金黃色葡萄球菌毒性因子之研究

The study of virulence factors from Staphylococcus aureus

葛芷彤(Jhih-Tong Ge)

指導教授 ： 劉佳宜

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系所/碩士(2015年)

https://doi.org/10.6831/TMU.2015.00043

摘要

金黃色葡萄球菌 (Staphylococcus aureus)是一種主要的病原菌，所導致的疾病範圍很廣，從輕微的皮膚感染到致命的敗血症，近期更出現抵抗抗生素的超級細菌MRSA，增加了治療的困難度。根據文獻研究指出位於金黃色葡萄球菌的毒性蛋白，能夠幫助細菌識別並吸附到宿主細胞，並促進細菌分泌毒素並入侵細胞造成感染，同時有利於保護細菌不被宿主的免疫系統攻擊。在本研究中主要探討參與金黃色葡萄球菌毒素分泌與吸附相關因子SaeR與SdgA，希望在未來能夠利用蛋白質結構來進行藥物設計。
金黃色葡萄球菌發展了一個重要的機制來傳遞訊息，就是雙因子調控系統。根據文獻指出金黃色葡萄球菌的SaeR/S雙因子調控系統 (Two component system, TCS)和金黃色葡萄球菌感染具有高度相關性。活化的SaeS會促使SaeR被磷酸，進而調控其下游目標基因的轉錄，最終將導致金黃色葡萄球菌會產生細胞毒素。
另一方面，金黃色葡萄球菌的細胞壁吸附蛋白中，特異性黏著素Microbial Surface Component Recognizing Adhesive Matrix Mol¬ecule 家族蛋白大多都含有大量絲胺酸-天門冬胺酸重複序列 (serine-aspartate repeats, SDRs)，而clumping factor A (ClfA)為其中一種重要的吸附因子。當ClfA蛋白中的SDR區被醣基化時，就能抵抗宿主細胞蛋白水解酶的攻擊，並且促使血液感染造成宿主死亡。現今證實，SDR蛋白可以被高度醣化且與SdgA、SdgB這兩種醣基轉移酶的作用有緊密的關係；文獻指出SdgA、SdgB醣化作用的機制其實是具有一定的順序性：SdgB會先將醣基的部分加到SDR蛋白上，再由SdgA做進一步的修飾。
在本研究中，我們從金黃色葡萄球菌的基因組DNA放大出saeR、sdgA這兩個致病基因片段，並將其分別構築到pET21a與pET28a。此載體中具有六個組胺酸組成的標籤序列 (6His tag)，目的是為了使標的蛋白能夠順利被表達與純化。利用大腸桿菌表現系統將標的蛋白順利被表現出來，並用Ni2+-chelating chromatography可獲得大量高純度SaeR、SaeR (129- 229)與SdgA重組蛋白質，並進行重組蛋白質的結晶條件篩選與SaeR (129-229)晶體繞射數據收集。最後根據晶體繞射分析結果，可以知道SaeR (129-229)結構具有3個α-螺旋和6個β-褶片。帶有組胺酸標籤的SdgA、SdgB和ClfA重組蛋白，會進行醣化試驗，我們得到一個SdgA初步結晶結果與在大腸桿菌菌體內的初步醣化結果，未來將需要進一步確認與探討這些初步結果。

關鍵字

金黃色葡萄球菌 ； 毒性因子 ； 雙因子調控系統 ； 醣基轉移酶

並列摘要

Staphylococcus aureus is a kind of main bacterial pathogens, which cause disease in wide ranges from slight infection of skin to fatal septicemias, and there is appealing superbug MRSA with antibiotic resistant, increasing the difficulty of therapy. According to the recent reviews, we know that various proteins on the surface of S. aureus can help bacteria recognize and adhere to host cells and promote bacteria to secrete endotoxin that will invade cells leading to infection. At the same time, those proteins benefit to protect bacteria from immune system of host cells. In this study, we focus on studying adhesive and virulence related proteins on S. aureus such as: SaeR and SdgA. In the future, the structure of these proteins can give us an insight on potential drug design.
Pathogen’s two-component gene-regulatory systems tightly control virulence and immuno-modulatory factors in response to environmental stimuli. The SaeR/S two-component system is a major regulator system which involve in virulence of S. aureus. Activated SaeS will then activate SaeR through phosphorylation, resulting in transcription initiation at the promoter of target gene to synthesis cytotoxins.
On the other hand, in the cell wall adhesion protein of S. aureus, the major specific adherin is Microbial Surface Component Recognizing Adhesive Matrix Molecule (MSCRAMMs Family). Proteins in the family have most amount serine-aspartate repeats (SDR), and clumping factor A (ClfA) is viewed as a kind of important pathogenicity factor. The latest research reported that the glycosylated SDR with higher proteolytic resistance and cause death of bloodstream infection hosts. Currently, studies demonstrated that SDR proteins can be highly glycosylated and are related to two glycosyltransferases: SdgA and SdgB. According to literature reviews, the mechanism of glycosylation of SdgA and SdgB is in proper order: SdgB transfers glycosyl groups to SDR proteins, and then SdgA performs further modifications.
In this study, we amplified the SaeR and SdgA DNA fragment from S. aureus genomic DNA and clone into pET21a and pET28a vector, respectively which contained 6 His tag that allowed our target protein to be further expressed and purified. We managed to express the target protein from Escherichia coli (E.coli). By using the Ni2+chelating chromatography, abundance of high purity SaeR, SaeR (129-229) and SdgA protein with His tag was purified. Then, we analyzed SaeR and SdgA protein by protein crystallization and glycosylation test. In conclusion, our study revealed the crystal structure SaeR (129-229) of contain 3 α-helices and 6 β-strands. SdgA, SdgB and ClfA recombinant proteins will be further analyze on glycosylation test.

並列關鍵字

Staphylococcus aureus ； SaeR ； SdgA

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