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研究生： 王琴雅
Wang, Chin-Ya
論文名稱： Prc在K1大腸桿菌逃離人類血清中補體擊殺所扮演的角色
The Role of Prc in Escherichia coli K1 Evasion of Complement-mediated Killing in Human Serum
指導教授： 張南山
Chang, Nan-Shan
鄧景浩
Teng, Ching-Hao
學位類別： 碩士
Master
系所名稱： 醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2011
畢業學年度： 99
語文別： 英文
論文頁數： 68
中文關鍵詞： K1大腸桿菌 、Prc蛋白酶 、補體系統 、免疫球蛋白Ｇ
外文關鍵詞： Escherichia coli K1, Prc, Complement, IgG
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    • K1大腸桿菌是革蘭氏陰性細菌中，最容易造成新生兒腦膜炎的致病菌。K1大腸桿菌引發的新生兒腦膜炎，大部分是藉由血液傳播感染，在細菌入侵到中樞神經系統之前，會藉由一些機制來逃離宿主免疫系統的攻擊，且在血液中大量複製造成菌血症。與K1大腸桿菌造成菌血症相關的致病因子，將有潛力成為標的因子用以發展預防或治療此疾病的新方法。Prc是細菌的蛋白酶，主要用來修飾細菌的蛋白質及使細菌生長在低鹽高溫的環境。之前的文獻中發現Prc及與它相似的蛋白CtpA會參與致病菌抵抗宿主細胞的作用，例如：Salmonella typhyimurium的Prc會讓它存活在巨噬細胞中。Brucella suis、Burkholderia mallei的CtpA會增加這些致病菌在老鼠體內感染的毒性及讓它們在巨噬細胞中存活。本文主要是想探討K1大腸桿菌在致病的過程中，Prc扮演的角色。prc基因剔除掉的K1大腸桿菌 (prc突變株) 在老鼠體內造成菌血症的能力及在血清中的生長皆下降，且會導致細菌表面上有較多補體蛋白C3b及membrane attack complex (MAC) 的黏附。這個結果顯示K1大腸桿菌在宿主體內造成嚴重的菌血症，是由於Prc會讓K1大腸桿菌逃離補體的攻擊。prc突變株在替代路徑移除掉的血清中生長會下降，在傳統路徑移除掉的血清中卻沒有這個現象。另外，prc突變株表面有較多免疫球蛋白Ｇ及補體蛋白C1q的直接黏附。這些顯示Prc會降低K1大腸桿菌表面免疫球蛋白Ｇ及補體蛋白C1q直接黏附的量，進而逃脫傳統路徑所活化的補體攻擊。與原本的K1大腸桿菌相比，prc突變株有明顯不同的外層膜蛋白型態，其中，部分不同的外層膜蛋白會被免疫球蛋白Ｇ辨識。另外，prc突變株會對MAC的攻擊較為敏感。這些代表prc基因剔除掉之後會導致K1大腸桿菌的外層膜蛋白型態改變，這樣的變化可能會造成免疫球蛋白Ｇ較容易辨識細菌表面及造成細菌對MAC的攻擊較為敏感。總結以上，Prc在K1大腸桿菌所引發的嚴重菌血症中扮演重要的角色，也許它可以拿來發展新的預防或是治療這個疾病的方法。

    Escherichia coli K1 (E. coli K1) is the most common gram-negative bacterium causing neonatal meningitis. E. coli meningitis develops as a result of hematogenous spread. Therefore, to survive and replicate in the blood are prerequisites for the pathogen to further infect the central nerve system. Thus, identification and characterization of bacterial factors required for E. coli K1 bacteremia may provide clues for development of novel preventive and therapeutic strategies against this E. coli-caused disease. Prc, a bacterial periplasmic protease, is involved in C-terminal processing of some bacterial proteins. This protease is required for survival of E. coli at 42 ºC under low osmolarity. Prc and its homologue CtpA contributes to the host-microbe interaction in many pathogenic bacteria. Prc of Salmonella typhyimurium is required for survival within macrophages. CtpA of Brucella suis and Burkholderia mallei contributes to these pathogens’ full virulence in mice in addition to survival within macrophages. Deletion of prc decreased the ability of E. coli K1 to induce bacteremia in mice and to survive in normal human serum. The prc mutant of E. coli K1 exhibited higher levels of C3b and membrane attack complex (MAC) deposition in the serum than its wild-type suggesting that Prc may contribute to E. coli K1 evasion of the complement-mediated killing in the serum so as to allow the bacteria to cause a high level of bacteremia in hosts. Deletion of prc decreased the survival of E. coli K1 in the alternative pathway-blocked serum, but did not affect the bacterial survival in the classical pathway-blocked serum. Besides, the prc mutant exhibited higher levels of IgG and direct C1q deposition on the bacterial surface. These results suggest that Prc may contribute to serum survival of E. coli K1 through facilitating bacterial evasion from direct binding of IgG and C1q to escape from attack by the antibody-dependent and -independent classical complement pathway. The prc mutant of E. coli K1 exhibited distinct outer membrane protein (OMP) profile compared to its wild-type strain, and the OMPs purified from the mutant recruited a higher level of IgG deposition than those of the wild-type. Besides, the prc mutant is more sensitive to　MAC than the wild-type E. coli K1. Therefore, the changed OMP profile caused by deletion of prc may be responsible for the mutant’s higher level of IgG binding on the surface and greater sensitivity to the attack mediated by MAC than its wild-type strain. In conclusion, Prc plays an important role for E. coli K1 to induce a high level of bacteremia. Thus, it may be a potential target for developing novel preventive and therapeutic intervention against E. coli-caused diseases.

    Abstract 1 誌 謝 4 Contents 6 Lists of Tables 9 Lists of Figures 10 Abbreviations 11 Introduction 13 1.1 Introduction of Escherichia coli 13 1.2 Pathogenesis of Bacterial Meningitis 13 1.3 The Complement Pathway 15 1.3.1 Activation of Complement System 15 1.3.2 Regulation of the Complement System 16 1.3.3 Virulence Factors of E. coli K1 Involved in Evasion of the Complement-mediated Killing 18 1.4 Introduction of Prc 18 Specific Aim 20 Materials and methods 21 2.1 Bacterial Strains and Culture Condition 21 2.2 Construction of the Plasmid pCL1920-prc 21 2.3 Normal Human Serum and Heat-inactivated Human Serum 21 2.4 Preparation of the IgG/IgM Double-depleted Serum 22 2.5 Mouse Model of E. coli Bacteremia 22 2.6 Serum Survival Assay 23 2.7 Flow Cytometry Analysis 24 2.7.1 Deposition of IgG, IgM, C1q, and MAC From Human Serum 24 2.7.2 Deposition of C3b From Human Serum 24 2.7.3 Deposition of Purified C1q 24 2.8 Western Blot Analysis 25 2.9 K1 Capsular Polysaccharide Determination 25 2.10 Purification and Analysis of Lipopolysaccharide (LPS) 26 2.11 Analysis of Bacterial Outer Membrane Protein 27 2.11.1 Purification and Quantification of Outer Membrane Protein 27 2.11.2 The Profile of the Outer Membrane Protein 27 2.11.3 Western Blot and Dot Blot Analysis for the Deposition of Human IgG on the Outer Membrane Protein 28 2.12 Statistical Analysis 28 Results 29 3.1 Deletion of prc decreased the ability of E. coli K1 to induce bacteremia in mice 29 3.2 The decreased ability of the prc mutant to cause bacteremia was not likely due to the fever of the infected animals 29 3.3 Prc was involved in E. coli K1 evasion of the complement-mediated killing in the human serum 30 3.4 Prc contributed to E. coli K1 resistance to serum-mediated killing by the classical complement pathway 31 3.5 Prc contributed to E. coli K1 evasion of serum killing mediated by the classical pathway activated through antibody-dependent and -independent manners 32 3.6 The contribution of Prc to E. coli K1 resistance to serum-mediated killing wasn’t associated with any secreted bacterial factors of E. coli K1 33 3.7 Deletion of prc in E. coli K1 wasn’t affect the expression of K1 capsular and lipopolysaccharide (LPS) 34 3.8 The outer membrane protein profile of the prc mutant was different from its wild-type RS218, and the outer membrane proteins derived from the mutant recruited a higher level of IgG deposition than those of the wild-type 34 3.9 The prc mutant of E. coli K1 was more sensitive to the attack of MAC than its wild-type 35 Discussion 36 Figures & Tables 39 References 59 Appendixes 66

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