Proteomic study of Helicobacter pylori under high-salt stress: Physicochemical characterization of neutrophil activating protein (NapA)

Helicobacter pylori (H. pylori) is a helix-shaped, microaerophilic Gram-negative bacterium, about 2 to 4 μm long with a diameter of about 0.5 μm. The bacterium can move through the stomach by means of its flagella, forming long-term stable colonies in the mucosa of stomachs. According to previous epidemiological studies, H. pylori infected stomachs of more than 50% of human population. Infection by H. pylori usually causes chronic gastritis without obvious symptoms; but in some strains producing specific virulence factors, they may lead to the development of ulcers or even gastric adenocarcinoma. It is known that H. pylori infection is the leading cause of gastric cancer and peptic-ulcer diseases. However, the factors involved in diseases caused by this bacterium are known to be complex and multi-factorial. Some human epidemiologic studies and animal models have confirmed that a high-salt dietary intake significantly increase the risk of gastritis and enhance the development of gastric carcinoma caused probably by H. pylori infection. o address the influence of high osmotic stress, we focus on the protein expression profile of H. pylori under high salt concentration by a proteomic approach. Clinical isolates of H. pylori from patients of gastric cancer (HC28) and duodenal ulcer (HD30) were grown in a normal BHI medium (0.5% NaCl) and hyper-osmotic stress (BHI medium supplemented with 2% NaCl) conditions for 24 h, followed by 2D electrophoresis, LC-MS/MS, MALDI-TOF-MS and bioinformatics database search/peptide-mass comparison. In this study, we identify some H. pylori proteins that were altered in response to high salt concentration in liquid culture media, including conspicuously a virulence factor of H. pylori, neutrophil activating protein (NapA), plus catalase, UreaA, UreaB, and 50S ribosomal proteins L7/L12. apA is a dodecameric protein consisting of 17 kDa monomers, and belongs to a member of Dps protein family. It acts as one of the major virulence factors in H. pylori infection. It has also been shown to play dual roles inside affected cells. First, it can elicit the cellular responses for the recruitment of human neutrophils and monocytes, the activation upon which immune cells would generate reactive oxygen radicals released from neutrophils and cause the inflammation locally. Secondly, NapA can sequester free ferrous ions in the cell to prevent the production of toxic hydroxyl radicals from Fenton reaction. n this study, we have also cloned and purified recombinant NapA by RT-PCR and overexpression of this protein factor. We characterized and compared the molecular sizes of NapA obtained from H. pylori of gastric cancer strain HC28 (HC28-NapA) and duodenum ulcer strain HD30 (HD30-NapA) under high-salt stress by using analytical ultracentrifuge (AUC) and fast performance liquid chromatography (FPLC). We found that the molecular mass of NapA can be reduced from 200 kDa to about 150 kDa in the presence of 2000 mM NaCl, suggesting that the subunits of NapA may dissociate into smaller aggregates under high-salt concentration. Furthermore, we also characterized the secondary and tertiary structures of NapA under different salt concentrations by circular dichroism (CD) and fluorescence spectroscopy. The binding stoichiometry between iron and NapA under different salt concentrations was also analyzed by using isothermal titration calorimetry (ITC) in order to reveal the quantitative binding relationship between iron and NapA.