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標題: Functional analysis and immunogenicity test of the Neisseria meningitidis outer membrane protein Ag473
作者: Liu, Yi-Lun
關鍵字: Neisseria meningitidis
出版社: 分子生物學研究所
摘要: 奈瑟氏腦膜炎雙球菌 (Neisseria meningitides, NM) 為一具有莢膜的革蘭氏陰性菌,根據其表面抗原莢膜多醣 (CPS) 的化學組成,可將之區分成 13 種血清群 (serogroup),其中以血清群 A、B、C、W135 及 Y 為主要的人類病原菌。 Ag473 為本實驗室新近發現的一個普遍存在於NM,帶有7個氨基酸的串聯重複序列(tandem repeat),功能未明,而具有成為疫苗潛力的表面脂蛋白質。為了探討 Ag473 可能的功能,本研究首先以ELISA、 FACS 及螢光顯微鏡觀察比較野生株Nm22209 與 Ag473 缺失株 (MT473) 之CPS、lipooligosaccharide (LOS) L3, 7, 9 以及外膜蛋白質 PorA。ELISA 的分析結果顯示兩者之 CPS、LOS 以及 PorA 之表現量沒有差異。但是 FACS 及螢光顯微鏡觀察卻發現,幾乎所有的 Nm22209 菌體都可偵測到 CPS,但大多數 MT473 的菌體上都偵測不到 CPS。根據此結果推測Ag473 缺失可能影響菌體與 CPS 結合之緊密度。螢光免疫染色觀察與人類細胞接觸之菌體表面抗原變化情形,發現 (1) 在感染的前 6 小時,黏附在細胞的菌體上無法偵測到 Ag473 的表現;在第 7 小時開始可偵測到表現 Ag473 的菌體,而且其數目隨感染時間之加長而增多;(2) 黏附在人類細胞上的 Nm22209,大部分的菌體都可偵測到 CPS,但並非均勻分布在菌體之表面。相對的,黏附的 MT473則幾乎都無法偵測到 CPS;(3) 以抗全菌之抗血清觀察發現黏附的 Nm22209 及 MT473 皆有絲狀結構,但兩者之抗原性 (antigenicity) 不同。此外,Nm22209 之黏附多成簇狀,而MT473 則呈分散狀,但後者在感染寄主細胞時其附著能力較 Nm22209 為佳。利用免疫沉澱法已搜尋到一個可能與 Ag473 有互動的蛋白質,由 MALDI-TOF 分析的結果推測,此蛋白質可能為 Opa (NM 與宿主細胞在黏附過程中的一個重要因子)或 RmpM。另一方面,為了探討脂化以及不同重複序列的 Ag473 變體之免疫性質是否有異,本研究以 Escherichia coli 表現 4 種變體以及兩種非脂化之 Ag473 重組蛋白質,並以所獲得的蛋白質進行小鼠免疫。初步結果顯示,上述重組蛋白質引發之抗體皆可辨識表現不同變體之NM,在兩種非脂化的變體中,以去除了 signal peptide之蛋白質效果最差,而將接受脂化的 Cys 突變為 Ser 之變體與對照組間並無明顯差異。此結果顯示,在維持 Ag473 之免疫特性方面,signal peptide 的存在比是否脂化為重要。本研究對於 Ag473 之了解提供了初步的探討,其確切之功能尚有待進一步的研究分析。
Neisseria meningitidis (NM) is a Gram negative, encapsulated bacterium which can be classified into 13 serogroups based on the chemical compositions of the capsular polysaccharide (CPS), with serogroups A, B, C, W135, and Y being the major human pathogens. Ag473, with 7-amino acid tandem repeats, is a lipoprotein of unknown functions newly identified in our laboratory. Expressed on the surface of all NM strains examined, this lipoprotein exhibits a good potential to serve as a vaccine component. In this study, CPS, LOS (lipooligosaccharides) L3, 7, 9, and outer membrane PorA in the wild-type Nm22209 and a mutant strain MT473 were examined by ELISA, FACS and fluorescence microscopy. The results from ELISA indicated that CPS, LOS and PorA are expressed at similar levels between the wild-type and the mutant strain. However, FACS showed that while almost all Nm22209 cells contain detectable amounts of CPS only a small portion of MT473 has CPS, suggesting that deficiency in Ag473 reduces the association between CPS and bacterial cells. Fluorescence microscopy revealed that 1) in the bacteria attached to the human epithelial cells, expression of Ag473 is undetectable until 7 hr post-infection, but expression levels increase as incubation is prolonged, 2) CPS is found on almost all of the attached wild-type bacteria while almost no CPS is detected on MT473, 3) filamentous structures with distinct antigenicity are observed in the attached bacteria of both strains. Furthermore, unlike Nm22209, which associates with the host cells in clustering, the mutant adheres dispersedly to the host cells in spite of a better adhesion. Immunoprecipitation in conjunction with MALDI-TOF analysis suggest Opa (a protein important during adhesion) or RmpM to be the protein possibly in close contact with Ag473. To investigate the effects of lipidation and numbers of the tandem repeat on the immunogenic attributes of Ag473, recombinant proteins of the four types of tandem repeats and the proteins with or without lipidation were expressed in E. coli and used to immunize mice. Preliminary results indicated that 1) each antiserum is able to recognize the NM strains expressing the different variants, 2) of the two non-lipidated immunogens, the protein without signal sequence exhibits weaker immunogenicity, and 3) a substitution of the Cys residue required for lipidation to Ser causes no effect on its immunogenicity. These results suggest that the signal sequence is more critical than lipidation in maintaining the immunogonicity of Ag473. The results presented in this thesis provide a preliminary understanding of Ag473 and further studies are necessary to define its function.
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