Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori

Abstract

Secreted proteins (the secretome) of the human pathogen Helicobacter pylori may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.

FIG. 1.

Protein compositions of whole-cell proteins (A) and secreted proteins (B) of H. pylori 26695. Two-dimensional gel electrophoresis was done with H. pylori harvested during exponential growth in liquid cultures with a protein-free medium. The numbers correspond to those in Table 1. Proteins were detected by silver staining. Spots 4_33 and 6_9 of the secretome were visualized only in Coomassie brilliant blue G-250-stained patterns. Their positions are marked by ellipses on the silver-stained standard patterns. Six abundant protein species that had putative cytosolic localizations and functions but that were not detected in the supernatants are marked with open arrowheads and labeled U_1 to U_6.

FIG. 2.

Complete sequence of vacuolating cytotoxin VacA (HP0887). Peptides with matching masses in tryptic digests of spot 2_2 or spot 5_2 are shown in bold. The mature exotoxin (27) (underlining) and the carboxy-terminal fragment of the passenger domain (double underlining) are also indicated.

FIG. 3.

Mass spectrometric characterization of spot 5_2. (A) Matrix-assisted laser desorption ionization mass spectrometric peptide mass fingerprint of a tryptic digest. All spots marked with an asterisk fit tryptic peptides of vacuolating cytotoxin VacA (HP0887) (Fig. 2). The peak designated CBB corresponds to Coomassie brilliant blue G-250. Peptide masses 1,018.46 and 1,174.57 fit peptides from aa 983 to 991 and from aa 982 to 991, respectively. (B) CID spectrum of a tryptic peptide with m/z = 587.8 (corresponding to a doubly-charged peptide with a mass of 1,174.6).