Comparative proteome analysis of Helicobacter pylori

Abstract

SummaryHelicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value.

Strategies for the enrichment and identification of basic proteins in proteome projects

“…Thirty-seven of the ORFs identified here have been characterized for the first time in H. pylori, with the remainder having been characterized in previous studies [39][40]. These results suggest that basic proteins are best analyzed using a combination of specialized methodologies including narrow range basic IPG 2-DE gels, and prefractionation combined with 2-DE, SDS-PAGE and multidimensional LC-MS/MS.…”

“…Therefore, we believe that over one-third of the H. pylori proteome will be difficult to resolve using standard 2-DE technology due to the prevalence of predicted proteins with extremely alkaline pI. Very few such basic proteins have been resolved in previous studies of the H. pylori proteome [39][40]. We therefore aimed to specifically examine such proteins using a range of commercially available and novel methodologies.…”

Role of a Bacterial Organic Hydroperoxide Detoxification System in Preventing Catalase Inactivation

“…pylori expresses abundant levels of catalase and AhpC proteins (19). The genetic and biochemical characterization of H. pylori catalase and AhpC has been performed in different laboratories (13, 20 -24).…”

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…The newly identified antigens are being used to develop vaccines, e.g. for pathogenic bacteria, such as H. pylori and M. tuberculosis [121][122][123][124]. New data on cell differentiation and on cell regulation can be obtained using the total cell protein approach, and new insights in functional genomics can be derived using these data.…”

“…For the elucidation of immunologically relevant proteins, the proteomes of M. tuberculosis and Heliobacter pylori were studied by high-resolution 2-DE and MALDI mass fingerprinting of the virulent and the attenuated strains [121][122][123]. From the mycobacterial unique spots, 36 were evaluated in a mouse model as DNA vaccines and one vaccine improved the efficacy of BCG vaccination [124].…”