Two-dimensional Blue Native/SDS Gel Electrophoresis of Multiprotein Complexes from Helicobacter pylori

Abstract

The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/ SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins. Molecular & Cellular Proteomics 6:193-206, 2007.

In Vivo Interactome of Helicobacter pylori Urease Revealed by Tandem Affinity Purification

“…Compared with the previous studies on the interactome of H. pylori using blue native two-dimensional gel or yeast twohybrid analysis (16,17) we found no interaction with incompatible cellular localization, i.e. between well defined cytoplasmic bait proteins and outer membrane or secreted proteins.…”

“…Analyses of H. pylori protein complexes by blue native two-dimensional gel electrophoresis (17) are difficult to interpret because the resolution of the gels is limited, and hence the definition of a complex is relatively subjective. The authors of the latter study identified 13 multicomplexes among which the only overlap was UreA-UreB.…”

“…We decided to attribute the status of "real interactor" to proteins that we reproducibly did not copurify with at least two baits belonging to non-overlapping protein complexes. The contaminants comprised a number of proteins previously noted as abundant in different proteomics studies (17,46) like chaperones such as HspA and HspB (the GrosES-EL homolog), DnaK, elongation factors such as EF-TU, several ribosomal proteins, proteases, and some other highly abundant proteins (e.g. acetone carboxylase).…”

“…However, the Y2H data are hampered by protein overexpression and by the use of a heterologous system for analysis, resulting in a relatively high false positive versus true positive ratio. Recently a protein interaction study based on the co-migration of proteins in a complex visualized by blue native/SDS-PAGE separation was performed in H. pylori, leading to results that are difficult to interpret because of the naturally limited protein resolution in this system (17). An alternative method, namely the tandem affinity purification (TAP) technique, has proven to be an efficient means to access multipartner protein complexes with fewer false positives and much better function predictions than from Y2H data (18 -20).…”

Differential Proteomics of Helicobacter pylori Associated with Autoimmune Atrophic Gastritis

“…Because of its effective role for HP survival, urease is one of the most abundant proteins synthesized by HP (41). This protein has been already described in HP proteomes obtained from different gastric diseases (24,53). In particular, Pyndiah et al (53) showed a higher intensity of urease B (UreB) in HP isolates from patients with chronic gastritis or GC compared with isolates from patients with DU.…”