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研究生： 林汶燕
LIM, BOON-YUEAN
論文名稱： 功能性分析線蟲體內IGLR-2對出血性大腸桿菌感染所扮演的角色
Functional Analysis of the Role of Caenorhabditis elegans IGLR-2 during Enterohemorrhagic E. coli Infection
指導教授： 陳昌熙
Chen, Chang-Shi
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學暨分子生物學研究所
Department of Biochemistry and Molecular Biology
論文出版年： 2016
畢業學年度： 104
語文別： 英文
論文頁數： 61
中文關鍵詞： 出血性大腸桿菌 、線蟲 、先天免疫 、模式辨認受體
外文關鍵詞： Enterohemorrhagic E. coli (EHEC), C. elegans, innate immunity, pattern recognition receptor (PRR)
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    • 出血性大腸桿菌為革蘭氏陰性桿菌，其主要血清型為O157:H7，人類通常透過攝入被污染的水源或食物而受到感染。當人類受到出血性大腸桿菌感染時會出現出血性下痢，溶血性尿毒症，甚至是腎衰竭等嚴重症狀。經過實驗室之前的研究發現線蟲 (Caenorhabditis elegans) 可被出血性大腸桿菌感染及毒殺，並會透過在演化上具有高度保留性的p38 MAPK先天免疫路徑來抵禦出血性大腸桿菌的感染。在多細胞生物體內具有一種會偵測病原或微生物相關分子模式及損傷相關分子模式並且會啟動體內免疫反應的接收器，稱為模式辨認受體。但是，線蟲用來偵測病原菌表面分子及可調控先天免疫路徑的模式辨認受體尚未被釐清。因此，我們將線蟲體內帶有模式辨認受體保守性結構域的基因建構成RNAi株庫並進行篩選。最後，我們發現在感染出血性大腸桿菌時，唯有iglr-2被抑制後與其對照組相比線蟲壽命有明顯縮短的結果，因測推測iglr-2為最具潛力的模式辨認受體。IGLR-2 帶有富含亮氨酸重複序列及免疫球蛋白的結構域，且其和馬 (E. caballus) 的類鐸受體二具有同源性。先前實驗室已利用CRISPR-Cas9的技術建立了iglr-2有缺失的突變蟲株，發現 iglr-2功能喪失的突變株對於血性大腸桿菌的感染比起野生型株N2顯得更敏感，而iglr-2過表現的蟲株則對出血性大腸桿菌具有抗性。因此，我們的目標是進一步了解線蟲體內iglr-2對於抵抗出血性大腸桿菌感染時所扮演的角色及其調控先天免疫路徑的機制。實驗結果發現線蟲的iglr-2的表現量與動物本身進食的情況與壽命並無直接關係。另外，iglr-2過表現的蟲株並不會對所有環境壓力刺激都具有抗性，是只有被致病菌感染時才出現的表徵。另外，會表現在神經細胞中的iglr-2缺失後，會使線蟲失去避開致病菌的能力。實驗中也觀察到原本對出血性大腸桿菌感染具有抗性的iglr-2過表現蟲株，在給予出血性大腸桿菌感染的同時抑制參與在p38 MAPK的基因後，蟲子的壽命會明顯縮短，然而，iglr-2突變株也有比較短命的結果，顯示了iglr-2與p38 MAPK都會幫助線蟲抵抗出血性大腸桿菌的感染但不是藉由共同路徑達到的結果。總結到目前的實驗結果，我們推測iglr-2這具潛力的模式辨認受體是參與在線蟲避開和抵禦出血性大腸桿菌感染過程所需的重要因子，並且是藉由活化在演化上具高度保留性的p38 MAPK或未知的先天免疫路徑而產生的效果。

    Enterohemorrhagic Escherichia coli (EHEC) is a gram-negative bacterium and the major serotype is O157:H7. Humans usually get infected with EHEC by uptaking EHEC contaminated water or food. EHEC infections can cause life-threatening diseases, such as hemolytic uremic syndrome (HUS) and hemorrhagic diarrhea, especially in young children and the elderly. Our previous studies demonstrated that EHEC infected and killed Caenorhabditis elegans. Moreover, C. elegans activated the p38 MAPK innate immune pathway to defend EHEC infection. The receptors for metazoans to detect pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) or damage-associated molecular patterns (DAMPs) and to activate immune response are called as pattern recognition receptors (PRRs). However, the C. elegans PRRs for bacterial pathogens detection and innate immunity regulation remain largely unknown. We therefore constructed a focused RNAi library to screen for C. elegans genes with PRRs conserved domains in previous. From the genetic screen, we identified the iglr-2 gene as a potential PRR candidate which showed significantly decreased in lifespan compared to its empty factor L4440 when it exposed to EHEC infection. IGLR-2 contains leucine-rich repeat (LRR) and immunoglobulin-like (Ig-like) domains and is homologous to the E. caballus (horse) Toll-like receptor 2. We generated iglr-2 deletion mutant by the CRISPR-Cas9 technology and found that the iglr-2 null mutant strain is more susceptible to EHEC infection compared to the parental wild-type N2 strain. Moreover, the iglr-2 overexpression strain is more resistant to EHEC. Here, we focused to identify the character and mechanism of innate immune regulation of iglr-2, which may be against EHEC infection. Previous study demonstrated that iglr-2 expression pattern was co-localized with some neurons such as PVD, OLL, AFD and AWB. From our results, C. elegans lost the ability to avoid pathogens when it lost the iglr-2 function specifically in neurons. The iglr-2 overexpression strain, which was more resistant to EHEC infection originally, showed more susceptibility to EHEC infection upon knockdown of the p38 MAPK cascade. And the iglr-2 null mutant showed the similar outcome. Furthermore, we found that iglr-2 did not control the physiological characteristics and responses to general stressors challenges in C. elegans. The iglr-2 overexpression strains are resistant to EHEC infection only; it does not confer a general resistant phenotype. Taken all together, our data suggested that iglr-2, a potential PRR, plays an important role in C. elegans to defend EHEC infection by activating the pathogen-avoidance behavior and immune responses via, at least in part, p38 MAPK pathway or controlling unidentified immune cascades.

    摘要 I Abstract III Acknowledgements V Introductions 1 Materials and methods 9 C. elegans and bacterial strains 9 Pathogen avoidance behavior assays 10 One generation RNA interference (RNAi) assays 10 Analysis of phospho-p38 expression level 11 Assays for general physiology 12 A. Pumping rate analysis 12 B. Developmental time 12 C. Body length analysis 12 D. Brood size analysis 12 Assays for general stresses 14 A. H２O２assays 14 B. CuSO４assays 14 C. Thermotolerance assays 14 D. Osmotic pressure assays 15 Results 16 The pathogen-avoidance behavior of the iglr-2 null mutant and iglr-2 overexpression strains to pathogens 16 The survival assays of the iglr-2 null mutant and iglr-2 overexpression strains during EHEC infection 17 The survival assays of p38 pathway knockdown in iglr-2 null mutant and iglr-2 overexpression strains during EHEC infection 19 The expression level of phospho-p38 (p-p38) in C. elegans 20 General physiology of the iglr-2 null mutant and iglr-2 overexpression strains 21 Pumping rate 21 Developmental time 21 Body length 22 Brood size 22 The susceptibility to general stresses of the iglr-2 null mutant and iglr-2 overexpression strains 23 Conclusions 25 Discussions 26 References 30 Tables 34 Table 1. C. elegans, bacterial strains and plasmids were used in this study. 34 Table 2. The statistics of survival curves 35 A. survival information of figure 2A and 2B 35 B. survival information of figure 3A and 3B 35 C. survival information of figure 3C and 3D 36 D. survival information of figure 3E and 3F 36 E. survival information of figure 3G and 3H 36 F. survival information of figure S1A and S1B 37 G. survival information of figure S1C and S1D 37 H. survival information of figure S1E and S1F 37 Figures 38 Fig. 1 The pathogen-avoidance behavior of the iglr-2 null mutant and iglr-2 overexpression strains to pathogens 38 Fig. 2 The survival assays of TLR pathway knockdown in iglr-2 null mutant and iglr-2 overexpression strains during EHEC infection 41 Fig. 3 The survival assays of p38 pathway knockdown in iglr-2 null mutant and iglr-2 overexpression strains during EHEC infection 43 Fig. 4 The expression level of phospho-p38 (p-p38) in C. elegans 48 Fig. 5 General physiology of the iglr-2 null mutant and iglr-2 overexpression strains 50 Fig. 6 The susceptibility to general stresses of the iglr-2 null mutant and iglr-2 overexpression strains 53 Fig. 7 Model of defending EHEC infection that regulated by IGLR-2 in C. elegans 57 Supporting information 58 Fig. S1 The survival assays of knockdowning different pathways in iglr-2 null mutant and iglr-2 overexpression strains during EHEC infection 58

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