The Primary Study Of Adaptive Protein On Helicobacter Pylori

Helicobacter pylori is the only pathogenic bacteria that can colonize and survive in acid microenvironment of human stomach for a long time. In vivo i ts growth is necessarily inhibited by kinds of adverse conditions. So only by starting a series of adaptive regulatory ? mechanisms, Helicobacter pylori can resist these stressing factors to survive and induce diseases. Morphologic change is one of its adaptive ways and Helicobacter pylori exsists in different form at diverse conditions. Protein is the last executor of vital activities and plays an important role in any token. Comparative proteomics is the ideal means to study the protein changes of stressed bacteria and its technologies in common use include two-dimensional electrophoresis (2-DE) gel image analysis, biomass spectrometry and bioinformatics. Bile acid is a kind of crude antibacterial substance in vivo. The study on the survivorship of Helicobacter pylori in bile is significant for revealing the relation between the Helicobacter pylori infection and duodenal ulcer. So we studied the protein changes of Helicobacter pylori stressed by bile acid. Filamentous form of Helicobacter pylori is another survival form besides its spiral and coccoid form. The research on the protein spectrum offilamentous form will help bring to light to its development. We have found flagellar filament cap has close relation to the formation of Helicobacter pylori structured community, however, the function of candidate proteins gotten from biomass spectrometry need to be tested further. So we constructed the deficient mutant Helicobacter pylori without flagellar filament cap and made a base for the research of this protein and the illustration of the forming mechanism of Helicobacter pylori structured community. The research contents and main results are as follows:1. The primary study of inhibition of bile acid on Helicobacter pylori and its corresponding protein changesBile stress is one of unfavorable conditions for the growth of Helicobacter pylori in vivo, but Helicobacter pylori can still exist in bile-rich duodenal bulb. So we inferred that the acidification of bile affected its inhibiting activity. To study the adaptive regulatory mechanism of Helicobacter pylori, at first we scanned the minimal inhibiting concentration of bile and acidified and centrifuged bile on Helicobacter pylori and observed the morphologic changes of Helicobacter pylori through scanning electron microscope, then we studied the protein changes of Helicobacter pylori after bile stress through 2-DE. We found that acidification of bile did reduce its inhibiting activity on Helicobacter pylori and Helicobacter pylori partly changed into coccoid form after stress. We compared the 2-DE map of bile stressing Helicobacter pylori with that of its normal form and detected 13 kinds of differential protein spots. In future, we will identify these proteins using biomass spectrometry and test their specific regulatory function in order to find new pathogenic factor or antibacterial target.2. The primary study of adhesion to gastric epithelial cells and protein expression changes on filamentous form of Helicobacter pyloriFilamentous form of Helicobacter pylori as a phase of its vital periodis probably one of its adaptive regulatory mechanism to survive in given condition. We have studied its reversion and specificity. In order to further study its pathogenicity and regulatory mechanism, we induced filamentous form model using aztreonam and observed its adhesion to gastric epithelial cells and the pathological changes of the cells through scanning electron microscope. In addition, we compared protein patterns of filamentous form with spiral form of Helicobacter pylori through 2-DE. The scanning electron microscope results showed that filamentous form of Helicobacter pylor can still adhere to gastric epithelial cells and lead to serious pathological changes such as shading and even disappearance of microvillus, which indicated that the filamentous form of Helicobacter pylori still has definite pathogenic ability. Through 2-DE we found the protein pattern of filamentous form of Helicobacter pylori is different from that of spiral form and found out 10 kinds of differential protein spots. The differential protein spots need to be identified through biomass spectrum and tested their genie function using functional genomic technologies to elucidate the adaptive regulary mechanism of filamentous form of Helicobacter pylori.3. The construction and identification of deficiency mutant of Helicobacter pylori without flagellar filament cap.Through our past work we found flagellar filament cap is connected with the formation of Helicobacter pylori structured community. Flagellar filament cap is coded by FLiD gene, participates the capping of flagellar filament and makes up flagellum hook with HAP1 (FlgK) and HAP3(FlgL). But its specific function in the formation of Helicobacter pylori structured community and pathogenic mechanism are not yet clear. The most dependable means to discern the function of a gene is to construct its deficiency mutant and observe the phenotype and function changes after gene knockout. We cloned the two regions of FLiD gene through PCR as the guidance ofhomologous recombination and the kannamycin resistantant gene from plasmid pUC18~K2 as the screening marker. These three fragments were engineered into plasmid pILL570 to construct the mutant vector with kannamycin and spectinomycin as the markers. Then the mutant vector was electrotransformed into Helicobacter pylori cells and followed by screening through kannamycin resistance. At last, the mutant strain was identified by PCR . We successfully constructed the mutant vector and restriction endonuclease analysis is equal with anticipant result. Using PCR identified that the mutant of Helicobacter pylori has not FLiD gene and other genes are not affected. This will be useful for clarify the role of FLiD gene in the formation of Helicobacter pylori structured community and its pathogenesis.