Proteome analysis of highly immunoreactive proteins of Helicobacter pylori.
BACKGROUND
Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori.


METHODS
The total complement of protein from seven strains of H. pylori was resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori-infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme-antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).


RESULTS
Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease beta-subunit UreB; elongation factor EF-Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains.


CONCLUSIONS
These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF-Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.
Lock
Robert A
RA
Western Australian Proteome Analysis Facility, Western Australian Biomedical Research Institute, Perth, WA, Australia.
Coombs
Geoffrey W
GW
McWilliams
Tracy M
TM
Pearman
John W
JW
Grubb
Warren B
WB
Melrose
Graham J H
GJ
Forbes
Geoffrey M
GM
D001426:Bacterial Proteins / Q000032:analysis* / Q000276:immunology*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005408:Flagellin / Q000032:analysis / Q000276:immunology; D006360:Heat-Shock Proteins / Q000032:analysis / Q000276:immunology; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D010444:Peptide Elongation Factor Tu / Q000032:analysis / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014510:Urease / Q000032:analysis / Q000276:immunology
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C112564:HspB protein, Helicobacter pylori; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D005408:Flagellin; C067138:flaA protein, bacteria; D014510:Urease; D010444:Peptide Elongation Factor Tu