SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023164612> ?p ?o ?g. }

Showing items 1 to 100 of ±137 with 100 items per page.

W2023164612 endingPage "15397" @default.
W2023164612 startingPage "15390" @default.
W2023164612 abstract "Infiltration of neutrophils and monocytes into the gastric mucosa is a hallmark of chronic gastritis caused by Helicobacter pylori. Certain H. pylori strains nonopsonized stimulate neutrophils to production of reactive oxygen species causing oxidative damage of the gastric epithelium. Here, the contribution of some H. pylori virulence factors, the blood group antigen-binding adhesin BabA, the sialic acid-binding adhesin SabA, the neutrophil-activating protein HP-NAP, and the vacuolating cytotoxin VacA, to the activation of human neutrophils in terms of adherence, phagocytosis, and oxidative burst was investigated. Neutrophils were challenged with wild type bacteria and isogenic mutants lacking BabA, SabA, HP-NAP, or VacA. Mutant and wild type strains lacking SabA had no neutrophil-activating capacity, demonstrating that binding of H. pylori to sialylated neutrophil receptors plays a pivotal initial role in the adherence and phagocytosis of the bacteria and the induction of the oxidative burst. The link between receptor binding and oxidative burst involves a G-protein-linked signaling pathway and downstream activation of phosphatidylinositol 3-kinase as shown by experiments using signal transduction inhibitors. Collectively our data suggest that the sialic acid-binding SabA adhesin is a prerequisite for the nonopsonic activation of human neutrophils and, thus, is a virulence factor important for the pathogenesis of H. pylori infection. Infiltration of neutrophils and monocytes into the gastric mucosa is a hallmark of chronic gastritis caused by Helicobacter pylori. Certain H. pylori strains nonopsonized stimulate neutrophils to production of reactive oxygen species causing oxidative damage of the gastric epithelium. Here, the contribution of some H. pylori virulence factors, the blood group antigen-binding adhesin BabA, the sialic acid-binding adhesin SabA, the neutrophil-activating protein HP-NAP, and the vacuolating cytotoxin VacA, to the activation of human neutrophils in terms of adherence, phagocytosis, and oxidative burst was investigated. Neutrophils were challenged with wild type bacteria and isogenic mutants lacking BabA, SabA, HP-NAP, or VacA. Mutant and wild type strains lacking SabA had no neutrophil-activating capacity, demonstrating that binding of H. pylori to sialylated neutrophil receptors plays a pivotal initial role in the adherence and phagocytosis of the bacteria and the induction of the oxidative burst. The link between receptor binding and oxidative burst involves a G-protein-linked signaling pathway and downstream activation of phosphatidylinositol 3-kinase as shown by experiments using signal transduction inhibitors. Collectively our data suggest that the sialic acid-binding SabA adhesin is a prerequisite for the nonopsonic activation of human neutrophils and, thus, is a virulence factor important for the pathogenesis of H. pylori infection. Colonization of the human stomach with Helicobacter pylori is accompanied by chronic active gastritis, which may lead to peptic ulcer disease, atrophic gastritis, and gastric adenocarcinoma (1Blaser M.J. Sci. Am. 1996; 274: 104-107Crossref PubMed Scopus (94) Google Scholar). To date a number of H. pylori virulence factors have been identified. Among these are the urease, the blood group antigen-binding adhesin (BabA), 1The abbreviations used are: BabA, blood group antigen binding adhesin; CL, chemiluminescence; DPI, diphenyleneiodonium; HP-NAP, neutrophil-activating protein of H. pylori; SabA, sialic acid-binding adhesin; VacA, vacuolating cytotoxin of H. pylori; wt, wild type; PBS, phosphate-buffered saline; GM1, Galβ3GalNAcβ4(NeuAcα3)Galβ4-Glcβ1Cer. the cag pathogenicity island, the vacuolating cytotoxin (VacA), and the H. pylori neutrophil-activating protein (HP-NAP). Binding of the bacterium to fucosylated host cell receptors is mediated by the BabA adhesin, an outer membrane protein of H. pylori (2Ilver D. Arnqvist A. Ögren J. Frick I.M. Kersulyte D. Incecik E.T. Berg D.E. Covacci A. Engstrand L. Borén T. Science. 1998; 279: 373-377Crossref PubMed Scopus (996) Google Scholar). The cag pathogenicity island encodes a type IV secretion system that enables translocation of the CagA protein into host cells, where the protein becomes tyrosine-phosphorylated and subsequently activates a eukaryotic phosphatase leading to dephosphorylation of host cell proteins and morphological changes (3Stein M. Rappuoli R. Covacci A. Achtman M. Suerbaum S. Helicobacter pylori: Molecular and Cellular Biology. Horizon Scientific Press, Norfolk, UK2001: 227-244Google Scholar). The VacA toxin induces formation of large cytoplasmic vacuoles in eukaryotic cells and causes alterations of tight junctions (4Montecucco C. de Bernard M. Microbes Infect. 2003; 5: 715-721Crossref PubMed Scopus (95) Google Scholar). VacA also forms anion-selective channels, which may be blocked by chloride channel inhibitors (5Szabo I. Brutsche S. Tombola F. Moschioni M. Satin B. Telford J.L. Rappuoli R. Montecucco C. Papini E. Zoratti M. EMBO J. 1999; 18: 5517-5527Crossref PubMed Scopus (241) Google Scholar, 6Tombola F. Carlesso C. Szabo I. de Bernard M. Reyrat R.M. Telford J.L. Rappuoli R. Montecucco C. Papini E. Zoratti M. Biophys. J. 1999; 76: 1401-1409Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). HP-NAP promotes the adhesion of neutrophils to endothelial cells and the production of reactive oxygen radicals (4Montecucco C. de Bernard M. Microbes Infect. 2003; 5: 715-721Crossref PubMed Scopus (95) Google Scholar, 7Evans Jr., D.J. Evans D.G. Takemura T. Nakano H. Lampert H.C. Graham D.Y. Granger D.N. Kvietys P.R. Infect. Immun. 1995; 63: 2213-2220Crossref PubMed Google Scholar). A prominent feature of the H. pylori-induced gastritis is an infiltration of neutrophils into the gastric epithelium (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar). Neutrophils play a major role in epithelium injury, because these cells have direct toxic effects on the epithelial cells by releasing reactive oxygen and nitrogen species and proteases (9Balk S.C. Youn H.S. Chung M.H. Lee W.K. Cho M.J. Ko G.H. Park C.K. Kasai H. Rhee K.H. Cancer Res. 1996; 56: 1279-1282PubMed Google Scholar, 10Yoshikawa T. Naito Y. Free Radic. Res. 2000; 33: 785-794Crossref PubMed Scopus (68) Google Scholar). An additional virulence factor of H. pylori bacterial cells is thus the neutrophil-activating capacity, i.e. the ability of certain H. pylori strains to activate human neutrophils in the absence of opsonins (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar). Strains with neutrophil-activating capacity are significantly more often isolated from patients with peptic ulcer disease (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar, 11Rautelin H. Blomberg B. Järnerot G. Danielsson D. Scand. J. Gastroenterol. 1994; 29: 128-132Crossref PubMed Scopus (71) Google Scholar, 12Danielsson D. Farmery S.M. Blomberg B. Perry S. Rautelin H. Crabtree J.E. J. Clin. Pathol. 2000; 53: 318-321Crossref PubMed Scopus (16) Google Scholar). The factor(s) of H. pylori responsible for the activation of neutrophils are heat-labile and dependent on whole nondisintegrated organisms (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar). Recently, preincubation with sialylated oligosaccharides demonstrated that the nonopsonic H. pylori-induced activation of human neutrophils occurs by lectinophagocytosis, i.e. recognition of sialylated glycoconjugates on the neutrophil cell surface by a bacterial adhesin leads to the phagocytosis and the oxidative burst reactions (13Teneberg S. Jurstrand M. Karlsson K-A. Danielsson D. Glycobiology. 2000; 10: 1171-1181Crossref PubMed Scopus (30) Google Scholar). To date, two sialic acid-binding proteins of H. pylori have been characterized: the sialic acid-binding adhesin SabA and the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAc-binding neutrophil-activating protein HP-NAP (14Mahdavi J. Sondén B. Hurtig M. Olfat F.O. Forsberg L. Roche N. Ångström J. Larsson T. Teneberg S. Karlsson K-A. Altraja S. Wadström T. Kersulyte D. Berg D.E. Dubois A. Peterson C. Magnusson K.-E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarström L. Borén T. Science. 2002; 297: 573-578Crossref PubMed Scopus (722) Google Scholar, 15Teneberg S. Miller-Podraza H. Lampert H.C. Jr Evans Evans D.G. Danielsson D. Karlsson K.-A. J. Biol. Chem. 1997; 272: 19067-19071Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). SabA is the sole factor responsible for binding of H. pylori bacterial cells to gangliosides, as recently demonstrated by using knock-out mutant strains devoid of the SabA adhesin or HP-NAP (16Roche N. Ångström J. Hurtig M. Larsson T. Borén T. Teneberg S. Infect. Immun. 2004; 72: 1519-1529Crossref PubMed Scopus (49) Google Scholar). A number of sialylated H. pylori-binding glycosphingolipids have been identified: sialyl-α3-neolactotetraosylceramide, sialyl-α3-neolactohexaosylceramide, sialyl-α3-neolactooctaosylceramide, the VIM-2 ganglioside and sialyl-dimeric-Lex glycosphingolipid (14Mahdavi J. Sondén B. Hurtig M. Olfat F.O. Forsberg L. Roche N. Ångström J. Larsson T. Teneberg S. Karlsson K-A. Altraja S. Wadström T. Kersulyte D. Berg D.E. Dubois A. Peterson C. Magnusson K.-E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarström L. Borén T. Science. 2002; 297: 573-578Crossref PubMed Scopus (722) Google Scholar, 16Roche N. Ångström J. Hurtig M. Larsson T. Borén T. Teneberg S. Infect. Immun. 2004; 72: 1519-1529Crossref PubMed Scopus (49) Google Scholar, 17Miller-Podraza H. Abul Milh M. Teneberg S. Karlsson K-A. Infect. Immun. 1997; 65: 2480-2482Crossref PubMed Google Scholar, 18Roche N. Ångström J. Larsson T. Teneberg S. Glycobiology. 2001; 11: 935-944Crossref PubMed Scopus (13) Google Scholar). Several of these H. pylori-binding gangliosides are also present in human neutrophils (19Stroud M.R. Handa K. Salyan M.E.K. Ito K. Levery S.B. Hakomori S-i. Reinhold B.B. Reinhold V.N. Biochemistry. 1996; 35: 758-769Crossref PubMed Scopus (83) Google Scholar, 20Stroud M.R. Handa K. Salyan M.E.K. Ito K. Levery S.B. Hakomori S-i. Reinhold B.B. Reinhold V.N. Biochemistry. 1996; 35: 770-778Crossref PubMed Scopus (103) Google Scholar). In addition, some H. pylori strains bind to polyglycosylceramides and glycoproteins of human neutrophils in a sialic acid-dependent binding manner (21Miller-Podraza H. Bergström J. Teneberg S. Abul Milh M. Longard M. Olsson B.-M. Uggla L. Karlsson K.-A. Infect. Immun. 1999; 67: 6309-6313Crossref PubMed Google Scholar). In the present study the role of some H. pylori virulence factors in the nonopsonic H. pylori-induced activation of human neutrophils was investigated. Human neutrophils were challenged with wild type H. pylori strains and isogenic deletion mutant strains lacking HP-NAP, BabA, SabA, VacA, or the 37-kDa fragment of VacA, followed by chemiluminescence measurement of the superoxide anions produced by the neutrophils. The nonopsonic adherence to and phagocytosis by neutrophils of wild type and mutant bacterial cells were examined at various time intervals as the appearance of visible macroscopic aggregation/agglutination of neutrophils and by microscopy of acridine orange stained smears. In addition, the effects of signal transduction inhibitors on H. pylori-induced neutrophil activation were studied, to identify intracellular signaling pathways required for H. pylori-induced neutrophil oxidative burst. H. pylori Strains, Culture Conditions, and Labeling—Characteristics of the H. pylori strains are presented in Table I. Strain NCTC 11637 was obtained from the National Collection of Type Cultures, London, UK, strain C-7050 from Professor T. Kosunen, Helsinki, Finland, and strain CCUG 17874 from the Culture Collection University of Göteborg. Strain J99 and the construction of the J99/SabA- mutant sabA(JHP662)::cam were described by Mahdavi et al. (14Mahdavi J. Sondén B. Hurtig M. Olfat F.O. Forsberg L. Roche N. Ångström J. Larsson T. Teneberg S. Karlsson K-A. Altraja S. Wadström T. Kersulyte D. Berg D.E. Dubois A. Peterson C. Magnusson K.-E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarström L. Borén T. Science. 2002; 297: 573-578Crossref PubMed Scopus (722) Google Scholar). The J99/BabA- mutant (babA::cam) and the J99/BabA-SabA- mutant (babA::cam sabA::kan) were constructed as previously described (2Ilver D. Arnqvist A. Ögren J. Frick I.M. Kersulyte D. Incecik E.T. Berg D.E. Covacci A. Engstrand L. Borén T. Science. 1998; 279: 373-377Crossref PubMed Scopus (996) Google Scholar, 14Mahdavi J. Sondén B. Hurtig M. Olfat F.O. Forsberg L. Roche N. Ångström J. Larsson T. Teneberg S. Karlsson K-A. Altraja S. Wadström T. Kersulyte D. Berg D.E. Dubois A. Peterson C. Magnusson K.-E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarström L. Borén T. Science. 2002; 297: 573-578Crossref PubMed Scopus (722) Google Scholar).Table ISummary of results from glycosphingolipid binding assaysH. pylori strainCharacteristics of wild type (wt) strainsSialic acid-binding capacityNCTC 11637VacA+/cagA+/BabA+/SabA++C-7050VacA-/cagA+/BabA+/SabA--J99 wtVacA+/cagA+/BabA+/SabA++J99/NAP-+J99/BabA-+J99/SabA--J99/BabA-SabA--CCUG 17874 wtVacA+/cagA+/BabA+/SabA++17874/VacA-+17874/p37-+ Open table in a new tab For construction of the HP-NAP knock-out mutant, designated J99/NAP-(napA::kan), the napA gene was amplified by PCR using the napA1F (forward) and napA1R (reverse) primers. The PCR fragment was cloned into the pBluescriptSK± EcoRV site (Stratagene, La Jolla, CA). The resulting plasmid was linearized with primers napA2F (forward) and napA2R (reverse), and then ligated with the kanamycin resistance (KanR) cassette from pILL600 (22Suerbaum S. Josenhans C. Labigne A. J. Bacteriol. 1993; 175: 3278-3288Crossref PubMed Google Scholar). The plasmid carrying the deleted napA was used for transformation of the J99 strain. For transformation the bacteria were grown for 24 h on agar plates before addition of 2 μg of plasmid DNA. After transformation the bacteria were grown on nonselective plates for 48 h to allow for the expression of antibiotic resistance and then transferred onto kanamycin-containing plates. The transformants were analyzed by PCR using primers napA3F and napA4R which verified that the KanR cassette was inserted into napA. Western blot analysis of napA mutants using anti-HP-NAP antibodies showed that the mutant strain was devoid of HP-NAP expression. The oligonucleotides used for PCR were: napA1F, 5′-TCAAGCCATAGCGGATAAGCT-3′; napA1R, 5′-TTGATAATGGCAAGGAAGTGGA-3′; napA2F, 5′-CACGATCGCATCCGCTTGCA-3′; napA2R, 5′-TTACCGTAACTTATGCGGATGAT-3′; napA3F, 5′-TGGTGTAGGATAGCGATCAAG-3′; and napA4R, 5′-TAATGTCATTCCACTTGTCTAAG-3′. The VacA knock-out mutant (17874/VacA-) and the mutant with deletion of the 37 kDa fragment of VacA (17874/p37-) were constructed as previously described (23Reyrat J.M. Lanzavecchia S. Lupetti P. de Bernard M. Pagliaccia C. Pelicic V. Charrel M. Ulivieri C. Norais N. Ji X. Cabiaux V. Papini E. Rappuoli R. Telford J.L. J. Mol. Biol. 1999; 290: 459-470Crossref PubMed Scopus (70) Google Scholar). For chromatogram binding experiments the bacteria were grown in a microaerophilic atmosphere at 37 °C for 48 h on Brucella medium (Difco Laboratories, Irvine, CA) containing 10% fetal calf serum (Harlan Sera-Lab Loughborough, UK) inactivated at 56 °C, and 1% BBL™ IsoVitalex enrichment (BD France S.A., Le Pont de Claix, France). The mutant strains J99/SabA- and J99/BabA- were cultured on the same medium supplemented with chloramphenicol (20 μg/ml). For the mutant strain J99/SabA-BabA- supplementation with chloramphenicol (20 μg/ml) and kanamycin (25 μg/ml) was used, whereas the 17874/VacA- and 17874/p37- strains were cultured on the above described medium supplemented with kanamycin (20 μg/ml). Bacteria were radiolabeled by the addition of 50 μCi of [35S]methionine (Amersham Biosciences) diluted in 0.5 ml of phosphate-buffered saline (PBS), pH 7.3, to the culture plates. After incubation for 12-72 h at 37 °C under microaerophilic conditions, the bacteria were harvested, centrifuged three times, and thereafter suspended to 1 × 108 colony forming units/ml in PBS. The specific activities of the suspensions were ∼1 cpm per 100 bacterial cells. Alternatively, the strains were grown in a microaerophilic atmosphere at 37 °C for 48 h on GC agar plates (GC II agar base, BBL, Cockeysville, MD) supplemented with 1% bovine hemoglobin (BBL), 10% horse serum, and 1% IsoVitaleX enrichment (BBL), without antibiotics for the wild type strains, and with antibiotics as above for the mutant strains J99/SabA-, J99/BabA-, J99/BabA-SabA-, 17874/VacA-, and 17874/p37-. The H. pylori organisms were collected in PBS and used in chemiluminescence and phagocytosis experiments as described below. Chromatogram Binding Assay—Glycosphingolipids were isolated and characterized by mass spectrometry, 1H NMR, and degradation studies, as described (24Karlsson K-A. Methods Enzymol. 1987; 138: 212-220Crossref PubMed Scopus (185) Google Scholar). De-sialylation was done by incubating the glycosphingolipids in 1% acetic acid (by volume) at 100 °C for 1 h. Thin-layer chromatography was performed on glass- or aluminum-backed silica gel 60 HPTLC plates (Merck, Darmstadt, Germany). Mixtures of glycosphingolipids (40 μg) or pure compounds (1-4 μg) were separated using chloroform/methanol/water (60:35:8, by volume) as solvent system. Chemical detection was accomplished by anisaldehyde (25Waldi D. Stahl E. Dünnschicht-Chromatographie. Springer-Verlag, Berlin1962: 496-515Google Scholar). Binding of 35S-labeled H. pylori to glycosphingolipids on thin-layer chromatograms was done as previously reported (26Ångström J. Teneberg S. Abul Milh M. Larsson T. Leonardsson I. Olsson B.-M. Ölwegård Halvarsson M. Danielsson D. Näslund I. Ljungh Å. Wadström T. Karlsson K.-A. Glycobiology. 1998; 8: 297-309Crossref PubMed Scopus (103) Google Scholar). Dried chromatograms were dipped for 1 min in diethylether/n-hexane (1:5, by volume) containing 0.5% (w/v) polyisobutylmethacrylate (Aldrich). After drying, the chromatograms were soaked in PBS containing 2% bovine serum albumin (w/v), 0.1% NaN3 (w/v), and 0.1% Tween 20 (by volume) for 2 h at room temperature. The chromatograms were subsequently covered with radiolabeled bacteria diluted in PBS (2-5 × 106 cpm/ml). Incubation was done for 2 h at room temperature, followed by repeated washings with PBS. The chromatograms were thereafter exposed to XAR-5 x-ray films (Eastman Kodak, Rochester, NY) for 12 h. Extraction of Membrane Proteins from Human Neutrophil Granulocytes—Membranes from fresh neutrophils were isolated as described previously (27Moore K.L. Stults N.L. Diaz S. Smith D.F. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (422) Google Scholar). The outer membrane fragment fraction was dissolved in 25 mm Tris-HCl containing 2.5% SDS and 1 mm EDTA, pH 8.0, heated to 95 °C for 10 min and centrifuged at 10,000 × g for 10 min. Electrophoresis and Binding of H. pylori—SDS-PAGE and staining were carried out with NuPAGE™ gels (Novex, San Diego, CA). Briefly, neutrophil membrane proteins samples in SDS sample buffer, with 50 mm dithiothreitol added, were heated to 95 °C for 5 min, and applied on a homogeneous 10% polyacrylamide gel. After electrophoresis, the gels were either stained with Coomassie Blue or electroblotted to polyvinylidene difluoride (0.2-μm) membranes. The polyvinylidene difluoride membrane was incubated in blocking solution, 3% bovine serum albumin, 50 mm Tris-HCl, 200 mm NaCl, 0.1% NaN3, pH 8.0, for 1.5 h. The membrane was then incubated with 35S-labeled H. pylori strain CCUG 17874 diluted in PBS for 2 h at room temperature and thereafter washed in a solution of 50 mm Tris-HCl, 200 mm NaCl, and 0.05% Tween 20, pH 8.0. After drying at room temperature, the membrane was exposed to XAR-5 x-ray films overnight. Reference bovine fetuin and bovine asialofetuin were purchased from Sigma. Human Neutrophil Granulocytes—Heparinized blood from healthy blood donors was used to prepare neutrophils by Ficoll-Paque (Amersham Biosciences) centrifugation in accordance with the method of Böyum (28Böyum A. Tissue Antigens. 1974; 4: 269-274Crossref PubMed Scopus (565) Google Scholar), slightly modified as described (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar). For each series of experiments on a particular day neutrophils were prepared and pooled from three blood donors of the same blood group (A Rh+ or O Rh+). Neutrophils were thus obtained from different blood donors at each experiment. They were suspended in PBS supplemented with MgCl2, CaCl2, glucose, and gelatin as previously described (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar). The purity and viability of the neutrophils exceeded 95%. Chemiluminescence Experiments—The oxidative burst of neutrophils challenged with nonopsonized whole and live H. pylori organisms was measured with luminol enhanced chemiluminescence (CL) as previously described (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar). To each test tube (LKB, Bromma, Sweden) were added 300 μl of PBS supplemented with MgCl2 and CaCl2, 100 μl of neutrophils (5 × 106/ml), 50 μlof10-5m luminol (Sigma), and finally 50 μl of nonopsonized H. pylori (5 × 108/ml). For the signal transduction inhibition experiments neutrophils (5 × 106/ml) were treated with 800 ng of pertussis toxin for 60 or 120 min at 37 °C, with wortmannin (5, 10, or 20 nm) for 5 min at 37 °C, or diphenyleneiodonium chloride (DPI, 10 μm) for 5 min at 37 °C, centrifuged, and resuspended in PBS supplemented with MgCl2 and CaCl2 before they were challenged with H. pylori cells of strain NCTC 11637 as described above. Pertussis toxin, wortmannin, and DPI were purchased from Calbiochem. For oligosaccharide inhibition experiments 50 μl of H. pylori (5 × 108/ml) were mixed with 50 μl of 3′-sialyllactose (IsoSep, Tullinge, Sweden) to receive final concentrations of 0.1-1.0 mm in the CL, for 15 min at 37 °C, and 100 μl of the mixture was thereafter transferred to the test tube for CL measuring. The oxidative bursts of the neutrophils were measured as luminol-enhanced chemiluminescence with a luminometer (LKB Wallac 1251, Turku, Finland), and the measurements were always started within 1 min after the bacterial suspension had been added. The assays were performed at 37 °C, and CL from each sample was measured at 60- to 90-s intervals during a period of 30-60 min, and data were stored in a computer for computerized calculations. This technique thus measures both the external and internal oxidative bursts of the nonopsonic phagocytosis by neutrophils, which was previously checked by quenching the external burst in the presence of catalase (2000 units/ml), and the internal one in the presence of azide (1 mm) and horseradish peroxidase (4 units/ml) as described by Lock and Dahlgren (29Lock R. Dahlgren C. APMIS. 1988; 96: 299-305Crossref PubMed Scopus (26) Google Scholar). The H. pylori strains NCTC 11637 (giving a strong and rapid CL response) and C-7050 (inducing no CL response) (8Rautelin H. Blomberg B. Fredlund H. Järnerot G. Danielsson D. Gut. 1993; 34: 599-603Crossref PubMed Scopus (132) Google Scholar) were included in each series of experiments as positive and negative controls, respectively. Adherence, Phagocytosis, and Neutrophil Agglutination Assays—To each test tube were added 350 μl of PBS supplemented with MgCl2 and CaCl2, 100 μl of neutrophils (5 × 106/ml), and 50 μl of nonopsonized H. pylori organisms (5 × 108/ml). Adherence to and phagocytosis by neutrophils of wild type and mutant H. pylori strains were examined by microscopy (see below) at the various time intervals of <2-5, 20-30, and 60-90 min, and for the appearance of visible, i.e. macroscopic agglutination (aggregation) of neutrophils by ocular inspection of the tubes at the same time intervals. For microscopic examination of adherence/phagocytosis assays, and the formation of neutrophil agglutinates/aggregates by H. pylori cells, 10 μl of the H. pylori/neutrophil mixture was smeared on a glass slide within an area of ∼2.5-3 mm2, air-dried, fixed in cold methanol for 5 min, washed in distilled water, and then stained with acridine orange as described by the manufacturer. The slides were inspected with a Zeiss fluorescence microscope for incident light with appropriate filter combinations at a magnification of 400× to look for bacteria that had adhered to neutrophils and/or were phagocytosed. The results obtained by this technique corresponded well to previously reported findings by electron microscopy (30Rautelin H. von Bonsdorff C.H. Blomberg B. Danielsson D. J. Clin. Pathol. 1994; 47: 667-669Crossref PubMed Scopus (11) Google Scholar). However, even though adherence of acridine orange-stained bacteria of H. pylori to the neutrophil cell membrane looks different from those that are obviously inside the cell, phagocytosis of an individual bacterial cell cannot be definitely separated from adherence with this technique. Adherence/phagocytosis have therefore been taken together and were graded as negative (-) when neutrophils were evenly dispersed with only occasionally adhered H. pylori cells; minor (+) or moderate (++) adherence/phagocytosis with 5 < 10 or 10 ≤ 20 bacterial cells, respectively, per neutrophil in representative fields of view; and heavy (+++) adherence/phagocytosis with >20 bacterial cells per neutrophil. In the signal transduction inhibition experiments, neutrophils were treated with pertussis toxin, wortmannin, and DPI in concentrations given above, and adherence to and phagocytosis by neutrophils of strain NCTC 11637, as well as visible macroscopic agglutination were examined at the time intervals <2-5, 20-30, and 60-90 min, and compared with untreated neutrophils challenged with the same strain. Adherence, phagocytosis, and neutrophil agglutination were graded as described above. Binding of H. pylori to Human Neutrophil Gangliosides Is Lost after Deletion of the SabA Adhesin—The sialic acid binding capacities of the wild type and deletion mutant H. pylori strains utilized in this study were evaluated by binding of the bacteria to glycosphingolipids separated on thin-layer chromatograms. The results are exemplified in Fig. 1 and summarized in Table I. The criterion used for sialic acid recognition was binding to the acid glycosphingolipid fraction of human neutrophils (Fig. 1, lane 2) with no binding after de-sialylation (Fig. 1, lane 3). Thus, while both the parent strains and their mutants bound to the nonacid reference glycosphingolipid gangliotetraosylceramide (Fig. 1, lane 1), binding to human neutrophil gangliosides was observed for all strains except the J99/SabA- mutant (Fig. 1C), the J99/BabA-SabA- mutant (not shown), and the C-7050 strain (not shown). H. pylori Also Bind to Human Neutrophil Membrane Proteins—Binding of SabA-expressing H. pylori strain CCUG 17874 to human neutrophil membrane proteins is shown in Fig. 2B. As reported previously (21Miller-Podraza H. Bergström J. Teneberg S. Abul Milh M. Longard M. Olsson B.-M. Uggla L. Karlsson K.-A. Infect. Immun. 1999; 67: 6309-6313Crossref PubMed Google Scholar), the bacteria bound to several proteins with apparent relative molecular masses between 40 and 70 kDa. Binding of H. pylori to Sialic Acid-carrying Neutrophil Receptors Is Necessary for Induction of the Oxidative Burst—The neutrophil-activating abilities of the wild type and deletion mutant H. pylori strains were investigated by luminol-enhanced chemiluminescence. Challenge of human neutrophils with the wild type H. pylori strains J99, CCUG 17874, and NCTC 11637 resulted in strong CL responses (Figs. 3, 4, 5, 6), although there was some strain to strain variation in the ability to induce the oxidative burst manifested by differences in peak values (millivolts) and time to reach peak (minutes). In most cases a biphasic response was observed, where the initial phase is due to activation of the plasma membrane (extracellular) NADPH oxidase, whereas the second phase represents activation of both plasma membrane and the intracellular NADPH oxidases. The extracellular and the intracellular production of H2O2 are linked to two separate pools of NADPH oxidase localized to the plasma membrane and granule membranes, respectively (31Karlsson A. Nixon J.B. McPhail L.C. J. Leukoc. Biol. 2000; 67: 396-404Crossref PubMed Scopus (170) Google Scholar).Fig. 4Oxidative burst activation of human neutrophil granulocytes challenged with nonopsonized wild type H. pylori strain C-7050 (C-7050) and strain J99 (J99 wt) and its isogenic mutants with deletions of HPNAP (J99/NAP-), the BabA adhesin (J99/BabA-), the SabA adhesin (J99/SabA-), and both the BabA adhesin and the SabA adhesin (J99/BabA-SabA-). The luminol enhanced chemiluminescence assay was done as described under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Oxidative burst activation of human neutrophils challenged with nonopsonized wild type H. pylori strain CCUG 17874 (CCUG 17874 wt) and it" @default.
W2023164612 created "2016-06-24" @default.
W2023164612 creator A5010330990 @default.
W2023164612 creator A5017472767 @default.
W2023164612 creator A5029157137 @default.
W2023164612 creator A5056893119 @default.
W2023164612 creator A5069892567 @default.
W2023164612 creator A5072639853 @default.
W2023164612 creator A5080581751 @default.
W2023164612 date "2005-04-01" @default.
W2023164612 modified "2023-10-11" @default.
W2023164612 title "The Sialic Acid Binding SabA Adhesin of Helicobacter pylori Is Essential for Nonopsonic Activation of Human Neutrophils" @default.
W2023164612 cites W1022571150 @default.
W2023164612 cites W1488299617 @default.
W2023164612 cites W1513238427 @default.
W2023164612 cites W1563690189 @default.
W2023164612 cites W1572372318 @default.
W2023164612 cites W1596377804 @default.
W2023164612 cites W1747612761 @default.
W2023164612 cites W1763394000 @default.
W2023164612 cites W1887520422 @default.
W2023164612 cites W1890454441 @default.
W2023164612 cites W1956725963 @default.
W2023164612 cites W1960281573 @default.
W2023164612 cites W1965117674 @default.
W2023164612 cites W1967210581 @default.
W2023164612 cites W1969334359 @default.
W2023164612 cites W1970674113 @default.
W2023164612 cites W1972427798 @default.
W2023164612 cites W1979171683 @default.
W2023164612 cites W1985126981 @default.
W2023164612 cites W1988046165 @default.
W2023164612 cites W1988984821 @default.
W2023164612 cites W1991172560 @default.
W2023164612 cites W1995733381 @default.
W2023164612 cites W2030013787 @default.
W2023164612 cites W2031775276 @default.
W2023164612 cites W2034153873 @default.
W2023164612 cites W2036953184 @default.
W2023164612 cites W2039905354 @default.
W2023164612 cites W2047440121 @default.
W2023164612 cites W2054604776 @default.
W2023164612 cites W2081743213 @default.
W2023164612 cites W2092190463 @default.
W2023164612 cites W2094168277 @default.
W2023164612 cites W2097011626 @default.
W2023164612 cites W2106489698 @default.
W2023164612 cites W2114581080 @default.
W2023164612 cites W2114798556 @default.
W2023164612 cites W2114988680 @default.
W2023164612 cites W2118899705 @default.
W2023164612 cites W2119422159 @default.
W2023164612 cites W2122842217 @default.
W2023164612 cites W2124726027 @default.
W2023164612 cites W2125099471 @default.
W2023164612 cites W2134252021 @default.
W2023164612 cites W2137937616 @default.
W2023164612 cites W2140016136 @default.
W2023164612 cites W2159093835 @default.
W2023164612 cites W2160261472 @default.
W2023164612 cites W2160803838 @default.
W2023164612 cites W2164891827 @default.
W2023164612 cites W2165393039 @default.
W2023164612 cites W2165516134 @default.
W2023164612 cites W2338966357 @default.
W2023164612 cites W2418348042 @default.
W2023164612 cites W2462191213 @default.
W2023164612 cites W4238292561 @default.
W2023164612 cites W53883719 @default.
W2023164612 doi "https://doi.org/10.1074/jbc.m412725200" @default.
W2023164612 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15689619" @default.
W2023164612 hasPublicationYear "2005" @default.
W2023164612 type Work @default.
W2023164612 sameAs 2023164612 @default.
W2023164612 citedByCount "105" @default.
W2023164612 countsByYear W20231646122012 @default.
W2023164612 countsByYear W20231646122013 @default.
W2023164612 countsByYear W20231646122014 @default.
W2023164612 countsByYear W20231646122015 @default.
W2023164612 countsByYear W20231646122016 @default.
W2023164612 countsByYear W20231646122017 @default.
W2023164612 countsByYear W20231646122018 @default.
W2023164612 countsByYear W20231646122019 @default.
W2023164612 countsByYear W20231646122020 @default.
W2023164612 countsByYear W20231646122021 @default.
W2023164612 countsByYear W20231646122022 @default.
W2023164612 countsByYear W20231646122023 @default.
W2023164612 crossrefType "journal-article" @default.
W2023164612 hasAuthorship W2023164612A5010330990 @default.
W2023164612 hasAuthorship W2023164612A5017472767 @default.
W2023164612 hasAuthorship W2023164612A5029157137 @default.
W2023164612 hasAuthorship W2023164612A5056893119 @default.
W2023164612 hasAuthorship W2023164612A5069892567 @default.
W2023164612 hasAuthorship W2023164612A5072639853 @default.
W2023164612 hasAuthorship W2023164612A5080581751 @default.
W2023164612 hasBestOaLocation W20231646121 @default.
W2023164612 hasConcept C104317684 @default.
W2023164612 hasConcept C179470777 @default.