Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22191
標題: Characterization of Staphylococcus aureus ATCC8095
金黃葡萄球菌 ATCC8095 之特性描述
作者: 洪敬晟
Hung, Ching-Cheng
關鍵字: Staphylococcus aureus
金黃葡萄球菌
ATCC8095
Spa
SrtA
ATCC8095
Spa
SrtA
出版社: 分子生物學研究所
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摘要: 金黃色葡萄球菌為一伺機性病原菌,會分泌許多種具超級抗原 ( superantigenic )特性的毒素,同時也會導致廣範圍的疾病,例如:食物中毒、肺炎、心內膜炎或敗血症等。過去的研究大多專注於臨床分離的菌株,首先為了瞭解 SA 適應不同環境的生理生態特徵,本研究使用 SDS-PAGE 針對分離自奶油派的 ATCC8095 及臨床分離的 SA 菌株,分析其胞外蛋白質圖譜。在 ATCC8095 中發現一蛋白質條帶在 55 kDa 較其它菌株明顯,此蛋白質條帶經由質譜儀及西方墨點法分析確認為 Staphylococcal protein A (Spa),接著分析 ATCC8095 及臨床分離株 (TC1057) 其 Spa 蛋白質在不同時間表現情形。ATCC8095 菌體相關連之 Spa 蛋白質,在對數期 (log phase) 開始增加而穩定期 (stationary phase) 逐漸減少;直至後穩定期 (post-exponential phase) 幾乎偵測不到,胞外的 Spa 蛋白質則可穩定的增加至後對數期,相對地, TC1057 菌體相關連之 Spa 蛋白質在穩定期仍維持著高表現量。已知 Spa 蛋白質會因 sortase (SrtA) 的作用,經由其 LPXTG motif 鑲嵌至細胞壁中,由 spa 及 srtA 的序列分析,顯示 ATCC8095 之 spa 所表現的蛋白質帶有典型的LPXTG 鑲嵌 motif ,但是其 srtA 基因在第96個密碼子具有一個無意義突變(nonsense mutation),產生了一個截斷的(truncated)蛋白質,此 srtA 的無意義突變或許是造成 ATCC8095 之胞外最大量蛋白質為 Spa 的原因,而此推測經由比較 RN4220 及其 srtA 突變株之 Spa 蛋白表現圖譜可獲得驗證。在上述的研究過程中,發現srtA 突變株與野生株相較之下,突變株之胞外 Spa 對 IgG 展現較低的結合能力,野生株其胞外 Spa 經由 pentaglycine 專一性的蛋白內切酶 lysostaphin 處理後,大幅降低其 IgG 結合能力,此結果指出 Spa 其 C 端與肽聚醣(peptidoglycan)的連結,使 Spa 具有較高的 IgG 結合能力。最近已發現 srtA 的突變會造成毒性下降,且可以誘發具保護性的免疫反應,而無論是 Spa 之 IgG 結合能力下降導致的毒性下降,或者可以誘發出具保護力的免疫反應,都值得進一步的研究。
Staphylococcus aureus (SA), an opportunistic pathogen capable of secreting multiple toxins with superantigenic activities, causes a wide spectrum of serious diseases including food poisoning, pneumonia, and septicemia. Studies have been focused mostly on clinical isolates. As an initial effort to investigate the ecophysiological traits associated to SA adaption to different environments, this study analyzed the extracellular protein profiles of the the SA strain ATCC8095 isolated from cream pie and clinical isolates by SDS-PAGE. A protein band around 55 kDa was found distinctly in ATCC8095 but not others. This protein was confirmed to be the Staphylococcal protein A (Spa) by mass spectrometry and Western blotting. Next, the temporal and spatial distributions of Spa protein in ATCC8095 and in one clinical isolate (TC1057) were examed. In strain ATCC8095, the cell-associated Spa levels increased during log phase but diminished gradually in stationary phase, and barely detectable in post-stationary phase, while the extracellular Spa steady increased to post-stationary phase. In contrast, the levels of cell-associated Spa in TC1057 maintained at the highest level in the stationary phase. It is known that Spa protein is anchored to the cell wall via the LPXTG motif and this process is catalyzed by the sortase A (SrtA). Sequence analysis of the spa and srtA genes revealed that the spa gene of ATCC8095 encodes a protein with typical LPXTG anchorage motif but the srtA gene has a nonsense mutation at codon 96 resulting in a truncated protein. The results indicate that a nonsense mutation in SrtA might responsible for the observation that Spa protein is the most abundant extracellular protein in ATCC8095. This speculation is confirmed by comparing the Spa expression patterns between the wild type and the srtA knockout RN4220 strains. In the course of the above studies, it was noted that the extracellular Spa (eSpa) protein of the srtA mutant strains exhibit lower IgG binding activities than that of wild type strains. The IgG binding activity of the eSpa from the wild type strain was reduced after treating with pentaglycine specific endopeptidase lysostaphin. This result indicates that the linking of the C-terminus of Spa to the peptidoglycan renders Spa higher affinity to IgG. Recently, a srtA mutant has been shown to be attenuated and can induce protective immunity. Whether the reduction of IgG binding activity of Spa contributing to the attenuation of virulence and induction of protective immune response were deserved further to investigate.
URI: http://hdl.handle.net/11455/22191
其他識別: U0005-0602201216242400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0602201216242400
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