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 ext racted during different in vitro growth phases were analyzed by one- and tw o-dimensional gel-electrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. B roth-cultured H. pylori cells were stained with an acridine-orange dye to m onitor the morphological status of the organism in 2-day-cultures, 96% of t he bacterial cells were spiral-shaped and four days later a morphological s witch to coccoid forms occurred. In 10-day cultures spiral-shaped forms wer e 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 cul tures. A protein corresponding in size to the heat shock protein (GroEI hom olog, 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 increas ed number of silver-stained spots of 8-day cultures tin average 250 spots) compared with protein extracted from 2-day cells tin 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 simi lar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultur es, suggesting that these proteins remain immunologically intact. Pooled se ra from infected patients absorbed with spiral-shaped cells showed an almos t total blocking of the antibody reactivity to extracted coccoid proteins i n 2-DE immunoblot. Eighteen spots were still visible, but this reactivity p robably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.