J Bacteriol Virol.  2012 Sep;42(3):211-223. 10.4167/jbv.2012.42.3.211.

Proteomic Analysis of Thiol-active Proteins of Helicobacter pylori 26695

• Park JW
• Song JY
• Hwang HR
• Park HJ
• Youn HS
• Seo JH
• Kang HL
• Lee KH
• Baik SC
• Lee WK
• Cho MJ
• Rhee KH

• KMID: 1434750
• DOI: http://doi.org/10.4167/jbv.2012.42.3.211

Abstract

Helicobacter pylori are a capnophilic bacterium, which colonize gastric mucosa and are resistant to acidic and oxidative damage. Thiol-active proteins subserve redox functions in tolerating oxidative stress and environmental toxicants, such as hydrogen peroxide and hypochlorous acid. We analyzed disulfide-containing proteins of H. pylori strain 26695. Active disulfide-containing proteins were separated by thiol-affinity chromatography, displayed with two-dimensional electrophoresis (2-DE), and identified by MALDI-TOF-MS. Thirty-five putative disulfide proteins, including AhpC (HP1563), GroEL (HP0011), and FrdB (HP0191), were identified in this study. In addition, 4 disulfide proteins of HypB, FusA, TufB, and AhpC showed enhanced intensities in the periplasmic space when compared with the pellet, suggesting that these proteins might play roles in the first redox system against environmental oxidative stresses. Disulfide-containing proteins identified in this study will provide the standard landscape for constructing the proteome components responsible for redox regulation of H. pylori.

Keyword

Helicobacter pylori; Thiol-active proteins; Proteome

MeSH Terms

Chromatography
Colon
Electrophoresis
Gastric Mucosa
Helicobacter
Helicobacter pylori
Hydrogen Peroxide
Hypochlorous Acid
Oxidation-Reduction
Oxidative Stress
Periplasm
Proteins
Proteome
Sprains and Strains
Hydrogen Peroxide
Hypochlorous Acid
Proteins
Proteome

Figure

• 

  Figure 1 SDS-PAGE analysis of flow-through and eluted fraction in the thiol-affinity chromatography applied to H. pylori 26695 whole cell extract that was subjected to alkylation and reduction. M, protein size marker; 1, the whole cell extract; 2, thiol-affinity-unbound protein, flow-through fraction; 3, thiol-affinity-bound proteins, eluted fraction. Proteins were resolved with 12% SDS-gels. The gel was stained with coomassie blue.

• 

  Figure 2 Two-dimensional gel electrophoresis profiles of the whole cell extract (A and C) and the eluted fraction of thiol-affinity chromatography (B and D) of H. pylori 26695. The protein mixture (200 µg) were separated on IPG strip of pH 5.0~8.0 and 6.0~11.0 by 13% SDS-PAGE and then visualized by silver staining. The spot numbers on the gels are given in Tigr locus name of Table 1.

• 

  Figure 3 Two-dimensional gel electrophoresis of periplasmic fraction (100 µg) of H. pylori strain 26695. The proteins were separated on an immobilized pH gradient strip and subsequently on a 13% SDS-PAGE gel. The gels were stained by silver staining and were scanned and processed using Fluor-S MutiImager system and the PDQUEST-2D Gel analysis software (Bio-rad, vesion 7.01, CA, USA). Locus No. represent protein spots showed >2-fold higher intensity in the periplasmic fraction (B) than in the pellet (A).

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