scholarly journals Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

2015 ◽  
Vol 197 (11) ◽  
pp. 1921-1930 ◽  
Author(s):  
Jennifer Tsang ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Basal Body ◽  
Protein Interactions ◽  
Transcript Levels ◽  
Content Type ◽  
Genes Encoding ◽  
H Pylori ◽  
Flagellar Biogenesis ◽  
Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

scholarly journals Helicobacter pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity

2015 ◽  
Vol 197 (11) ◽  
pp. 1886-1892 ◽  
Author(s):  
Jennifer Tsang ◽  
Takanori Hirano ◽  
Timothy R. Hoover ◽  
Jonathan L. McMurry
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Kinase Domain ◽  
Optical Biosensing ◽  
Content Type ◽  
High Affinity ◽  
Flagellar Assembly ◽  
Flagellar Biogenesis

ABSTRACTFlagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. InHelicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes,flaBandflgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). TheKD(equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon= 2.9 × 104M−1s−1) and slow-off (koff= 6.2 × 10−4s−1) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANTsequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANTbinding did not stimulate FlgS autophosphorylationin vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.IMPORTANCEThe high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.

scholarly journals Helicobacter pylori-Induced Disruption of Monolayer Permeability and Proinflammatory Cytokine Secretion in Polarized Human Gastric Epithelial Cells

2013 ◽  
Vol 81 (3) ◽  
pp. 876-883 ◽  
Author(s):  
Maria Fiorentino ◽  
Hua Ding ◽  
Thomas G. Blanchard ◽  
Steven J. Czinn ◽  
Marcelo B. Sztein ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Tight Junction ◽  
Proinflammatory Cytokines ◽  
Barrier Function ◽  
Cytokine Secretion ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
Monolayer Permeability ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection of the stomach is related to the development of diverse gastric pathologies. The ability ofH. pylorito compromise epithelial junctional complexes and to induce proinflammatory cytokines is believed to contribute to pathogenesis. The purpose of this study was to use anin vitrohuman gastric epithelial model to investigate the ability ofH. pylorito affect permeability and the extent and polarity of the host inflammatory response. NCI-N87 monolayers were cocultured with live or heat-killedH. pylorior culture supernatants. Epithelial barrier function was measured by transepithelial electric resistance (TEER) analysis, diffusion of fluorescein isothiocyanate (FITC)-labeled markers, and immunostaining for tight junction proteins. Supernatants from both apical and basolateral chambers were tested for cytokine production by multiplex analysis.H. pyloricaused a significant decrease in TEER, an increased passage of markers through the infected monolayer, and severe disruption and mislocalization of ZO-1 and claudin-1 proteins. Cell viability was not altered byH. pylori, indicating that loss of barrier function could be attributed to a breakdown of tight junction integrity. Significantly high levels of cytokine secretion were induced by either viable or heat-killedH. pylori.H. pyloriaffects monolayer permeability of polarized human gastric epithelial cells. Proinflammatory cytokines were secreted in a polarized manner, mostly basolaterally. Live bacteria are required for disruption of tight junctions but not for the induction of cytokine secretion. The NCI-N87 cell line provides an excellent model for thein vitrostudy ofH. pyloripathogenesis and the epithelial cell host response to infection.

scholarly journals A Genome-WideHelicobacter pyloriMorphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Desirée C. Yang ◽  
Kris M. Blair ◽  
Jennifer A. Taylor ◽  
Timothy W. Petersen ◽  
Tate Sessler ◽  
...  
Keyword(s):  
Cell Wall ◽  
Helicobacter Pylori ◽  
Light Scattering ◽  
Protein Interactions ◽  
Cell Morphology ◽  
Cell Shape ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Content Type ◽  
H Pylori

ABSTRACTEvident in its name, the gastric pathogenHelicobacter pylorihas a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations ofH. pyloricell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, bothcsd2andcsd7mutants show the same enhancement of PG tetra-pentapeptide cross-linking ascsd1mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable inccmAmutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modifyH. pylori’s cell morphology.IMPORTANCEThe stomach ulcer and cancer-causing pathogenHelicobacter pylorihas a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.

scholarly journals Helicobacter pylori Flagellar Hook-Filament Transition Is Controlled by a FliK Functional Homolog Encoded by the Gene HP0906

2005 ◽  
Vol 187 (16) ◽  
pp. 5742-5750 ◽  
Author(s):  
Kieran A. Ryan ◽  
Najma Karim ◽  
Mulugeta Worku ◽  
Charles W. Penn ◽  
Paul W. O'Toole
Keyword(s):  
Helicobacter Pylori ◽  
Enteric Bacteria ◽  
Hook Length ◽  
Chloramphenicol Resistance ◽  
Functional Homolog ◽  
Flagellar Proteins ◽  
H Pylori ◽  
Flagellar Biogenesis ◽  
Control Protein ◽  
Flagellar Genes

ABSTRACT Helicobacter pylori is a human gastric pathogen which is dependent on motility for infection. The H. pylori genome encodes a near-complete complement of flagellar proteins compared to model enteric bacteria. One of the few flagellar genes not annotated in H. pylori is that encoding FliK, a hook length control protein whose absence leads to a polyhook phenotype in Salmonella enterica. We investigated the role of the H. pylori gene HP0906 in flagellar biogenesis because of linkage to other flagellar genes, because of its transcriptional regulation pattern, and because of the properties of an ortholog in Campylobacter jejuni (N. Kamal and C. W. Penn, unpublished data). A nonpolar mutation of HP0906 in strain CCUG 17874 was generated by insertion of a chloramphenicol resistance marker. Cells of the mutant were almost completely nonmotile but produced sheathed, undulating polyhook structures at the cell pole. Expression of HP0906 in a Salmonella fliK mutant restored motility, confirming that HP0906 is the H. pylori fliK gene. Mutation of HP0906 caused a dramatic reduction in H. pylori flagellin protein production and a significant increase in production of the hook protein FlgE. The HP0906 mutant showed increased transcription of the flgE and flaB genes relative to the wild type, down-regulation of flaA transcription, and no significant change in transcription of the flagellar intermediate class genes flgM, fliD, and flhA. We conclude that the H. pylori HP0906 gene product is the hook length control protein FliK and that its function is required for turning off the σ54 regulon during progression of the flagellar gene expression cascade.

scholarly journals In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Naturally Occurring Tricyclic Sesquiterpene, against Helicobacter pylori Infection

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Y. F. Xu ◽  
D. W. Lian ◽  
Y. Q. Chen ◽  
Y. F. Cai ◽  
Y. F. Zheng ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Resistance ◽  
Clinical Isolates ◽  
Potential Candidate ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Patchouli Alcohol ◽  
H Pylori

ABSTRACT This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activities and potential underlying mechanism of patchouli alcohol (PA), a tricyclic sesquiterpene. In the in vitro assay, the capacities of PA to inhibit and kill H. pylori were tested on three standard strains at different pH values and on 12 clinical isolates. The effects of PA on H. pylori adhesion (and its alpA, alpB, and babA genes), motility (and its flaA and flaB genes), ultrastructure, and flagellation were investigated. Moreover, the H. pylori resistance to and postantibiotic effect (PAE) of PA were determined. Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were examined. Results showed that MICs of PA against three standard strains (pH 5.3 to 9) and 12 clinical isolates were 25 to 75 and 12.5 to 50 μg/ml, respectively. The killing kinetics of PA were time and concentration dependent, and its minimal bactericidal concentrations (MBCs) were 25 to 75 μg/ml. In addition, H. pylori adhesion, motility, ultrastructure, and flagellation were significantly suppressed. PA also remarkably inhibited the expression of adhesion genes (alpA and alpB) and motility genes (flaA and flaB). Furthermore, PA treatment caused a longer PAE and less bacterial resistance than clarithromycin and metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, inhibit gastritis, and suppress the expression of inflammatory mediators (COX-2, interleukin 1β, tumor necrosis factor alpha, and inducible nitric oxide synthase [iNOS]). In conclusion, PA can efficiently kill H. pylori, interfere with its infection process, and attenuate gastritis with less bacterial resistance, making it a potential candidate for new drug development.

scholarly journals Two Predicted Chemoreceptors of Helicobacter pylori Promote Stomach Infection

2002 ◽  
Vol 70 (10) ◽  
pp. 5877-5881 ◽  
Author(s):  
Tessa M. Andermann ◽  
Yu-Ting Chen ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Growth Defect ◽  
In Vitro Growth ◽  
Wild Type ◽  
Encoded Proteins ◽  
Genes Encoding ◽  
H Pylori

ABSTRACT Helicobacter pylori must be motile or display chemotaxis to be able to fully infect mammals, but it is not known how this chemotaxis is directed. We disrupted two genes encoding predicted chemoreceptors, tlpA and tlpC. H. pylori mutants lacking either of these genes are fully motile and chemotactic in vitro and are as able as the wild type to infect mice when they are the sole infecting strains. In contrast, when mice are coinfected with the H. pylori SS1 tlpA or tlpC mutant and the wild type, we find more wild type than mutant after 2 weeks of colonization. Neither strain has an in vitro growth defect. These results suggest that the tlpA- and tlpC-encoded proteins assist colonization of the stomach environment.

scholarly journals Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori

2008 ◽  
Vol 74 (7) ◽  
pp. 2095-2102 ◽  
Author(s):  
Ivo G. Boneca ◽  
Chantal Ecobichon ◽  
Catherine Chaput ◽  
Aurélie Mathieu ◽  
Stéphanie Guadagnini ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Shuttle Vector ◽  
Expression System ◽  
Gene Promoter ◽  
Start Codon ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Kanamycin Cassette ◽  
H Pylori

ABSTRACT The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

scholarly journals Hydrogen Peroxide-Mediated Oxygen Enrichment Eradicates Helicobacter pylori In Vitro and In Vivo

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Jia Di ◽  
Jun Zhang ◽  
Lei Cao ◽  
Ting-ting Huang ◽  
Jun-xia Zhang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Helicobacter Pylori ◽  
Bacterial Cell ◽  
Cell Membranes ◽  
Enriched Environment ◽  
Mongolian Gerbils ◽  
Content Type ◽  
H Pylori

ABSTRACT Helicobacter pylori is an important risk factor for gastric ulcers. However, antibacterial therapies increase the resistance rate and decrease the eradication rate of H. pylori. Inspired by the microaerophilic characteristics of H. pylori, we aimed at effectively establishing an oxygen-enriched environment to eradicate and prevent the recurrence of H. pylori. The effect and the mechanism of an oxygen-enriched environment in eradicating H. pylori and preventing the recurrence were explored in vitro and in vivo. During oral administration and after drug withdrawal, H. pylori counts were evaluated by Giemsa staining in animal cohorts. An oxygen-enriched environment in which H. pylori could not survive was successfully established by adding hydrogen peroxide into several solutions and rabbit gastric juice. Hydrogen peroxide effectively killed H. pylori in Columbia blood agar and special peptone broth. Minimum inhibition concentrations and minimum bactericidal concentrations of hydrogen peroxide were both relatively stable after promotion of resistance for 30 generations, indicating that hydrogen peroxide did not easily promote resistance in H. pylori. In models of Mongolian gerbils and Kunming mice, hydrogen peroxide has been shown to significantly eradicate and effectively prevent the recurrence of H. pylori without toxicity and damage to the gastric mucosa. The mechanism of hydrogen peroxide causing H. pylori death was related to the disruption of bacterial cell membranes. The oxygen-enriched environment achieved by hydrogen peroxide eradicates and prevents the recurrence of H. pylori by damaging bacterial cell membranes. Hydrogen peroxide thus provides an attractive candidate for anti-H. pylori treatment.

scholarly journals Helicobacter pylori Eradication by Sitafloxacin-Lansoprazole Combination and Sitafloxacin Pharmacokinetics in Mongolian Gerbils and ItsIn VitroActivity and Resistance Development

2011 ◽  
Vol 55 (9) ◽  
pp. 4261-4266 ◽  
Author(s):  
Tatsuo Yamamoto ◽  
Tomomi Takano ◽  
Wataru Higuchi ◽  
Akihito Nishiyama ◽  
Ikue Taneike ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antimicrobial Agents ◽  
Therapeutic Effects ◽  
Mongolian Gerbils ◽  
Maximum Serum ◽  
Resistance Development ◽  
Content Type ◽  
Maximum Serum Concentration ◽  
H Pylori

ABSTRACTA total of 293 strains ofHelicobacter pylori, including strains resistant to levofloxacin, clarithromycin, metronidazole, or amoxicillin, were examined forin vitrosusceptibility to 10 antimicrobial agents. Among these agents, sitafloxacin (a fluoroquinolone) showed the greatest activity (MIC90, 0.06 μg/ml), with high bactericidal activity and synergy in sitafloxacin-lansoprazole (a proton pump inhibitor) combination. In a Mongolian gerbil model with aH. pyloriATCC 43504 challenge, marked eradication effects were observed at ≥1 mg/kg for sitafloxacin, ≥10 mg/kg for levofloxacin, and ≥10 mg/kg for lansoprazole, reflecting MIC levels for each agent (0.008, 0.25, and 2 μg/ml, respectively). The therapeutic rates were 83.3% for the sitafloxacin (0.3 mg/kg)-lansoprazole (2.5 mg/kg) combination and 0% for either sitafloxacin or lansoprazole alone. The maximum serum concentration (Cmax) of sitafloxacin was 0.080 ± 0.054 μg/ml at 30 min, when orally administered at 1 mg/kg. The simultaneous administration of lansoprazole resulted in no difference. In the resistance development assay, MICs of levofloxacin increased 64- to 256-fold withgyrAmutations (Ala88Pro and Asn87Lys), while MICs of sitafloxacin only up to 16-fold with the Asn87Lys mutation. The data suggest that sitafloxacin exhibited superior anti-H. pyloriactivity with low rates of resistance developmentin vitroand that, reflecting highin vitroactivities, sitafloxacin-lansoprazole combination exhibited strong therapeutic effects in Mongolian gerbils with aCmaxof sitafloxacin that was 10-fold higher than the MIC value at a 1-mg/kg administration.

scholarly journals In VivoExpression of Helicobacter pylori Virulence Genes in Patients with Gastritis, Ulcer, and Gastric Cancer

2011 ◽  
Vol 80 (2) ◽  
pp. 594-601 ◽  
Author(s):  
Francisco Avilés-Jiménez ◽  
Adriana Reyes-Leon ◽  
Erik Nieto-Patlán ◽  
Lori M. Hansen ◽  
Juan Burgueño ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Environmental Conditions ◽  
Virulence Genes ◽  
Content Type ◽  
Ulcer Disease ◽  
Type Iv ◽  
H Pylori

ABSTRACTThe best-studiedHelicobacter pylorivirulence factor associated with development of peptic ulcer disease or gastric cancer (GC) rather than asymptomatic nonatrophic gastritis (NAG) is thecagpathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host epithelial cells. Here we used real-time reverse transcription-PCR (RT-PCR) to measure thein vivoexpression of genes on thecagPAI and of other virulence genes in patients with NAG, duodenal ulcer (DU), or GC.In vivoexpression ofH. pylorivirulence genes was greater overall in gastric biopsy specimens of patients with GC than in those of patients with NAG or DU. However, sincein vitroexpression ofcagAwas not greater inH. pyloristrains from patients with GC than in those from patients with NAG or DU, increased expression in GCin vivois likely a result of environmental conditions in the gastric mucosa, though it may in turn cause more severe pathology. Increased expression of virulence genes in GC may represent a stress response to elevated pH or other environmental conditions in the stomach of patients with GC, which may be less hospitable toH. pyloricolonization than the acidic environment in patients with NAG or DU.

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