BSP相似重複序列家族蛋白在轉醣鏈球菌所引起的感染性心內膜炎中細菌逃脫免疫逃脫攻擊所扮演的角色及機制

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Title:	BSP相似重複序列家族蛋白在轉醣鏈球菌所引起的感染性心內膜炎中細菌逃脫免疫逃脫攻擊所扮演的角色及機制 BSP-like repeat family proteins in bacterial immune evasion in Streptococcus mutans-induced infective endocarditis
Authors:	廖千晴 Liao, Chien-Ching
Contributors:	醫學科學研究所碩士班鍾筱菁
Keywords:	感染性心內膜炎;轉糖鏈球菌;自體溶體素蛋白;Ahp蛋白 Infective endocarditis;Streptococcus mutans;AtlA protein;Ahp protein
Date:	2023-06-20
Issue Date:	2023-12-15 14:05:28 (UTC+8)
Abstract:	轉糖鏈球菌(Streptococcus mutans)是人類口腔中常見的細菌也是細菌群中比例佔多數的，是造成蛀牙(Dental carries)主要致病菌。此細菌經由口腔手術進入血液也能造成短暫性的菌血症，這些進入血液中的細菌其表面蛋白能幫助細菌躲避宿主免疫細胞的攻擊，能順利附著在受損的心臟瓣膜上，形成贅疣引發感染性心內膜炎(Infective endocarditis; IE)。從實驗室過去的研究結果顯示，S. mutans 的自溶素(Autolysin; AtlA)，可以透過與血漿纖維蛋白結合增加細菌逃避嗜中性白血球的吞噬。另外，在S. mutans中找到與AtlA同樣屬於BSP相似重複序列家族蛋白，為Ahp。在過去感染性心內膜炎動物模型的結果顯示，atla和ahp的缺失皆會降低S. mutans在血液存活率，從而降低心內膜炎。因此，在本研究中我們設定了兩個目標。首先，確定了S. mutans於人類血漿作用下在180-245kDa形成了含有Ahp蛋白的複合物，經檢驗結果得知其中含Complement C3及Complement factor H等補體蛋白。為了瞭解其中的機制以及差異，首先建構了ahp剔除株及ahp-bsp domain剔除株。根據結果顯示，在ahp缺失的突變菌株中補體因子iC3b和FH與野生株相比有明顯得減少。而值得注意的是，在ahp-bsp domain缺失的突變菌株中發現補體因子iC3b和FH與野生株相比則是相似的。所以我們認為S. mutans與C3及FH的結合位置並非在BSP domain上。接著透過全血殺菌實驗得知Ahp蛋白對S. mutans的重要性，但該階段的分子機制還不是很清楚。另外，已知鈣離子可以引起AtlA蛋白N端的移除，進而促進AtlA的成熟及功能提升。在實驗室過去研究發現鈣離子無法在vick剔除株中引發成熟的AtlA。我們透過交聯蛋白測試尋找負責AtlA的成熟的蛋白?系統。在初步結果中發現一個含有AtlA的蛋白聚合物會在細胞壁中形成，經檢驗結果我們建構clpl剔除株及gtfB剔除株。在添加鈣離子與無添加的結果顯示兩種基因的缺失不成熟的AtlA確實減少了，至於其中確切的機制將有待後續更多研究來確認。 Streptococcus mutans is a common bacterium that exists in the human oral cavity and often times may enter into the circulatory system via open wound in the mouth. Upon entrance, bacteria cells may overcome innate immune attack by expressing certain virulence protein to compromise the immune system. In some cases, these persistent bacterial strains may adhere onto damaged heart valves, forming vegetation and cause infective endocarditis (IE). Our previous reports demonstrated that S. mutans protein autolysin (Autolysin; AtlA) enhances biofilm formation on the heart valves through mediating extracellular DNA (eDNA) release and resists neutrophil killing via fibronectin binding in blood plasma. Interestingly, we identified another protein Ahp that contains BSP domain repeats which is also present in AtlA. By using in vivo rat experimental IE model, results showed that deletion of AtlA and Ahp reduces S. mutans blood survival. Subsequently, reduced ability of the bacteria to cause endocarditis. These results is parallel with ex vivo whole blood bactericidal assay. Surprisingly, we discovered that BSP domain in Ahp contributes to deposition of the complement factor H and might be a key to bacterial survival in blood, but the insights are still remains unclear. Apart from that, mature AtlA is important in biofilm formation and eDNA release, AtlA maturation requires calcium ions as reported previously. However, we observed that cell wall anchored AtlA also interacts with other proteins inside the cell, this includes an upstream sensor protein GtfB and ClpL. How these protein engage in AtlA maturation and pathogenesis remains obscure. Ultimately, both BSP containing AtlA and Ahp are crucial factors for S. mutans in biofilm formation, blood survival and vegetation expansion in IE.
Description:	碩士指導教授:鍾筱菁委員:林秋烽委員:吳珊瑩委員:鍾筱菁
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Medical Sciences] Dissertation/Thesis

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