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研究生： 廖怡君
Liao, Yi-Jyun
論文名稱： eco60-63 operon 降低大腸桿菌K1 RS218在人類血清中存活的機制
The mechanism of reduced human serum survival of Escherichia coli K1 RS218 by the eco60-63 operon
指導教授： 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： 2015
畢業學年度： 103
語文別： 英文
論文頁數： 66
中文關鍵詞： K1大腸桿菌 、eco60-63基因組 、補體活化
外文關鍵詞： Escherichia coli K1, the eco60-63 operon, complement activation
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    • K1大腸桿菌是新生兒腦膜炎感染中常見的致病菌，而菌血症是發展這類腦膜炎一個重要的進程，因此鑑定K1大腸桿菌毒性因子在血液中的角色將有助於我們了解細菌的致病機轉，以發展策略對抗疾病。RS218為K1大腸桿菌，eco60-63基因組被發現位於此菌一個幫助發展腦膜炎的質體pRS218上，此基因組已知在早期尿道感染扮演重要角色，然而根據我們先前實驗結果，細菌表達此基因組會降低血清中的存活率，因此在本篇研究，我的目的是要探討致病性大腸桿菌表達此基因組 (eco60-63+ RS218) 比不表達株 (eco60-63- RS218) 受到較嚴重血清擊殺的原因。
    在血清中，eco60-63+ RS218會黏附比eco60-63- RS218較高量的C3b和MAC，因此可推測eco60-63+ RS218比eco60-63- RS218遭受較高量血清補體攻擊。抑制血清中古典和替代路徑後，會降低eco60-63+ RS218和eco60-63- RS218的血清存活差距，且eco60-63+ RS218比eco60-63- RS218表面有較多C1q、IgG和properdin的黏附，由結果可推測細菌表達eco60-63基因組會活化較高量補體反應中抗體依賴型古典及替代路徑。此外我們發現表達eco60-63 operon會降低RS218 K1的莢膜表達以及增加對membrane attack complex (MAC) 的敏感性，此可能是造成表達株有較低血清存活率的原因。表達eco60會降低RS218的血清存活能力，但表達基因組中的其他基因不會降低細菌的血清存活率，由此可知eco60在eco60-63+ RS218和eco60-63- RS218有差別的血清擊殺上有參與角色。
    由以上結果我們得知eco60-63基因組會妨礙細菌在serum中的存活，但RS218的毒性質體仍保留此operon，因此未來仍需探討是否eco60-63基因組對大腸桿菌在腦膜炎其他致病過程上有所貢獻。在本篇研究中，經由了解eco60-63基因組在K1大腸桿菌serum中存活的角色，將使我們對K1大腸桿菌的感染有進一步的了解，並有助於發展策略對抗此類感染。

    Escherichia coli (E. coli) K1 is one of the most common pathogens causing neonatal meningitis. Since induction of bacteremia is a key step for the development of E. coli meningitis, characterizing the roles of the bacterial virulence factors when the pathogen is in the bloodstream would facilitate our understanding of the pathogenesis of this disease and contribute to the development of strategies against this disease. The eco60-63 operon encoded by pRS218, a virulence plasmid of the E. coli K1 strain RS218 known to be involved in the pathogenesis of E. coli meningitis. This operon has been shown to play an important role in the early stage of urinary tract infection (UTI) caused by E. coli. However, our previous study showed that the expression of eco60-63 decreased the ability of RS218 to survive in the bloodstream and serum. Thus, in the present study I further investigated why the pathogenic E. coli strain expressing this operon (eco60-63+ RS218) reduced resistance to serum killing than the strain without this operon (eco60-63- RS218).
    In normal human serum (NHS), eco60-63+ RS218 exhibited higher levels of C3b and membrane attack complex (MAC) deposition on the surface than eco60-63- RS218, suggesting that eco60-63+ RS218 encounters a higher level of complement-mediated attack in NHS than eco60-63- RS218. Blocking the activation of the classical or alternative complement pathways in NHS significantly decreased the difference of these two strains’ serum survival. Also, eco60-63+ RS218 exhibited higher levels of C1q, IgG and properdin deposition in NHS than eco60-63- RS218. These findings suggested that eco60-63+ RS218 triggers stronger activation of the antibody-dependent classical and alternative complement pathways than eco60-63- RS218. Moreover, expressing the eco60-63 operon may decrease RS218’s K1 capsule expression and increase this bacterium’s sensitivity to attack of membrane attack complex (MAC). The phenotypes induced by this operon may also contribute to the decreased serum survival caused by eco60-63 expression. In addition, expressing the eco60 gene alone decreased RS218’s serum survival ability, while the expression of the other individual genes in the operon did not decrease the bacterium’s serum survival. This suggested that eco60 plays a role in the differential serum killing of eco60-63+ RS218 and eco60-63- RS218.
    Although the eco60-63 operon hindered NMEC RS218 to survive in the serum, RS218’s virulence plasmid still retains the operon. A further study is required to determine whether the eco60-63 operon contributes to other pathogenesis steps of E. coli meningitis. The knowledge obtained from this study facilitates our understanding of E. coli pathogenesis, which may contribute to the development of novel strategies against E. coli-caused infections in the future.

    Abstract I 中文摘要 IV 誌謝 VI Contents VII Lists of tables X Lists of figures XI Abbreviations XII Introduction 1 1.1 Introduction of E. coli 1 1.2 Neonatal bacterial meningitis 2 1.3 Pathogenesis of E. coli meningitis 2 1.4 Virulence plasmid and NMEC pathogenesis 3 1.4.1 Brief introduction of virulence plasmid 3 1.4.2 Relationship of NMEC plasmid and bacterial pathogenesis 4 1.5 Introduction of the eco60-63 operon on pRS218 5 1.6 The complement pathway 6 1.6.1 Activation of complement system 6 1.6.2 Regulation of complement activation 8 1.6.3 Virulence factors of NMEC K1 in evasion of the complement system 9 Specific aim 11 Materials and methods 12 2.1 Bacterial strains and culture condition 12 2.2 Normal human serum, heat-inactivated human serum and modified human serum 12 2.3 Serum survival 13 2.4 Detection of protein deposition by flow cytometry 13 2.4.1 Deposition of IgG, properdin, C1q and MAC from human serum 13 2.4.2 Deposition of C3b from human serum 14 2.4.3 The protein binding assays with the purified C1q 14 2.5 Detection of ribonuclease (RNase) leakage 15 2.6 Western blot analysis 15 2.7 LPS extraction and silver stain 15 2.8 Analysis of capsular gene expression 16 2.8.1 RNA extraction and DNA digestion 16 2.8.2 Reverse transcription polymerase chain reaction (PCR) 18 2.8.3 Real-time PCR 18 2.9 Statistical analysis 18 Results 19 3.1 The expression of the eco60-63 operon facilitates bacterium serum killing mediated by the heat-labile complement system 19 3.2 The classical and the alternative pathways were involved in differential serum killing of eco60-63+ RS218 and eco60-63- RS218 20 3.3 The expression of eco60-63 operon in E. coli K1 triggered higher activation levels of the antibody-dependent classical and alternative complement pathways 20 3.4 The expression of the eco60-63 operon increased the bacterial sensitivity to MAC 21 3.5 The expression of the eco60-63 operon increased E. coli membrane permeability and decreased K1 capsule expression 22 3.6 The eco60 of bacteria plays a role in the differential serum killing on eco60-63+ RS218 and eco60-63- RS218 23 Discussion 25 Figures& Tables 30 References 42 Appendixes 51

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