臺灣博碩士論文加值系統

出血性大腸桿菌主要血清型為O157:H7，能經由食物或水源傳播感染人類，進而導致出血性腹瀉、溶血性尿毒症等症狀發生。在我們過去的研究中，發現出血性大腸桿菌能寄殖並複製於秀麗隱桿線蟲(Caenorhabditis elegans) 的腸道當中，進行感染及毒殺。已有許多研究發現參與在出血性大腸桿菌寄殖的毒力因子，然而對於參與其中的宿主因子尚且不多。在我們的研究當中，我們利用甲基磺酸乙酯(EMS)突變劑篩選對於出血性大腸桿菌寄殖於線蟲腸道中所參與的宿主因子。我們利用餵食帶有綠色螢光(GFP)的出血性大腸桿菌，篩選出32株突變株具有”腸道寄殖增加”(enhanced intestinal colonization, INC)的表現型。出乎意料之外地，我們發現其中四株擁有INC表現型的突變株，卻對於出血性大腸桿菌有較強的耐受能力，這個現象違背我們對於細菌感染的現有知識，因此，我們利用單核苷酸多態性分析方法(SNP mapping)，以及次世代定序(NGS)，分析這四株突變株的突變位點，透過生資分析平台CloudMap，我們得到了23個候選基因並透過RNA干擾以及突變株確認該突變基因為unc-89。unc-89已知能調控肌細胞組成，其腸道寄殖增加知表現型，可能因咽部細胞突變受損所致。值得一提的是，細菌迴避行為以及活化hlh-30先天免疫反應，可能與unc-89增加對於出血性大腸桿菌的耐受性有關。整體來說，此研究顯示，在線蟲中unc-89因咽部細胞受損導致腸道寄殖性的增加，卻也同時促進先天性免疫反應，以提升對於出血性大腸桿菌感染之耐受性。我們相信本篇研究可為抵禦出血性大腸桿菌感染之未來研究發展帶來新的曙光。

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes acute hemorrhagic diarrhea and hemolytic uremic syndrome through water and food. In our previous study, we found that EHEC colonized and replicated in the intestinal lumen of C. elegans during the infection. Several researches have identified the key bacterial factors required for EHEC colonization; however, the host factors involved in EHEC colonization remain unclear. Here, we applied a forward genetic screen to identify the host factors involved in EHEC colonization in C. elegans. We have isolated 32 Ethyl methanesulfonate (EMS)-induced mutants conferred the enhanced intestinal colonization (Inc) phenotype under the infection of GFP-labeled EHEC. Surprisingly, in the susceptibility test of these mutants, we found four mutants exhibited a significant tolerant phenotype to EHEC infection compared to the wild type C. elegans. Given that the phenotype with enhancement of intestinal colonization yet tolerance to EHEC infection is contradictory to our current knowledge about bacterial infection, we therefore turned our attentions to identify the mutant genes by single-nucleotide polymorphism (SNP) mapping and Next-generation sequencing (NGS). By using the CloudMap, a useful bioinformatics tool, we identified 23 candidate genes and confirmed unc-89 as inc-1 by complementation test, RNAi knockdown and mutant worms. unc-89, known for muscle cell assembly, caused the enhancement of bacterial colonization in the intestine may due to pharyngeal muscle defect. On top of that, the tolerance to EHEC in unc-89 may relate to bacterial avoidance behavior and activated hlh-30-mediated innate immune response. Taken together, we demonstrated unc-89 enhanced the intestinal colonization due to the pharyngeal muscle defect yet enhanced the tolerance of EHEC through activated innate immunity in C. elegans. We believed our study may gain insights on the novel aspects of immunity in bacterial infection.

中文摘要 i
Abstract ii
誌謝 iv
Introduction 1
Material and Methods 4
Bacterial and Nematode Strains 4
Bacteria (E. coli OP50 and E. coli O157:H7 EDL933) Killing Assay 4
INC (enhanced intestinal colonization) phenotype assay 5
Two Generation RNA Interference (RNAi) 5
Colony Forming Units (CFU) 6
SNP Mapping 7
Next Generation Sequencing (NGS) 7
Measurement of Bacterial Avoidance Behavior 8
HLH-30 nuclear localization of unc-89 KD in OP433 8
Results 10
YQ032, YQ087, YQ106 and YQ269 strains performed INC phenotype yet conferred tolerance to EHEC infection. 10
There were two complementation groups (inc-1 and inc-2) among four mutant allele. 11
unc-89 caused INC phenotype yet conferred tolerance to EHEC infection in RNAi knockdown and mutant alleles. 13
Bacterial avoidance behavior by unc-89 may limit bacterial accumulation in intestine and cause survival extension. 16
unc-89 mutation may induce HLH-30 nuclear localization 17
Conclusions and Discussion 19
Tables 22
Table 1. Bacterial and nematode strains and plasmids used for this study 22
Table 2. SNP mapping of YQ269 (inc-1) crossed with Hawaiian strain CB4856 23
Table 3. Twenty-three candidate genes were predicted by CloudMap platform on Galaxy website….. 25
Figures 26
Fig. 1 YQ032, YQ087, YQ106 and YQ269 strains performed INC phenotype yet conferred tolerance to EHEC infection. 27
Fig. 2 The INC phenotype of eleven mutants. 28
Fig. 3 The INC phenotype of worms knockdown in seventeen candidate genes 29
Fig. 4 The INC phenotype of F1 progeny from YQ269 crossed with unc-89(e1469). 30
Fig. 5. The INC phenotype of unc-89 mutants and RNAi knockdown in rrf-3. 33
Fig. 6 EDL933 colonization is increased in YQ269, unc-89 and phm-2. 34
Fig. 7. unc-89 mutants performed tolerance to EHEC infection. 36
Fig. 8 The defect of pharyngeal muscle in unc-89 and phm-2 may lead to the enhancement of EHEC colonization 38
Fig. 9 unc-89 mutants display bacterial avoidance behavior. 40
Fig. 10 Survival of unc-89 in small or large bacterial lawns. 42
Fig. 11. The INC phenotype of innate immune genes knockdown in inc-1(wf132). 43
Fig. 12 HLH-30 nuclear localization in the unc-89 RNAi knockdown assay. 45
Fig. 13 unc-89 was predicted as inc-1 by NGS analysis. 46
Reference 47

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