Genetic regulation of virulence factors contributing to colonization and pathogenesis of helicobacter pylori

by Baker, Patrick Ericson

Abstract (Summary)
The urease enzyme of the gastric pathogen Helicobacter pylori catalyzes the degradation of urea into ammonia and carbon dioxide. Urease is believed to protect the bacterium from gastric acid. Urease activity of H. pylori is essential for colonization of the stomach. An objective of this dissertation was to correlate the level of urease expression with loss of colonization of H. pylori isolated from infected piglets and subjected to rounds of in vitro passage. Using slot blot and reverse transcription-polymerase chain reaction (RT-PCR) in a thermocycler with real-time analysis, urease RNA was determined to be constitutively expressed regardless of the number of in vitro passages. Determination of urease activity of the isolates by the phenol-hypochlorite assay confirmed the RNA data. Lewis x (Lex) and Lewis y (Ley) antigens of Helicobacter LPS undergo phase variation during in vitro growth due to slip-strand mispairing of polycytidine (poly(C)) tracts in the alpha-3-fucosyltransferase genes, futA and futB, that specify the Lewis antigen phenotype. Hence, another objective of this dissertation is to evaluate whether the on/off status of futA and futB via slip-strand mispairing determines Lex and Ley phenotype. The lengths of the poly(C) regions were analyzed by PCR. The Lewis phenotypes of the samples were determined by immunoblots and enzyme-linked immunoabsorbent assay (ELISA) using anti-Lex and anti-Ley antibodies. This study failed to correlate futA/B expression with Lex expression. Because both futB and Ley were expressed in all strains, no relationship could be determined. However, futB is sufficient for Lewis x/y synthesis. The final objective of this dissertation was to evaluate the ability of H. pylori LPS and its O-antigen to modulate the CD4+-dependent host immune response to H. pylori. This was performed by culturing splenocytes from uninfected and SS1-infected C57Bl/6 mice and T-bet-deficient mice. The cultures were stimulated with sonicate preparations and LPS samples from the parental and O-antigen-deficient bacterial strains. The data from this study suggests that Lewis antigens cannot alone account for the strong stimulation of the immune system and may even down-regulate the inflammatory response to H. pylori based on the higher stimulation of interferon-ã of SS1:0826 LPS compared to SS1 LPS.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:helicobacter pylori urease alpha 3 fucosyltransferase beta 1 4 galactosyltransferase slip strand mispairing gene regulation gram negative bacteria lipopolysaccharide lewis histo blood group antigens x y immunology


Date of Publication:01/01/2003

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