Analysis For Adhesive Factors Of Helicobacter Pylori Animal Mode Strain SS1

Helicobacter pylori (H. pylori) is a Gram-negative, microaerobic bacterium. This main pathogenic agent of upper digestive diseases chronically infects the gastric mucosa of more than half of all people worldwide, and the infection rate is much higher in developing countries. Besides that, once infected, most people will carry this bacterium for decades until it is been eradicated. H. pylori strains have host and tissue tropism. So most clinical strains were emptied in short period of time after inoculating and could not induce inflammatory reaction and other diseases.In 1996, Andrew Lee and his colleagues got a H.pylori strain with good colonizing ability after screening a range of fresh clinical isolates and named it Sydney Strain 1(SS1). Experiment data of this strain fulfills the Lausanne criteria and can be repeated in other laboratories. SS1 and its parent strain 10700 colonized in C57BL/6 with high level of 10~7 and 10~6 cfu/g animal stomachs respectively. Genomic typing revealed few modifications of genes during the process of mouse adaptation. We know little about the genome of SS1 itself and the changes during the course of adaptation.H.pylori strains used in this study is 700392 bought from ATCC, pre- and postmouse strains( 10700 and SS1) cultured in vitro less than seven times. SS1 and 10700 were compared for searching changes during adaptation, while 700392 was used as control for poor colonization.SS1 and 10700 were cultured on plates. Whole cell proteins were prepared by precipitation with TCA and acetone and separated using 2-DE. The spots of the differential expressed proteins between SS1 and 10700 were cut for zymohydrolysis and identified using MALDI-TOF-MS/MS. No proteins were found only in SS1 or 10700. Eleven down-regulated spots presented ten proteins. Four were related to anti-oxidation, five were enzymes associated with metabolism, and the last one is a putative protein HPAG0942.Colonization ability is determined by many factors, such as the characteristic of strains, the kind of mouse, and the stomach circumstances. Components involved in interaction between H.pylori strains and mouse gastric mucous were studied. Gastric mucous fixed with PLP fixative was incubated with whole cell proteins of SS1 and 700392 respectively. Proteins enrichment on C57BL/6 mouse gastric mucous and epithelium were eluted by buffer containing SDS. The eluent were separated with 2-DE and all eyeable spots were cut for identification. Eight proteins were identified in both eluents: HP0175, HP1286, 48 kDa antigen(HP0599), Chain A ofγ-GGT, Chain B ofγ-GGT, alkyl hydroperoxide reductase, Catalase, and isocitrate dehydrogenase. Most of these were related with inflammation. Among them, HP0175 and GTT can induce apoptosis of AGS cell and HP0175 interacts with TLR-4 on surface of AGS. Proteins identified only in eluent from SS1 were putative aldo-keto reductase and 3-ketoacyl-(acyl-carrier-protein) reductase. Much more putative aldo-keto reductase expressed in SS1 than in 700392 may be helpful for colonization.Function of common component HP1286 is unknown. In this study, HP0175 and HP1286 without signal peptide were cloned into pET32a(+) and expressed in E.coli BL21. Recombinant proteins were purified by nickel-affinity resin. Before adding proteins into culture medium of adherent AGS cells in 24-well plates, all proteins were desalting and sterilized by filtration. By detecting apoptosis of AGS cell, it is obvious that His-HP0175 and His-HP1286 can induce much more serious cell death in a time-dependent manner than tag protein. In current study, there were not many changes between SS1 and its primary strain 10700, just a few proteins related to anti-oxidation and metabolism. Maybe some high expressed proteins were beneficial for colonization, such as putative aldo-keto reductase. We supposed that no obvious changes happened in the course of adaptation and the good colonizing ability is inborn to some extent, then the mouse-adaptation strengthened the ability. HP1286 which induced apoptosis of AGS cell in vitro was one of many microstructures of H.pylori, and many microstructures lead to the fact that H.pylori infection can induce apoptosis of gastric epithelium cell.