Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori.
Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.
Nilsson
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Department of Infectious Diseases and Medical Microbiology, University of Lund, Sweden. ingrid.nilsson@mmb.lu.se
Utt
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Nilsson
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Ljungh
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Wadström
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D000165:Acridine Orange; D001426:Bacterial Proteins / Q000032:analysis*; D015153:Blotting, Western; D004396:Coloring Agents; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D016480:Helicobacter pylori / Q000737:chemistry* / Q000254:growth & development / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D016622:Silver Staining; D013194:Staining and Labeling
D001426:Bacterial Proteins; D004396:Coloring Agents; D008565:Membrane Proteins; D000165:Acridine Orange