Clustering of clinical strains of Helicobacter pylori analyzed by two-dimensional gel electrophoresis

Abstract

Strain variations of Helicobacter pylori have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether H. pylori expresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). H. pylori strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot patterns was observed between the strains, but a dendrogram analysis revealed that some strains within each disease group clustered together. Eight proteins were sequenced and found in the H. pylori genome sequence. 2-D PAGE is a useful method for studies of protein expression and for highlighting the extensive strain variation that H. pylori exhibits.

FIG. 1

2-D PAGE of two clinical isolates of H. pylori. About 10 μg of protein derived from water extracts of one Ca strain (A) and one Du strain (B) was focused on 110-mm IPG strips and separated on 8 to 18% gradient gels. Circled spots (1 and 3 to 8) exemplify proteins that were absent in certain strains or differed in intensity between strains. Spots 2 and 9 are indicated by arrows. The pI scale and the molecular weight (in thousands) are indicated at the bottom and to the left of each panel, respectively.

FIG. 2

Two dendrograms showing the relationships among 12 H. pylori strains. Dendrogram A is produced by the method of average linkage (unweighted pair group method with averages), and B is produced by the method of neighbor joining. Both dendrograms are based on calculation method 2. At the bottom, the percentages of similarity are indicated, and to the right, there are strain identity numbers.

FIG. 3

2-D PAGE followed by immunoblot of one Ca and one Du strain of H. pylori. Whole-cell protein extracts were focused on 180-mm 3-10 nonlinear IPG strips, separated on 12 to 14% gradient gels, and visualized by silver staining (A and B) or transferred to PVDF membranes and blotted using sera from Ca (C and F) or Du (D and E) patients. For silver staining and blots, 20 and 40 μg of protein, respectively, were applied on each strip. Some of the highly immunogenic proteins are circled (for use as internal standards), and differences in protein pattern and/or immunogenicity are indicated by arrowheads. The rectangles (spots 9 and 10) indicate strain-specific differences. A pI scale is given for panels A and B, and markers for molecular weight (in thousands) are given for panel A.