Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori

Abstract

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.

Helicobacter pylori: immunoproteomics related to different pathologies

“…The publication of the genomic sequence of three H. pylori strains has assisted in the proteomic analysis and subsequent identification of antigens: strain 26695, isolated from a patient with superficial gastritis [36], strain J99, obtained from a patient with duodenal ulcer (DU) disease [37] and strain HPAG1, isolated from a patient with chronic atrophic gastritis [38]. Several groups have used this approach to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development, as well as to investigate potential associations between specific immune responses and manifestations of disease, revealing a high variability of humoral recognition pattern among different patients and H. pylori isolates [39][40][41][42][43][44][45][46][47]. In each study, there was considerable inter-individual variation that may be partially related to specific antigen patterns of the various H. pylori strains that infected the different patients [48].…”

vaccine development: Facing the challenge

“…However, two novel protective antigens that fulfill all criteria were identified using this process (HP0410 and HP0231). The ever-increasing information on microbial genomes and a comprehensive list of more than 600 immunoreactive antigens described through proteomic approaches (Kimmel et al, 2000;Krah et al, 2004;Nilsson et al, 2000) now allow us to up-date and refine these criteria. At least three antigens, NapA, HP0410 (putative neuraminyl lactose-binding hemagglutinin homologue) and HP0231 (homologue of DsbA and DsbC, which have been described to oxidize protein thiols and have a potential role in the periplasmic folding of proteins in E. coli, (Bessette et al, 2001)) fulfill all criteria.…”

Identification of antigens for the development of a subunit vaccine against Photobacterium damselae ssp. piscicida

“…It has been used for the identification of antigenic proteins such as those of Helicobacter pylori [14,15], Chlamydia trachomatis [16], Edwardsiella tarda [17,18], Edwardsiella ictaluri [19], Shigella flexneri [20], Borrelia garinii [21], and Nisseria meningitides [22].…”