Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment

Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application

Mimotopes selected with a neutralizing antibody against urease B from Helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination

<p>Abstract</p> <p>Background</p> <p>Urease B is an important virulence factor that is required for <it>Helicobacter pylori </it>to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of <it>H. pylori </it>infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes <it>in vivo</it>.</p> <p>Results</p> <p>The urease B gene was obtained (GenBank accession No. <ext-link ext-link-id="DQ141576" ext-link-type="gen">DQ141576</ext-link>) and the recombinant pGEX-4T-1/UreaseB protein was expressed in <it>Escherichia coli </it>as a 92-kDa recombinant fusion protein with glutathione-S-transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced immune responses were antigen specific.</p> <p>Conclusions</p> <p>The present work demonstrated that phage-displayed mimotopes were accessible to the mouse immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising approach for the development of a vaccine for the diagnosis and treatment of <it>H. pylori </it>infection.</p

Immunoregulatory properties of Helicobacter pylori derived molecules

Helicobacter pylori (H. pylori) is a micro-aerophilic, spiral-shaped Gram-negative bacterium which colonises the human stomach of approximately 50% of the population worldwide (1). The infection is asymptomatic in the vast majority of cases, however, about 10-15% result in peptic ulcer disease and 1-2% result in gastric adenocarcinoma. H. pylori prevalence in developed countries is decreasing as the incidence of asthma and allergy is increasing. H. pylori infections are usually established in early childhood when the immune system is developing and at a common age for asthma onset. The infection induces cellular immune responses of types known to inhibit those that drive allergy. Previously, we showed that H. pylori secreted component stimulated CD4+CD25hi regulatory T cells (Tregs) expressing the anti-inflammatory cytokine interleukin-10 (IL-10) more strongly than whole cell lysate. SDS-PAGE was used to resolve the H. pylori and a whole-cell lysate to determine which proteins from the supernatant were enriched and Mass spectrometry (MALDI-TOF) was used to identify the major proteins by peptide mass fingerprinting (PMF). From the result of the MALDI-TOF, vacuolating cytotoxin A (VacA) catalase (KatA), γ- Glutamyl transpeptidase (GGT) and Peptidyl prolyl cis-trans isomerases (PPT) were selected based on the few pieces of evidence of their immunoregulatory abilities, their solubility and potential to be cloned and purified in the past. The main aim of this study is to investigate candidate H. pylori protein factors that are involved in the induction of the immunoregulatory response and how these effects could be utilised in the treatment of allergy and autoimmune response. Four H. pylori candidate proteins (VacA, KatA, GGT and PPT), previously selected on the basis of their immunoregulatory potential, were used in in vitro, in vivo or ex vivo investigations. Three of these candidate proteins factors, KatA, GGT and PPT were first investigated in vivo. To do this the genes for each H. pylori candidate protein were cloned and expressed in ClearColi® BL21(DE3) E. coli to minimise effects from LPS. The proteins were purified and characterised. LPS content in the recombinant proteins was assayed, using an E-TOXATE assay, and shown to be <0.1 EU/ml. Jurkat T-cells, THP-1 monocytic cells and AGS epithelial were incubated for 1 hour with 10, 25 and 50 µg/ml of the recombinant proteins, prior to activation with PMA/Ionomycin, LPS or TNF-. After 24 hours of treatment IL-2 (Jurkat cells), IL-6 (THP-1) and IL-8 concentrations were quantified by ELISA. All three proteins induced a dose-dependent reduction in cytokine production, compared to controls treated only with PMA/Ionomycin, LPS or TNF-. KatA most strongly suppressed IL-2 secretion by Jurkat cells (79.5% reduction with 50 g/ml, p<0.05), whereas GGT was most effective in suppressing IL-6 from THP-1 cells (68.07% reduction with 50 µg/ml, p<0.05), there were no changes in the IL-8 production in AGS. There was no accompanying decrease in cell viability. Likewise, VacA was investigated in vivo and ex vivo to compare the Treg response induced in vivo, by H. pylori mutants expressing different forms of VacA. Groups of 18 female C57BL/6 mice were infected orally with isogenic H. pylori SS1 mutants expressing the s1/i1 or s2/i2 form of VacA. A control group received plain Brucella broth as a placebo. Mice were killed at 3-, 6- and 9-weeks post-infection and their infection status confirmed. Spleen cells were isolated, stimulated with mitogens for 6 hours and stained with fluorochrome-conjugated antibodies. Treg populations were quantified by flow cytometry. Treg cells were purified and assayed for suppressive functional activity in vitro. Mitogen stimulation resulted in significantly increased frequencies of IL-10+ Tregs at all time-points and all groups (p<0.001). No statistically significant differences were found in frequencies of IL-10+ Tregs between the groups. There were also no differences in the functional suppressive activity of purified Tregs. Despite previously finding markedly increased Treg populations in peripheral blood from infected patients, we were unable to find increased Treg numbers in the spleen of infected mice. It is recommended that further investigation frequencies of Tregs in the gastric mucosa of the mice should be studied. H. pylori infection exerts immunomodulation, through the Treg induction notwithstanding some of the protein factors such as GGT and KatA also have a direct immune regulatory effect on the immune and could be harnessed for the development of anti-inflammatory therapy

Vaccine Development

Vaccination is the most effective and scientifically based means of protection against infectious diseases, especially in this era of the COVID-19 pandemic. This book examines several issues related to the development of vaccines against viral, bacterial, and parasitic infections

Immunoregulatory properties of Helicobacter pylori derived molecules

Helicobacter pylori (H. pylori) is a micro-aerophilic, spiral-shaped Gram-negative bacterium which colonises the human stomach of approximately 50% of the population worldwide (1). The infection is asymptomatic in the vast majority of cases, however, about 10-15% result in peptic ulcer disease and 1-2% result in gastric adenocarcinoma. H. pylori prevalence in developed countries is decreasing as the incidence of asthma and allergy is increasing. H. pylori infections are usually established in early childhood when the immune system is developing and at a common age for asthma onset. The infection induces cellular immune responses of types known to inhibit those that drive allergy. Previously, we showed that H. pylori secreted component stimulated CD4+CD25hi regulatory T cells (Tregs) expressing the anti-inflammatory cytokine interleukin-10 (IL-10) more strongly than whole cell lysate. SDS-PAGE was used to resolve the H. pylori and a whole-cell lysate to determine which proteins from the supernatant were enriched and Mass spectrometry (MALDI-TOF) was used to identify the major proteins by peptide mass fingerprinting (PMF). From the result of the MALDI-TOF, vacuolating cytotoxin A (VacA) catalase (KatA), γ- Glutamyl transpeptidase (GGT) and Peptidyl prolyl cis-trans isomerases (PPT) were selected based on the few pieces of evidence of their immunoregulatory abilities, their solubility and potential to be cloned and purified in the past. The main aim of this study is to investigate candidate H. pylori protein factors that are involved in the induction of the immunoregulatory response and how these effects could be utilised in the treatment of allergy and autoimmune response. Four H. pylori candidate proteins (VacA, KatA, GGT and PPT), previously selected on the basis of their immunoregulatory potential, were used in in vitro, in vivo or ex vivo investigations. Three of these candidate proteins factors, KatA, GGT and PPT were first investigated in vivo. To do this the genes for each H. pylori candidate protein were cloned and expressed in ClearColi® BL21(DE3) E. coli to minimise effects from LPS. The proteins were purified and characterised. LPS content in the recombinant proteins was assayed, using an E-TOXATE assay, and shown to be <0.1 EU/ml. Jurkat T-cells, THP-1 monocytic cells and AGS epithelial were incubated for 1 hour with 10, 25 and 50 µg/ml of the recombinant proteins, prior to activation with PMA/Ionomycin, LPS or TNF-. After 24 hours of treatment IL-2 (Jurkat cells), IL-6 (THP-1) and IL-8 concentrations were quantified by ELISA. All three proteins induced a dose-dependent reduction in cytokine production, compared to controls treated only with PMA/Ionomycin, LPS or TNF-. KatA most strongly suppressed IL-2 secretion by Jurkat cells (79.5% reduction with 50 g/ml, p<0.05), whereas GGT was most effective in suppressing IL-6 from THP-1 cells (68.07% reduction with 50 µg/ml, p<0.05), there were no changes in the IL-8 production in AGS. There was no accompanying decrease in cell viability. Likewise, VacA was investigated in vivo and ex vivo to compare the Treg response induced in vivo, by H. pylori mutants expressing different forms of VacA. Groups of 18 female C57BL/6 mice were infected orally with isogenic H. pylori SS1 mutants expressing the s1/i1 or s2/i2 form of VacA. A control group received plain Brucella broth as a placebo. Mice were killed at 3-, 6- and 9-weeks post-infection and their infection status confirmed. Spleen cells were isolated, stimulated with mitogens for 6 hours and stained with fluorochrome-conjugated antibodies. Treg populations were quantified by flow cytometry. Treg cells were purified and assayed for suppressive functional activity in vitro. Mitogen stimulation resulted in significantly increased frequencies of IL-10+ Tregs at all time-points and all groups (p<0.001). No statistically significant differences were found in frequencies of IL-10+ Tregs between the groups. There were also no differences in the functional suppressive activity of purified Tregs. Despite previously finding markedly increased Treg populations in peripheral blood from infected patients, we were unable to find increased Treg numbers in the spleen of infected mice. It is recommended that further investigation frequencies of Tregs in the gastric mucosa of the mice should be studied. H. pylori infection exerts immunomodulation, through the Treg induction notwithstanding some of the protein factors such as GGT and KatA also have a direct immune regulatory effect on the immune and could be harnessed for the development of anti-inflammatory therapy

CHARACTERISATION OF ¿-GLUTAMYL TRANSPEPTIDASE AND ELUCIDATING ITS ROLES IN THE PATHOGENESIS OF HELICOBACTER PYLORI

Ph.DDOCTOR OF PHILOSOPH

Targeted identification of glycosylated proteins in the gastric pathogen helicobacter pylori (Hp)

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen\u27s ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host\u27s stomach. Although Hp\u27s glycans are linked to pathogenesis, Hp\u27s glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp\u27s glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp\u27s azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Interaction of Helicobacter pylori with glycosylated salivary proteins

Since the discovery of Helicobacter pylori (H. pylori) in 1983 enormous progress has been made in determining the pathogenesis of this microbe in gastric disease. While the way of transmission is still under dispute, it is generally accepted that H. pylori must reach the stomach via the oral cavity. During this passage it comes into contact with salivary components. However, there are only few studies about interactions of H. pylori with salivary components and no study about the influence of saliva on H. pylori exists so far. The immediate aim of this thesis was to search for possible interactions of H. pylori with glycosylated salivary proteins, by developing an appropriate methodological approach and to characterize the molecular basis of such interactions. In order to utilize two-dimensional gel electrophoresis (2-DE) and subsequent transfer of separated proteins onto nitrocellulose membrane (blotting) for a high resolution bacterial overlay, a proteome analysis of human whole saliva (WS), parotid, and submandibular-sublingual (SMSL) secretions was performed in the first part of this thesis. Salivary secretions were subjected to 2-DE and spots were analyzed by matrix-assisted laser desorption/ionization-MS. Altogether 131 protein spots were identified in WS, 53 spots were identified in SMSL secretion and 43 spots in the parotid secretion. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic proline-rich proteins (PRPs), the proline-rich glycoprotein and mucin MUC7 could be mapped for the first time in a 2-D gel of human saliva. An intra-individual comparison (comparison of three different collection days) and an inter-individual comparison (comparison of four different individuals) of spot pattern showed moreover a sufficient reproducibility of 2-D salivary proteom maps for subsequent adhesion studies. Second part of the thesis was the characterization of the H. pylori wild-type strain J99A and isogenetic mutants, lacking either the BabA- and/or SabA-adhesins. This was performed by using a bacterial overlay technique with fluorescence-labeled bacteria on immobilized (neo)glycoproteins. Interaction between the adhesin BabA and the H-1-, Lewis b- and related fucose-containing antigens could be confirmed. The previously described interaction of H. pylori with terminal alpha-2,3-linked sialic acids could be shown too. The use of a sabA mutant and sialidase treatment of glycoconjugate arrays showed for the first time that the adherence of H. pylori to laminin is mediated by the sialic acid-binding adhesin, SabA. In addition, it could be shown that the adhesion to salivary mucin MUC5B is mainly associated with the BabA adhesin, and to a lesser extent with the SabA adhesin. It turned out that the adhesion of H. pylori to fibronectin and lactoferrin persisted in the babA/sabA double mutant. This binding could be abolished by denaturation but not by deglycosylation. Therefore, it was suggested that this interaction may depend on the recognition of unknown receptor moieties by one or more additional unknown bacterial surface components. The third and final part of the thesis was the performance of adhesion studies on human saliva. The characterized H. pylori mutants and the J99 wild-type were applied to blots of 1-D and 2-D gels of human saliva by bacterial overlay. Three receptor molecules of H. pylori detected by 1-D overlay could successfully be identified by MALDI-MS, confirming the binding of H. pylori to MUC5B, MUC7 and gp-340. With the help of the mutants also the responsible adhesins for binding could be determined. The 2-D overlay revealed novel salivary receptors for H. pylori. Identification of these receptors was achieved by comparison of the overlay membrane with the established proteome maps of human saliva. Binding of H. pylori to the proline-rich glycoprotein was detected for the first time and assigned to the activity of the BabA adhesin. The SabA adhesin was found responsible for binding to other newly detected receptor molecules, including carbonic anhydrase VI, secretory component (poly-Ig-receptor), parotid secretory protein and zinc-alpha-2-glycoprotein. In conclusion, this thesis combined for the first time successfully a proteomics approach with the bacterial overlay technique. This method showed not only the binding of H. pylori to salivary proteins but facilitated the identification of respective receptor molecules considerably. Because this technique is also suitable for other bacteria a widely applicable tool for studying adhesion of bacteria was developed