J Bacteriol Virol.  2014 Jun;44(2):177-187. 10.4167/jbv.2014.44.2.177.

Proteome Analysis of a Catalase-deficient Isogenic Mutant of Helicobacter pylori 26695

• Kang HL
• Lee SG
• Park JS
• Song JY
• Cho MJ
• Baik SC
• Youn HS
• Seo JH
• Rhee KH
• Lee WK

• KMID: 2168693
• DOI: http://doi.org/10.4167/jbv.2014.44.2.177

Abstract

Helicobacter pylori, a gram-negative bacterium, is a causative agent of gastroduodenal diseases of human. Human immune system produces harmful reactive oxygen species to kill this bacterium that locates the microaerophilic mucous layer. H. pylori harbors various antioxidant enzymes including SodB, KatA and AhpC to protect the oxygen toxicity. We removed the catalase gene (katA) from H. pylori 26695 genome, and the change of profile of the gene expression of the mutant was analyzed by high resolution 2-DE followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), tandem MS and microarray analysis. Eleven and 37 genes were upregulated and downregulated in the mutant respectively, either transcriptionally or translationally. Expression level of pfr and hp1588 that were decreased on protein level in the mutant was confirmed by RT-PCR analysis.

Keyword

Helicobacter pylori; Antioxidant; katA

MeSH Terms

Catalase
Gene Expression
Genome
Helicobacter pylori*
Humans
Immune System
Mass Spectrometry
Microarray Analysis
Oxygen
Proteome*
Reactive Oxygen Species
Catalase
Oxygen
Proteome
Reactive Oxygen Species

Figure

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  Figure 1. Construction of plasmids for knock-out mutant.

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  Figure 2. Confirmation of katA deficiency. top, H. pylori 26695; bottom, katA mutant.

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  Figure 3. Growth curve of H. pylori 26695 and isogenic mutants at thin layer culture condition.

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  Figure 4. Comparison of normalized gel images of wild type and kat isogenic mutant. The proteins were separated on an IPG strip of pH 5.0∼8.0 and subsequently on a 12.5% SDS-PAGE and then detected by silver staining. The original gel size was 18×20×0.15. (A) Wild type. (B) kat isogenic mutant.

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  Figure 5. Comparison of expression of genes in wild type H. pylori and in katA mutant. (A) RT-PCR analysis of pfr and hp1588. (B, C) expression of two genes. Levels were measured by 2-DE, microarray and RT-PCR.

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