This proposal is to isolate, purify, and characterize biochemically, immunochemically, and at the molecular genetic level, surface fimbrial proteins which contribute to the ability of an extremely common pathogen of humans, Helicobacter pylori, to adhere to gastric epithelial cells and ulcerative tissue and produce disease. This spiral bacterium colonizes and is associated with disease of the upper gastrointestinal tract of a large number of individuals during their life-span. The diseases include gastritis, and gastric and duodenal ulcers. Since the diseases associated with H. pylori colonization are among the most common which require medical consultation, intervention, and therapy, there is clearly a pressing need for an improved understanding of the mechanisms by which H. pylori colonizes the host. This understanding should lead to the development of improved therapeutics, and potentially to the development of a vaccine to prevent colonization of the upper gastrointestinal tract by H. pylori. Proteins contributing to the ability of this organism to specifically adhere to gastric mucosal epithelial cells and extracellular matrix proteins and so colonize its niche are both excellent targets for therapeutic strategies, and primary candidates for inclusion in a vaccine. The fimbrial adhesion proteins of H. pylori will be isolated from the cell surface by shearing and purified to homogeneity by ammonium sulfate fractionation, gel filtration chromatography, and ion-exchange and reverse phase chromatography. The biochemical structure of the proteins will be characterized by amino acid composition and N-terminal sequence analysis, peptide mapping, isoelectric point determination, and solution molecular weight determination. Secondary structure predictions will be made by analysis of Circular Dichroism spectra. The antigenic structure of the H. pylori adhesion and matrix protein binding proteins will be analyzed with antibodies raised to purified adhesion protein, antibodies raised to intact native proteins on H. pylori cells, and when appropriate, with monoclonal antibodies. Antigenically cross-reactive surface exposed epitopes will be identified with the aid of hydrolytic and proteolytic cleavage experiments, and fine epitope mapping using solid phase peptide synthesis. Purified fimbriae, and peptides carrying these epitopes will be isolated or synthesized to evaluate their possible use as protective immunogens. Selected parallel studies will be performed with the fimbrial adhesions of Helicobacter mustelae and Helicobacter felis where animal models are currently available. Oligonucleotide probes will be constructed to selected adhesion proteins, and the structural genes coding for each adhesion will be cloned and the nucleotide sequence of the gene determined. Finally, shuttle vector and natural transformation systems will be employed to generate mutations in the structural genes of the various Helicobacter adhesion proteins. This will allow the role of the proteins in cell attachment, extracellular matrix protein binding, and colonization to be confirmed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI029927-02
Application #
3144939
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1992-05-01
Project End
1995-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Victoria
Department
Type
DUNS #
209567957
City
Victoria
State
BC
Country
Canada
Zip Code
V8 5-C2
Exner, M M; Doig, P; Trust, T J et al. (1995) Isolation and characterization of a family of porin proteins from Helicobacter pylori. Infect Immun 63:1567-72
Huang, J; O'Toole, P W; Doig, P et al. (1995) Stimulation of interleukin-8 production in epithelial cell lines by Helicobacter pylori. Infect Immun 63:1732-8
Doig, P; Trust, T J (1994) Identification of surface-exposed outer membrane antigens of Helicobacter pylori. Infect Immun 62:4526-33
Doig, P; Austin, J W; Trust, T J (1993) The Helicobacter pylori 19.6-kilodalton protein is an iron-containing protein resembling ferritin. J Bacteriol 175:557-60