臺灣博碩士論文加值系統

化膿性鏈球菌是一種重要的人類病原體，會導致多種的疾病，並會分泌許多蛋白質去影響宿主和病原體交互作用。化膿性鏈球菌感染的過程中，會導致傷口環境的酸化，形成膿瘡或壞死性病變，但是目前很少研究提到化膿性鏈球菌在酸性環境下如何調控毒性蛋白質的表現，增加細菌的入侵能力。因此，本研究的目的是要找出受酸性影響的化膿性鏈球菌分泌性蛋白質。首先，我們收集在中性條件(pH7.5)和微酸性條件(pH6.0)培養的化膿性鏈球菌分泌蛋白質樣品。進一步量測其生長曲線，發現化膿性鏈球菌在中性和酸性環境下的生長曲線並無太大差異。但經由一維膠體電泳分析，可以明顯看出中性和酸性環境下蛋白質有表現量上的差異。利用堆疊膠體電泳(stacking gel)將中性和酸性環境下取得的蛋白質回收，再以非標記串聯式質譜分析方法得到中性和酸性環境下的蛋白質體資訊。在本研究當中，我們總共鑑定到172個蛋白質受到環境酸化的影響，在連續兩個時間點以上的表現量有相同的顯著性差異，其中篩選出45 (26%)個蛋白質被預測可能是分泌蛋白質。根據過去研究的分析比對，分別對應到21個蛋白質與免疫抗性、營養源的攝取、對宿主的黏附力、導致凝血功能障礙及組織損傷等致病機制相關，其中已知受酸化刺激會誘導表現的蛋白質：化膿性鏈球菌熱源性外毒素B和線毛相關蛋白也在本研究中被觀察到。本研究鑑定到許多受酸化影響的化膿性鏈球菌致病相關分泌性蛋白質，此非標記蛋白質體的分析數據對於瞭解化膿性鏈球菌感染時引起的環境酸化的相關機轉是很重要的。

Streptococcus pyogenes is an important human pathogen that causes a broad range of diseases. S. pyogenes secretes a number of proteins involved in host-pathogen interactions. The acidic environment in the infected site can trigger S. pyogenes to generate an abscess or a necrotic lesion. But, little is known about how environmental acidification regulates expression of many virulence factors in S. pyogenes for severe infections. Therefore, the objective of this study was to identify the acid-influenced secreted proteins of S. pyogenes. First, the secretome samples of S. pyogenes were collected from neutral culture conditions (pH7.5) and from acidic culture condition (pH6.0) at designed sampling time points. The optical density was measured for establishment of growth curve. The growth curves were similar between neutral and acidic culture conditions. The protein patterns on SDS-PAGE show dissimilarity between the secretomes from acidic and neutral conditions. The secretome samples were cleaned up by a stacking gel and were analyzed by label-free LC-MS/MS analysis. The dynamic label-free LC-MS/MS profiling identified 172 proteins which are influenced by environmental acidification at least two consecutive time points. Among these, 45 (26%) the identified proteins are predicted secreted proteins. According to pathogenesis evidences, 21 proteins are pathogenesis-related protein and can influence immune evasion, nutrient acquisition, cell adhesion, hemolysis, and tissue damage. The known acid-induced proteins, SpeB and pilus-related protein are also observed in this study. In this study, we identified many acid-influenced secreted proteins related to Streptococcus pyogenes pathogenesis. Our label-free proteomic data is crucial for realizing mechanisms related to environmental acidification of group A streptococcal infection.

中文摘要...II
ABSTRACT...III
誌謝...IV
英文縮寫對照表...V
第一章 緒論...1
1.化膿性鏈球菌...1
1.1化膿性鏈球菌及其病理介紹...1
2.化膿性鏈球菌分泌性蛋白質與環境的關係...2
2.1分泌性蛋白所扮演的角色...2
2.2疾病造成皮膚傷口的酸化現象...2
2.3在酸性環境下轉錄物的調控...2
3.利用蛋白質體技術分析細菌分泌性蛋白體的運用...2
3.1蛋白質體學...2
3.2非標記蛋白質體定量方法在分泌蛋白質上的應用...3
第二章 研究目的...4
第三章 研究方法與材料...5
1.研究架構簡述...5
2.分泌性蛋白體收集...7
2-1化膿性鏈球菌菌株...7
2-2培養化膿性鏈球菌於添加蛋白酶抑制劑的培養液...7
2-3蛋白體樣本的收集...7
3.分泌性蛋白體樣本前處理...7
3-1蛋白體樣本濃度測定...7
3-2超高速離心濃縮及去鹽...7
3-3一維膠體電泳(SDS-PAGE)...8
3-4銀染(Silver stain)...8
3-5堆疊膠體電泳(Stacking gel)...8
3-6膠體內酵素水解(In gel digestion)...9
4.質譜分析...10
4-1高效液相層析串聯質譜儀(LC-MS/MS)...10
4-2非標記定性與定量軟體分析(MaxQuant)...10
第四章 結果與討論...12
1.分泌性蛋白體樣本...12
1-1化膿性鏈球菌的培養...12
1-2一維膠體凝膠電泳...14
2.蛋白質樣本分析...16
2-1蛋白質樣本分析與MAXQUANT的定量與定性...16
2-2受到環境酸化影響的蛋白質...18
2-3分泌性蛋白體定量分析...20
2-4 45個分泌性蛋白質...36
2-5蛋白質樣本組成分析...40
3.蛋白質體的定量分析與差異表現量的毒性因子找尋...41
3-1差異表現量毒性因子的找尋與文獻探討...41
3-2差異表現量毒性因子與文獻的驗證...45
第五章 結論...46
第六章 參考文獻...47
自述...51

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