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系統識別號 U0026-0812200911173152
論文名稱(中文) A群鏈球菌熱原性外毒素B抑制巨噬細胞吞噬死亡細胞機制之探討
論文名稱(英文) Streptococcal Pyrogenic Exotoxin B-mediated Inhibition of Apoptotic Cell Clearance by Macrophages
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 92
學期 2
出版年 93
研究生(中文) 吳岳穎
研究生(英文) Yueh-Ying Wu
電子信箱 kellypepe@yahoo.com.tw
學號 s4691111
學位類別 碩士
語文別 中文
論文頁數 75頁
口試委員 口試委員-莊偉哲
口試委員-黃智雄
指導教授-林以行
口試委員-黎煥耀
中文關鍵字 鏈球菌  吞噬作用  熱原性外毒素 
英文關鍵字 SPE B  Protein S 
學科別分類
中文摘要   在先天性免疫系統中,死亡細胞快速地被巨噬細胞清除,對於維持細胞的恆定扮演了重要的角色。在先前的研究中指出,血清中的蛋白質S (protein S) 會媒介巨噬細胞去清除死亡細胞。而我們發現,由A群鏈球菌所分泌的外毒素,熱原性外毒素B (SPE B),會切割蛋白質S,並影響其生物功能。化膿性鏈球菌 (Streptococcus pyogenes) 為人類常見的傳染性致病菌之一,能造成非常廣泛性的疾病,輕者如咽喉炎、小膿泡疹,嚴重者則會造成致死性的併發症,如壞疽性肌膜炎、毒性休克症候群和菌血症。另外,此菌也會造成感染後的併發症,如急性風濕熱和急性腎絲球炎。SPE B為眾多在A群鏈球菌致病過程中參與的致病因子之一,而在過去的研究中指出,SPE B 這個cysteine protease的活性存在與否對於造成較嚴重的病症是有相關性的。在本實驗中,我們證實了熱原性外毒素B,會抑制蛋白質S所媒介的吞噬作用。首先,我們觀察到由血清所媒介巨噬細胞吞噬死亡細胞的作用會受到熱原性外毒素B的抑制。另外,熱原性外毒素B會去切割蛋白質S並且造成蛋白質S所媒介的吞噬作用受到抑制,而這樣的作用結果是和熱原性外毒素B的劑量與作用時間呈現正相關性的。再者,這些現象都與熱原性外毒素B的蛋白脢活性有關,因為在熱原性外毒素B的活性突變蛋白C192S,熱原性外毒素A,以及受到蛋白脢抑制劑E64去活性的熱原性外毒素B,其吞噬死亡細胞的過程都沒有受到抑制。接著我們進一步去研究熱原性外毒素B在蛋白質S上的切割位置,我們推測在蛋白質S的C端胺基酸序列615和616的SK,可能為熱原性外毒素B第一個切割的切位。我們認為當C端受到切割的蛋白質S,可能已經影響了其媒介吞噬作用的功能。除此之外,我們也發現受到切割的蛋白質S,會喪失其與死亡細胞之間的結合作用。根據以上的實驗結果,我們可以推測熱原性外毒素B在A群鏈球菌的感染過程中可能的新致病機轉,主要的影響是血清中的蛋白質S會因為熱原性外毒素B的切割,進而喪失了媒介巨噬細胞清除死亡細胞的功能,而可能導致免疫反應的缺陷,進而造成更嚴重的病程發展。
英文摘要   Clearance of apoptotic cells rapidly by phagocytes is important in innate immunity to maintain cell homeostasis. Serum-derived protein S-mediated apoptotic cell clearance by macrophages was recently demonstrated. In this study, we show that streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease produced by Streptococcus pyogenes, causes an inhibitory effect on protein S-mediated phagocytosis. Streptococcus pyogenes is a Gram-positive bacterium that can cause a wide range of diseases, including uncomplicated pharygitis, bacteremia, necrotizing fasciitis, and streptococcal toxin shock syndrome (STSS). This bacterium is also responsible for nonsuppurative sequelae, such as acute rheumatic fever and acute glomerulonephritis. There is vast repertoire of virulence factors that may participate in group A streptococcal (GAS) infections. SPE B, a streptococcal cysteine protease, may play an important role in severe GAS pathogenesis. We found that the ability of murine peritoneal macrophage phagocytosis of apoptotic cells promoted by serum was inhibited in the presence of SPE B. Interestingly, SPE B cleaved protein S directly via dose- and time-dependent manner and reduced protein S-mediated macrophage phagocytosis of apoptotic cells. These effects caused by SPE B were dependent on its protease activity as compared with SPE B mutant C192S, SPE A, or SPE B inactivation by cysteine protease inhibitor E64 pretreatment. Analysis of SPE B-specific cutting site and the cleaved protein S sequencing suggest that the C-terminal site of protein S, SK (a.a. 615 and 616), acts as the initial site of SPE B cleavage function. The loss of support on macrophage phagocytosis by protein S actually occurred while C-terminal site was disrupted by SPE B. Further studies demonstrated that cleaved protein S lost the binding ability to the surface of apoptotic cells. Therefore, a novel pathogenic role of SPE B is proposed that SPE B causes protein S destruction, followed by the inhibition of apoptotic cell clearance by macrophages.
論文目次 中文摘要
英文摘要
致謝
目錄
圖目錄
縮寫檢索表

緒論
特定目標與實驗設計
實驗材料
A. 實驗動物
B. 細菌株
C. 儀器與藥劑試劑
D. 耗材
實驗方法
A. 細胞培養
B. SPE B及其突變株C192S重組蛋白的表現及純化
C. 蛋白質定量
D. 蛋白質電泳
E. 銀染
F. 分離小鼠腹腔巨噬細胞
G. 誘導細胞凋亡並標示綠色螢光
H. 吞噬能力測試
I. 凋亡細胞與protein S結合情形偵測
J. 收集細菌培養之培養液

結果
1. ProSPE B與突變蛋白C192S的純化
2. SPE B降低血清媒介巨噬細胞吞噬死亡細胞的作用
3.SPE B降低protein S媒介巨噬細胞吞噬死亡細胞的作用
4. SPE B所造成的protein S切割
5. SPE B降低protein S與死亡細胞的結合
6. SPE B降低C4BP與死亡細胞的結合
7. 細菌培養液降低血清中protein S與死亡細胞的結合
討論
參考文獻

附錄
自述
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