幽门螺杆菌适应性蛋白的初步研究
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摘要
幽门螺杆菌(Helicobacter pylori,H. pylori)作为唯一能在人体胃内酸性环境中定植并长期生存的致病菌,其生长在体内必然会受到各种不利条件的影响,H. pylori通过启动一系列的适应性调节机制来抵抗这些胁迫因素以达到保种生存并导致疾病的目的。形态学变化是H. pylori的适应性调控机制之一,在不同的胁迫条件下H. pylori可发生不同的形态学变化,蛋白质是生命活动的最终执行者,无论何种表征蛋白质在其中将起重要作用,比较蛋白质组学是研究病原菌在各种胁迫因素下蛋白质表达差异的理想工具,现今较普遍使用的技术体系包括双向凝胶电泳,质谱分析及生物信息学分析。胆汁是机体重要的天然抗病原菌物质,研究H. pylori在胆汁中的存活能力,对于揭示H. pylori感染与十二指肠溃疡的发生规律有重要意义,为此我们研究了H. pylori在胆汁胁迫下的蛋白表达状态。丝状体是H. pylori除螺杆状和球形体以外的一种成活形式,对丝状体蛋白表达谱的研究,有助于揭示其发生规律。我们曾发现鞭毛丝帽蛋白与H. pylori结构性群体结构形成密切相关,然而,对生物质谱筛选的候选蛋白尚需作进一步的功能验证,为此,我们构建了H. pylori结构性群体形成相关蛋白鞭毛丝帽蛋白基因缺失突变株,为进一步验证此蛋白的功能及阐明H. pylori结构性群体的形成机制奠定工作基础。主要研究内容及研究结果如下:
一、胆汁对幽门螺杆菌的抑制及其蛋白质表达谱的初步研究
胆汁胁迫是人体内抑制H. pylori生长的不利条件之一,许多研究也表明胆汁能够抑制H. pylori的生长,但H. pylori仍能够在胆汁丰富的十二指肠球部存在,而对其他肝胆系统的感染较少见,因此我们推测胆汁的酸化影响其对H. pylori的抑制活性。为了研究H. pylori对胆汁胁迫的适应性调
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 of
filamentous 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 period
is 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 of
homologous 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.
一、胆汁对幽门螺杆菌的抑制及其蛋白质表达谱的初步研究
胆汁胁迫是人体内抑制H. pylori生长的不利条件之一,许多研究也表明胆汁能够抑制H. pylori的生长,但H. pylori仍能够在胆汁丰富的十二指肠球部存在,而对其他肝胆系统的感染较少见,因此我们推测胆汁的酸化影响其对H. pylori的抑制活性。为了研究H. pylori对胆汁胁迫的适应性调
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 of
filamentous 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 period
is 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 of
homologous 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.
引文
1. Blaser MY, et al. Hypothesis: the changing relationships of Helicobacter pylori and human: implications for health and disease. J Infect Dis, 1999, 179: 1523-1531.
2. Malaty HM, Nyren O. Epidemiology of Helicobacter pylori infection. Helicobacter. 2003; 8 Suppl 1: 8-12.
3.朱兴彦。会议简讯,中华医学信息导报,2003,01期。
4. Washburn MP, Yates JR 3rd. Analysis of the microbial proteome. Curt Opin Microbiot, 2000, 3(3): 292-297.
5. Mannazzu I, Guerra E, Ferretti R, et al. Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha. Biochim Biophys Acta, 2000, 1475(2): 151-156
6. Leboeuf C, Leblanc L, iuffray Y. Characterization of the ccpi gene of Enterococcus faecalis: identification of starvation-inducible proteins regulated by ccpA. J Bacteriol. 2000, 182(20): 5799-5806.
7. intelmann H, Scharf C, Hecker M. Phosphate starvation-inducible proteins of Bacillus subtitis: proteomics and transcriptional analysis. J Bacteriol. 2000, 182(16): 4478-4490.
8.陈敏章,胡伏莲,周殿元,等.幽门螺杆菌感染的基础与临床.北京:中国科学技术出版社,1997,405-411.
9. Masaharu Itoh, Kaori Wada, Satoko Tan, et al. Antibacterial action of bile acids against Helicobacter pylori and against its ultrastructural morphology: effect of unconjugated dihydroxy bile acid. J Gastroenterol, 1999, 34: 571-576.
10. bavid Y. Graham, M.D.M.A.C.G, Michael S. Osato. H. pylori in the pathogenesis of duodenal ulcer: interaction between duodenal acid load, bile and H. pylori. The American J of Gastroenterology. 2000, 95: 87-91.
11.贾继辉,于修平,郭辉玉,等.幽门螺杆菌结构性群体形成的研究.中华微生物和免疫学杂志,2003,23:507-512。
12.贾继辉,陈春燕,于修平,等.幽门螺杆菌球形休眠体形成的比较蛋白质组学研究.中华微生物和免疫学杂志,2003,10:288-291。
13.贾继辉.张茂修.周亚滨等,幽门螺杆菌丝状体的形成和回复性的研究.山东大学学报(医学版),2004,42:1-4。
14. George Toole, Heidi B Kaplan, Roherto Kolter. Biofilm formation as microbial development. Annu Rev Microbiol, 2000, 54: 49-56.
15. Chen X. Structural identification of a bacterial quorum-sensing signal containing boron. Nature, 2002, 287: 1973-1979.
16. Koji Yonekura, Saori Maki, David Gene Morgan, et al. The bacteria flagella cap as the rotary promoter of flagellin self assembly. Science, 2000, 290: 2148-2157.
2. Malaty HM, Nyren O. Epidemiology of Helicobacter pylori infection. Helicobacter. 2003; 8 Suppl 1: 8-12.
3.朱兴彦。会议简讯,中华医学信息导报,2003,01期。
4. Washburn MP, Yates JR 3rd. Analysis of the microbial proteome. Curt Opin Microbiot, 2000, 3(3): 292-297.
5. Mannazzu I, Guerra E, Ferretti R, et al. Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha. Biochim Biophys Acta, 2000, 1475(2): 151-156
6. Leboeuf C, Leblanc L, iuffray Y. Characterization of the ccpi gene of Enterococcus faecalis: identification of starvation-inducible proteins regulated by ccpA. J Bacteriol. 2000, 182(20): 5799-5806.
7. intelmann H, Scharf C, Hecker M. Phosphate starvation-inducible proteins of Bacillus subtitis: proteomics and transcriptional analysis. J Bacteriol. 2000, 182(16): 4478-4490.
8.陈敏章,胡伏莲,周殿元,等.幽门螺杆菌感染的基础与临床.北京:中国科学技术出版社,1997,405-411.
9. Masaharu Itoh, Kaori Wada, Satoko Tan, et al. Antibacterial action of bile acids against Helicobacter pylori and against its ultrastructural morphology: effect of unconjugated dihydroxy bile acid. J Gastroenterol, 1999, 34: 571-576.
10. bavid Y. Graham, M.D.M.A.C.G, Michael S. Osato. H. pylori in the pathogenesis of duodenal ulcer: interaction between duodenal acid load, bile and H. pylori. The American J of Gastroenterology. 2000, 95: 87-91.
11.贾继辉,于修平,郭辉玉,等.幽门螺杆菌结构性群体形成的研究.中华微生物和免疫学杂志,2003,23:507-512。
12.贾继辉,陈春燕,于修平,等.幽门螺杆菌球形休眠体形成的比较蛋白质组学研究.中华微生物和免疫学杂志,2003,10:288-291。
13.贾继辉.张茂修.周亚滨等,幽门螺杆菌丝状体的形成和回复性的研究.山东大学学报(医学版),2004,42:1-4。
14. George Toole, Heidi B Kaplan, Roherto Kolter. Biofilm formation as microbial development. Annu Rev Microbiol, 2000, 54: 49-56.
15. Chen X. Structural identification of a bacterial quorum-sensing signal containing boron. Nature, 2002, 287: 1973-1979.
16. Koji Yonekura, Saori Maki, David Gene Morgan, et al. The bacteria flagella cap as the rotary promoter of flagellin self assembly. Science, 2000, 290: 2148-2157.