title: Comparative proteomics analysis of sarcosine insoluble outer membrane proteins from clarithromycin resistant and sensitive strains of Helicobacter pylori.
abstract: Helicobacter pylori causes disease manifestations in humans including chronic gastric and peptic ulcers, gastric cancer, and lymphoid tissue lymphoma. Increasing rates of H. pylori clarithromycin resistance has led to higher rates of disease development. Because antibiotic resistance involves modifications of outer membrane proteins (OMP) in other Gram-negative bacteria, this study focuses on identification of H. pylori OMP's using comparative proteomic analyses of clarithromycin-susceptible and -resistant H. pylori strains. Comparative proteomics analyses of isolated sarcosine-insoluble OMP fractions from clarithromycin-susceptible and -resistant H. pylori strains were performed by 1) one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein separation and 2) in-gel digestion of the isolated proteins and mass spectrometry analysis by Matrix Assisted Laser Desorption Ionization-tandem mass spectrometry. Iron-regulated membrane protein, UreaseB, EF-Tu, and putative OMP were down-regulated; HopT (BabB) transmembrane protein, HofC, and OMP31 were up-regulated in clarithromycin-resistant H. pylori. Western blotting and real time PCR, respectively, validated UreaseB subunit and EF-Tu changes at the protein level, and mRNA expression of HofC and HopT. This limited proteomic study provides evidence that alteration of the outer membrane proteins' profile may be a novel mechanism involved in clarithromycin resistance in H. pylori.
authors.affiliation: Department of Clinical Investigation, William Beaumont Army Medical Center, 5005 Piedras Street, El Paso, TX, 79920-5001, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Smiley
authors.forename: James
authors.initials: J
authors.lastname: Bailey
authors.forename: Mahadevan
authors.initials: M
authors.lastname: Sethuraman
authors.forename: Norberto
authors.initials: N
authors.lastname: Posecion
authors.forename: M
authors.initials: M
authors.lastname: Showkat Ali
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D015153:Blotting, Western; D017291:Clarithromycin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D016480:Helicobacter pylori / Q000737:chemistry* / Q000187:drug effects*; D006801:Humans; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D020543:Proteome; D017291:Clarithromycin