significantly co-precipitated with TLR5. Interestingly, we found H. pylori flagellins bind to the surface of gastric epithelial cells like FliC, although they do not bind to or stimulate TLR5. Based on the physical interaction of H. pylori flagellins and FliC with human gastric epithelial cells, we further analyzed transcriptional regulation by H. pylori flagellin in these host cells using microarray analysis. The result showed that H. pylori flagellins modulate host cell gene expression, and many of the identified regulation events overlap with the genes regulated by FliC. These findings imply that H. pylori flagellins do play a role in gene regulation of host cells probably through still unknown factors or receptors, although they do not trigger TLR5-related signaling pathways. The results of our study suggest that, in addition to the low immune-stimulatory activity of H. pylori LPS, the evolutionary reduction in stimulating activity of H. pylori flagellins on the local innate immune responses in the stomach in vivo might be a further strategy of this chronic mucosal pathogen to evade and minimize deleterious host responses, thereby promoting life-long persistence in the host, and possibly contributing to cancerogenesis.},
  subject      = {Helicobacter pylori},
  language  = {en}
}