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Proteomic analysis of the function of sigma factor ?54 in Helicobacter pylori survival with nutrition deficiency stress in vitro.

ABSTRACT: H. pylori can survive under a nutrition-deficient environment. During infection and transmission, H. pylori is confronted with nutrient limitation and the bacterium requires rapid alteration in gene expression for survival under stress conditions. However, the mechanism underlining this regulation remains unknown. A previous study showed that ?(54) is an important regulation factor for H. pylori survival in the nutrition-deficient environment. Our results show that the expression of ?(54) (rpoN) is significantly induced in the stationary phase (nutrition deficiency) and the rpoN mutant showed a significantly lower viability than wild-type H. pylori in the late stationary phase. Thus, ?(54) is involved in H. pylori survival during nutrient limitation. We used comparative proteomics to analyze the protein differentiation between wild-type and rpoN mutant during the stationary phase. With depleted nutrients, ?(54) can slow the process of proliferation by negatively regulating genes involved in energy metabolism and biosynthesis and enhance stress-resistant ability by positively regulating genes involved in protein fate and redox reaction. Especially, NapA positively regulated by ?(54) plays an important function in H. pylori survival both in the stationary phase and in water, and the latter situation would be beneficial for bacterial in vitro transmission. Our investigations give new light on the adaptive regulation of H. pylori under stress conditions.

SUBMITTER: Sun Y

PROVIDER: S-EPMC3755968 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Proteomic analysis of the function of sigma factor σ54 in Helicobacter pylori survival with nutrition deficiency stress in vitro.

Sun Yundong Y Liu Shuang S Li Wen W Shan Yuqun Y Li Xinpeng X Lu Xingxiao X Li Yan Y Guo Qing Q Zhou Yabin Y Jia Jihui J

PloS one 20130828 8

H. pylori can survive under a nutrition-deficient environment. During infection and transmission, H. pylori is confronted with nutrient limitation and the bacterium requires rapid alteration in gene expression for survival under stress conditions. However, the mechanism underlining this regulation remains unknown. A previous study showed that σ(54) is an important regulation factor for H. pylori survival in the nutrition-deficient environment. Our results show that the expression of σ(54) (rpoN) ...[more]

PMID: 24015282

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Project description:BackgroundSerine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins.MethodsThe in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of β-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy.ResultsWe demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of β-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage.ConclusionHere we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

Dataset Information

Proteomic analysis of the function of sigma factor ?54 in Helicobacter pylori survival with nutrition deficiency stress in vitro.

Publications

Proteomic analysis of the function of sigma factor σ54 in Helicobacter pylori survival with nutrition deficiency stress in vitro.

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