Confirmation of adherence Protein Hemagglutinin sub-unit Pili with MW 49.6 kDa Helicobacter pylori on Mice Gastric Epithelial Cell (original) (raw)
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Characterization of a Helicobacter pylori neutrophil-activating protein
Infection and Immunity, 1995
Helicobacter pylori-associated gastritis is mainly an inflammatory cell response. In earlier work we showed that activation of human neutrophils by a cell-free water extract of H. pylori is characterized by increased expression of neutrophil CD11b/CD18 and increased adhesiveness to endothelial cells. The work reported here indicates that the neutrophil-activating factor is a 150,000-molecular-weight protein (150K protein). Neutrophil proadhesive activity copurified with this protein, which is a polymer of identical 15K subunits. Specific antibody, prepared against the purified 15K subunit, neutralized the proadhesive activity of the pure protein and of water extracts obtained from different strains of H. pylori. The gene (napA) for this protein (termed HP-NAP, for H. pylori neutrophil-activating protein) was detected, by PCR amplification, in all of the H. pylori isolates tested; however, there was considerable strain variation in the level of expression of HP-NAP activity in vitro....
Antigenic proteins of Helicobacter pylori of potential diagnostic value
Asian Pacific journal of cancer prevention : APJCP, 2013
Helicobacter pylori antigen was prepared from an isolate from a patient with a duodenal ulcer. Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcers, gastric ulcers and gastritis (n=30). As controls, three kinds of sera without detectable H. pylori IgG antibodies were used: 30 from healthy individuals without history of gastric disorders, 30 from patients who were seen in the endoscopy clinic but were H. pylori culture negative and 30 from people with other diseases. OFF-GEL electrophoresis, SDS-PAGE and Western blots of individual serum samples were used to identify protein bands with good sensitivity and specificity when probed with the above sera and HRP-conjugated anti-human IgG. Four H. pylori protein bands showed good (≥ 70%) sensitivity and high specificity (98-100%) towards anti-Helicobacter IgG antibody in culture- positive patients sera and control sera, respectively. The identities of the antigenic proteins were elucidated by m...
H. pylori Virulence Factors: Influence on Immune System and Pathology
Mediators of Inflammation, 2014
Helicobacter pylori is the most widespread chronic bacterial agent in humans and is well recognized for its association with ulcer disease and gastric cancer, with both representing major global health and socioeconomic issues. Given the high level of adaptation and the coevolution of this bacterium with its human host, a thorough and multidirectional view of the specific microbiological characteristics of this infection as well as the host physiology is needed in order to develop novel means of prevention of therapy. This review aims to pinpoint some of these potentially important angles, which have to be considered mutually when studying H. pylori's pathogenicity. The host's biological changes due to the virulence factors are a valuable pillar of H. pylori research as are the mechanisms by which bacteria provoke these changes. In this context, necessary adhesion molecules and significant virulence factors of H. pylori are discussed. Moreover, metabolism of the bacteria, one of the most important aspects for a better understanding of bacterial physiology and consequently possible therapeutic and prophylactic strategies, is addressed. On the other hand, we discuss the recent experimental proofs of the "hygiene hypothesis" in correlation with Helicobacter's infection, which adds another aspect of complexity to this infection.
Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology
Journal of Immunological Methods, 2002
Cell surface proteins of the human gastric pathogen Helicobacter pylori, reference strain CCUG 17874, were extracted with acid glycine and fractionated by heparin affinity chromatography. The extracts were subsequently analysed using Ž . high-resolution two-dimensional gel electrophoresis 2-DE and immunoblotting. Four proteins of low molecular masses Ž . 25-30 kDa stained by Coomassie R-350, were identified by peptide ESI-MSrMS sequencing after in-gel tryptic digestion. The identified proteins were recognised by sera from H. pylori-infected patients. Two of them are now described for the first time as immunogenic proteins of which one protein was determined to be distinct from all H. pylori proteins previously described. In addition, the specificity of the identified peptides was evaluated using both 1-D and 2-D immunoblotting against a panel of sera from patients with various bacterial infections. The present identification of highly specific antigens of H. pylori will encourage the improvement of serological diagnostic tests to diagnose and monitor H. pylori infection. q
FEMS Microbiology Letters, 2000
We describe a new procedure allowing the generation and detection of immunogenic antigens from Helicobacter pylori via the hemolysin secretion apparatus of Escherichia coli. The gene (or gene fragment) encoding the H. pylori protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyA s ). These fusion proteins are secreted efficiently by E. coli. This new approach allows the identification of immunodominant antigens by using sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. Three immunodominant antigens bearing the ureB (urease B-subunit), flaA (flagellin A-subunit), and an unknown ORF (HP0888) encoding an E. coli FecE analogous protein fused to hlyA s were identified and characterized. ß : S 0 3 7 8 -1 0 9 7 ( 0 0 ) 0 0 1 5 7 -9
Inhibition of H. pylori colonization and prevention of gastritis in murine model
World Journal of Microbiology and Biotechnology, 2012
Helicobacter pylori is a Gram-negative spiral bacterium that colonizes human gastric mucosa causing infection. In this study aiming at inhibition of H. pylori infection we made an attempt to evaluate immunogenicity of the total (UreC) and C-terminal (UreCc) fragments of H. pylori urease. Total UreC and its C-terminal fragment were expressed in E. coli. Recombinant proteins were analyzed by SDS-PAGE and western blot and then purified by Ni-NTA affinity chromatography. Female C57BL6/j mice were immunized with the purified proteins (UreC and UreCc). Antibody titers from isolated sera were measured by ELISA. Immunized mice were then challenged by oral gavage with live H. pylori Sydney strain SS1. Total of 109 CFU were inoculated into stomach of immunized and unimmunized healthy mice three times each at one day interval. Eight weeks after the last inoculation, the blood sample was collected and the serum antibody titer was estimated by ELISA. Stomach tissues from control and experimental animal groups were studied histopathologically. UreC and UreCc yielded recombinant proteins of 61 and 31 kDa respectively. ELIZA confirmed establishment of immunity and the antibodies produced thereby efficiently recognized H. pylori and inhibited its colonization in vivo. Pathological analysis did not reveal established infection in immunized mice challenged with H. pylori. The results support the idea that UreC and UreCc specific antibodies contribute to protection against H. pylori infections.
The use of a 49.6 kDa pili protein of Helicobacter pylori for serological diagnosis in mice
Bali Medical Journal
Bacterial culture is currently not recommended for routine evaluation of H. pylori infection in humans. For this reason, serological techniques play a critical role in diagnosing H. pylori infection in humans, particularly for initial pre-endoscopy or pre-treatment screening in dyspeptic patients. However, several current "in-office" tests appear to be less accurate or would need further validation before being recommended for use in a primary clinical care level. Preparation of specific and immunogenic antigens is an important step to improve the sensitivity and specificity of serologic assays, particularly for confirming the peptic ulcer status. The purpose of this study was to use a sub-unit protein of H. pylori with a molecular mass of about 49.6 kDa for the evaluation of H. pylori infection in mice. Fifty mice were orally infected with live H. pylori, and a similar number of mice were only orally given sterile phosphate-buffered saline (PBS). This process was repeated three times with a three-day interval between each administration, and blood samples were collected before and after each infection. The sera were tested using dot blot and ELISA. Seroconversion was detected two weeks after infection, and ELISA showed 98% for both its sensitivity and specificity. This study has indicated that the 49.6 kDa subunit pili proteins recognized homologous antibodies against the microorganism. Further studies are required to confirm the reaction of this protein against serum originated from H. pylori-infected human.
Journal of Experimental Medicine
The mechanism by which Helicobacter eylori, a noninvasive bacterium, initiates chronic antral gastritis in humans is unknown. We now show that H. Fflori rdeases products with chemotactic activity for monocytes and neutrophils. This chemotactic activity was inhibited by antisera to either H. ~lori whole bacteria or H. pylor/-derived urease. Moreover, surface proteins extracted from H. pylori and purified H. ~lori urease (a major component of the surface proteins) exhibited dose-dependent, antibody-inhibitable chemotactic activity. In addition, a synthetic 20--amino acid peptide from the NHx-terminal portion of the 61-kD subunit, but not the 30-kD subunit, of urease exhibited chemotactic activity for monocytes and neutrophils, localizing the chemotactic activity, at least in part, to the NH2 terminus of the 61-kD subunit of urease. The ability of leukocytes to chemotax to H. i;,ylori surface proteins despite formyl-methionyl-leucyl-phenylalanine (FMLP) receptor saturation, selective inhibition of FMLP-mediated chemotaxis, or preincubation of the surface proteins with antiserum to FMLP indicated that the chemotaxis was not FMLP mediated. Finally, we identified H. eylori surface proteins and urease in the lamina propria of gastric antra from patients with H. pylori-associated gastritis but not from uninfected subjects. These findings suggest that H. f,ylori gastritis is initiated by mucosal absorption of urease, which expresses chemotactic activity for leukocytes by a mechanism not involving N-formylated oligopeptides.
Proteins Released by Helicobacter pylori In Vitro
Journal of Bacteriology, 2002
Helicobacter pylori may contribute to gastric inflammation and epithelial damage. An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis. The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n ؍ 6). Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium. Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry. Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNAbinding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion. These proteins may be implicated in H. pylori-induced effects on the gastric epithelium.
Identification of erythrocyte-binding antigens in Helicobacter pylori
Journal of General Microbiology, 1992
The surface antigens of Helicobacter pylori conferring erythrocyte-binding activity were obtained by adsorption onto formaldehyde-treated dog and goat erythrocytes from supernatant fractions of sonicated bacteria and elution using a high concentration of NaCl. The desorbed material was analysed by SDS-PAGE and immunoblotting with anti-whole-cell serum to agar-grown bacteria which had been absorbed with broth-grown, non-haemagglutinating cells (haemagglutination-associated antiserum). Two polypeptides with molecular masses of 25 and 59 kDa were revealed as erythrocyte-binding antigens. Strains which agglutinated both dog and goat erythrocytes possessed both these erythrocyte-binding antigens, whereas an antigenically cross-reactive 24 kDa polypeptide was present in a strain which only agglutinated goat erythrocytes. Haemagglutinin material was extracted from H.pylori using n-octylglucopyranoside and purified by Sepharose chromatography and sucrose density gradient ultracentrifugation. The purified extract directly agglutinated erythrocytes in a neuraminyl-lactose-sensitive and neuraminidasesensitive manner. The 59kDa polypeptide was not present in the purified haemagglutinin preparation. The haemagglutination-associated antiserum reacted strongly with the 25 kDa polypeptide band which was the most prominent polypeptide band on analysis of the purified haemagglutinin preparation by SDS-PAGE and silver staining. Thus, H.pylori possesses at least two adhesins, one of which recognises a N-acetylneuraminic acid (a2-3) moiety of receptors, the other being of unknown receptor specificity. Differences in the antigenicity and molecular masses of these adhesins in individual strains may underlie differences in receptor-binding specificities and haemagglutination profiles.