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標題: Proteomic analysis of outer membrane proteins of Neisseria meningitidis, Neisseria gonorrhoeae and Neisseria lactamica
作者: 黃一民
Huang, Yi-Min
關鍵字: Neisseria meningitidis;奈瑟氏菌;Proteomics;outer membrane protein;蛋白質體;外膜
出版社: 分子生物學研究所
引用: 洪雅琪 (2005) 以免疫蛋白質體學之策略鑑定差異表現之微量蛋白質。國立中興大學分子生物研究所,碩士論文。 Agne`s Perrin, Ste′phane Bonacorsi, Etienne Carbonnelle, Driss Talibi, Philippe Dessen, XavierNassif, and Colin Tinsley. 2002. Comparative Genomics Identifies the Genetic Islands That Distinguish Neisseria meningitidis, the Agent of Cerebrospinal Meningitis, from Other Neisseria Species. Infection and immunity. p. 7063–7072 Baart G. J., Willemsen M, Khatami E, de Haan A, Zomer B, Beuvery E. C., Tramper J, Martens D. E. 2008. Modeling Neisseria meningitidis B metabolism at different specific growth rates. Biotechnol Bioeng. 101(5):1022-35. Bernardini G, Braconi D, Santucci A. 2007. The analysis of Neisseria meningitidis proteomes: Reference maps and their applications. Proteomics. 7(16):2933-46 Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. 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The majority of genes in the pathogenic Neisseria species are present in non-pathogenic Neisseria lactamica, including those designated as ''virulence genes''. BMC Genomics. 7:128 Mignogna G, Giorgi A, Stefanelli P, Neri A, Colotti G, Maras B, Schininà M. E. 2005. Inventory of the proteins in Neisseria meningitidis serogroup B strain MC58. J Proteome Res. 4(4):1361-70. Nsofor MN, Ryals PE, Champlin FR. 2006. Subcellular distribution of Plp-40, a lipoprotein in a serotype A strain of Pasteurella multocida. Biochim Biophys Acta.8:1160-1166. Pannekoek Y, Huis in ''t Veld R, Hopman CT, Langerak AA, Speijer D, van der Ende A. 2009. Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis. FEMS Microbiol Lett. 294(2):216-24. Rabilloud T, Carpentier G, Tarroux P. 1988. Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. Electrophoresis. 9(6):288-91. Sierra GV, Campa HC, Varcacel NM, Garcia IL, Izquierdo PL, Sotolongo PF, et al. Vaccine against group B Neisseria meningitidis: protection trial and mass vaccination results in Cuba. NIPH Ann 1991;14(2):195–207 [discussion 208–10]. Tettelin,H., Saunders,N.J., Heidelberg,J., Jeffries,A.C., Nelson,K.E., Eisen,J.A., Ketchum,K.A., Hood,D.W., Peden,J.F., Dodson,R.J., Nelson,W.C., Gwinn,M.L., DeBoy,R., Peterson,J.D., Hickey,E.K., Haft,D.H., Salzberg,S.L., White,O., Fleischmann,R.D., Dougherty,B.A., Mason,T., Ciecko,A., Parksey,D.S., Blair,E., Cittone,H., Clark,E.B., Cotton,M.D., Utterback,T.R., Khouri,H., Qin,H., Vamathevan,J., Gill,J., Scarlato,V., Masignani,V., Pizza,M., Grandi,G., Sun,L., Smith,H.O., Fraser,C.M., Moxon,E.R., Rappuoli,R. and Venter,J.C. 2000. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science. 287 (5459), 1809-1815. Thomas E. Vaughana,∗, Paul J. Skippb, C. David O’Connorb, Michael J. Hudsona, Richard Viponda, Michael J. Elmorea, Andrew R. Gorringe. 2006. Proteomic analysis of Neisseria lactamica and Neisseria meningitides outer membrane vesicle vaccine antigens. Vaccine. 5277–5293 Wise G. E., Lin F. 1991. Transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride membranes. J Biochem Biophys Methods. 22(3):223-31. Wu H. J., Wang A. H., Jennings M. P. 2008. Discovery of virulence factors of pathogenic bacteria. Curr Opin Chem Biol. 12(1):93-101.
Both Neisseria meningitidis and Neisseria gonorrhoeae are the sole pathogens in the Neisseria species. N. meningitidis lives at human nasopharynx, causes opportunistic infection leading to meningitis and septicemia, but N.gonorrhoeae colonizes the human genitals and causes gonorrhea. Neisseira lactamica also lives at human nasopharynx but it is not normally pathogenic. These three neisseria species have genetic similarity but are different at pathogenicity or adaptability. Previous research used comparative genomics to explore the genes which are related to pathogenicity, however these genes are not organized in large coromosomal islands. A later research showed that most virulence genes in the pathogenic Neisseria species are present in non-pathogenic N. lactamica. In this research, we used proteomic strategies to search for outer membrane proteins that may be associated with bacterial pathogenicity or adaptability. First, we prepared the crude outer membrane preparation and constructed 2 dimentional electrophoresis maps. After that, we focus on the region of map composed mainly by small molecular weight proteins and compared the expression patterns between these three species. MALDI-TOF and LC/MS are used for identification of target spots that may be involved in bacterial pathogenicity or adaptability. According to mass spectrometry results and public genome sequence data, we can prepare recombinant protein by E.coli expression system and then use immune mice to prepare polyclonal antibody. Thus, we can use Western blotting and 2D-Western to check protein expression patterns and confirm the spot distribution on the 2DE map respectively. Spot 97 is identified as Eda (4-hydroxy-2-oxoglutarate aldolase) and functionally involved in metabolic process including pyruvate and glutamine family amino acid biosynthesis. According to 2DE map Eda was predicted to express in pathogenic N. meningitidis and N.gonorrhoeae only, not in commensal N. lactamica. Immunoblotting results showed that Eda has similar Mr between these three species but lower pI in N. meningitidis and N. lactamica. Spot 1 is identified as Ppi (Peptidyl-prolyl cis-trans isomerase) and functionally involved in protein translation and modification. According to 2DE map Ppi was predicted to express in pathogenic N. meningitidis and N.gonorrhoeae only, not in commensal N. lactamica. Immunoblotting results showed that Ppi has similar Mr and pI between N. meningitidis and N.gonorrhoeae but lower Mr in N. lactamica. Spot 18 is identified as Hypothetical protein NMB1500 and unknown function, but it contains a motif belonging to universal sress protein (USP) family. According to 2DE map Hypo NMB1500 was predicted to express in N.gonorrhoeae only. Immunoblotting results showed that Hypo NMB1500 containing multiple spots with closely related Mr and pI, and also expressed in all three species. However, Hypo NMB1500 has lower pI in N. meningitidis and N. lactamica and slightly Mr changes between these species. Spot 11 is identified as atpC (F0F1 ATP synthase subunit epsilon) and functionally involved in ATP synthesis. According to 2DE map atpC was predicted to express in pathogenic N. meningitidis and N.gonorrhoeae only, not in commensal N. lactamica. Immunoblotting results showed that atpC has different Mr and pI between N. meningitidis and N.gonorrhoeae but not detected in N. lactamica. In this research, we compared protein expression patterns of four target proteins between N. meningitidis, N.gonorrhoeae and N. lactamica. We found that even though protein sequences are highly conserved between species, but Mr or pI of expressed proteins can be different. That is why 2DE map comparison and immunoblotting result are not consistent. Therefore, future works are needed to explore if post-translational modifications are the major cause of Mr or pI changes, and if these four proteins are associated with bacterial pathogenicities or adaptability. Also, it is important to confirm if atpC does not express in N. lactamica.

奈瑟氏菌中,只有 Neisseria meningitidis (NM) 和 Neisseria gonorrhoeae (NG) 具致病性,NM 棲息於人類鼻咽並可伺機感染侵入宿主導致腦膜炎及敗血症,而NG 則感染人體生殖道導致淋病,Neisseria lactamica (NL) 和 NM 同樣棲息於人類鼻咽中,但不具致病性。NM, NG 以及 NL 遺傳上相似度高,卻具備不同的致病特徵以及適應性,前人利用比較基因體學探討與致病特徵的差異有關的基因,發現大部分與致病相關的基因在 NM 染色體中不會聚集成 genetic island 的形式,此外進一步之研究發現大部分致病基因,同樣存在於非致病的 NL 中。本篇研究改由蛋白質體學的策略著手,分析外膜組成中可能與菌種致病性或適應性有關之蛋白,首先萃取外膜組成建立二維電泳圖譜,針對以小分子蛋白為主的區域進行分析,比對 NM, NG 以及 NL 之差異,挑選出可能與致病性或適應性有關的差異蛋白點,利用質譜儀鑑定其身份後,將對應的基因轉殖至 E.coli 製備重組蛋白,再透過免疫小鼠製備多株抗體,得到抗體後利用 Western 以及 2D-Western 確認目標蛋白在菌種間表現的情形,以及確認在二維電泳圖譜上分佈的位置。Eda (4-hydroxy-2-oxoglutarate aldolase)為 spot 97 之身份,其功能與代謝相關,參與 pyruvate 以及 glutamine family 胺基酸之生合成,根據二維電泳圖譜比對預測其只在具致病性的 NM 及 NG 中表現,在非致病的 NL 不表現。利用抗體確認後顯示雖然 Eda 在菌種間表現蛋白之分子量相同,但在 NM 以及 NL 中 pI 較小,因此事實上在三個菌種中皆有表現。Ppi (Peptidyl-prolyl cis-trans isomerase) 為 spot 1 之身份,其功能與蛋白質之轉譯及修飾作用相關,亦可促進蛋白之折疊 (folding),根據二維電泳圖譜比對預測其只在致病性的 NM 及 NG 中表現,在非致病的 NL 不表現。利用抗體確認後顯示 Ppi 在 NM, NG 中表現蛋白點之分子量及 pI相當,在 NL中表現蛋白之分子量較小,因此其實 Ppi 在三個菌種皆有表現。Hypothetical protein NMB1500 為 spot 18 之身份,其功能目前未知,但其序列中包含一段與 Universal stress protein (USP) 相似的 motif ,根據二維電泳圖譜比對預測其只在感染生殖道之 NG 中表現,在 NM 以及 NL 皆不表現。利用抗體確認後顯示 Hypo NMB1500 在二維電泳圖譜中具有多個蛋白點,集中分佈於分子量及 pI 相近的區域,在三個菌種皆有表現,但分佈區域之 pI 於 NM, NL 較小且表現蛋白之分子量在三個菌種皆略有差異。atpC (F0F1 ATP synthase subunit epsilon) 為 spot 11 之身份,其功能與合成 ATP 及質子運輸有關,根據二維電泳圖譜比對預測其只在具致病性的 NM 及 NG 中表現,在非致病的 NL 不表現。利用抗體確認後顯示在 NM 及 NG 中皆有表現但分子量及 pI 皆不同,無法偵測到在 NL 中的表現情形。本篇論文分析四個目標蛋白在菌種間表現之差異,雖然蛋白序列相似,但不同菌種的表現蛋白皆具有分子量或 pI 之差異,導致圖譜比對結果與實際結果不一致,是否轉譯後修飾作用為影響之主因,以及與致病性及適應性的相關性值得後續研究,而 atpC 在 NL 是否確實不表現也值得進一步確認。
其他識別: U0005-1208200918043300
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