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.The widespread human pathogen Helicobacter pylori is a major cause of gastric and duodenal ulcers and gastric cancer (48, 53). To identify factors of H. pylori that are potentially involved in pathogen-host interactions (11), proteome analysis has been successfully used by numerous groups (4,7,14,17,21,22,24,29,37,50). Secreted proteins (the secretome) may be of special importance, since these proteins come into direct contact with host compartments; however, technical difficulties have led to somewhat contradictory results and have prevented a comprehensive analysis (8,19,25,34,42,51). H. pylori is commonly cultivated in rich media complemented with various additions of serum containing numerous foreign proteins that are difficult to resolve from extracellular H. pylori proteins. Only a few studies have used protein-free media (1,19,26,38,49,51), and growth is usually much slower in such media. Moreover, H. pylori is particularly prone to spontaneous autolysis (34), resulting in the nonspecific release of numerous proteins; the latter makes the interpretation of protein patterns obtained from culture supernatants difficult.In this study, we optimized culture conditions for minimal autolysis, adapted a precipitation method for the optimal recovery of extracellular proteins, resolved the various secreted proteins by two-dimensional gel electrophoresis, and identified 26 protein species. Based on a comparison of the intensities of staining of specific protein species in supernatants and wholecell samples, we obtained a semiquantitative estimate for secretion selectivity. Among the secreted proteins were several redox-active enzymes, various components of the flagellar apparatus, three fragments of the vacuolating cytotoxin VacA, the serine protease and chaperone HtrA, and several previously uncharacterized proteins that are potential targets for therapy and vaccine development. To our knowledge, this is the first comprehensive analysis of the H. pylor...

Tip-α (hp0596 Gene Product) Is a Highly Immunogenic Helicobacter pylori Protein Involved in Colonization of Mouse Gastric Mucosa

“…The protein was also absent in 33 protein species which constitute the H. pylori 2665 secretome established by 2-D electrophoresis [8]. Obtained data do not rule out the possibility that Tip-a is unleashed by the pathogen in vivo during infection, especially that the predicted 3-D protein model suggests its soluble character.…”

“…Comparison of the provided data presents difficulties, due mainly to the genetic diversity of H. pylori as well as to methodological differences. There have been a number of significant attempts undertaken which were aimed at resolving surface or membrane proteins [5,8]. In each assay new immunoreactive proteins were identified and some of them have been tested as potential vaccine candidates.…”

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

“…Antibody staining indicated that urease A, catalase, and a homologue of HP0410, the flagellar sheath protein, are on the surface (25,31,33,34). MsrA, the cell-binding factor 2, HP0231, HP1098, HP1350, HtrA, carbonic anhydrase, and ␥-glutamyltranspeptidase have been found to be secreted into the extracellular medium (35,36). The large overlap between surface-exposed, firmly bound proteins (this study) and previously characterized secreted proteins is consistent with the concept of re-adsorption of released proteins at the H. pylori surface in vitro (25,26).…”

Analysis of Automatically Generated Peptide Mass Fingerprints of Cellular Proteins and Antigens from Helicobacter pylori 26695 Separated by Two-dimensional Electrophoresis

“…At present this technology seems to be limited to the identification of about 500 -700 spots of the H. pylori pattern. To identify a higher percentage of the open reading frames at the proteome level, prefractionations (6,8,14), complementary technologies (15), or improvements in the sensitivity of mass spectrometry and 2-DE methods are promising. Another attempt is to evaluate the data obtained by PMFs more comprehensively.…”

“…Immunologically relevant proteins were searched in the complete cellular proteins (1), surface proteins (6,7), and secreted proteins (8) and by immunoproteomics (9 -13). These studies used classical proteomics combining high resolution two-dimensional electrophoresis and peptide mass fingerprinting.…”