中文摘要
大腸桿菌O157:H7是一種會隨著食物或水源傳播感染的出血性大腸桿菌，此致病菌會導致人們腹瀉，出血性結腸炎，溶血性尿毒症甚至腎衰竭等症狀。現今，對於出血性大腸桿菌感染之治療方式仍限制於給予支持性照護來減輕症狀及併發症的預防。目前科學家們已經利用許多模式動物來進行出血性大腸桿菌的研究，然而，在自然情況下感染出血大腸桿菌且能夠進行遺傳操作之模式生物的缺乏下，阻礙了出血性大腸桿菌感染於活體內遺傳方法之整體性研究。在此，我們利用了自然環境下會被出血性大腸桿菌感染的可遺傳操作生物—秀麗隱桿線蟲(Caenorhabditis elegans)，來研究宿主對出血性大腸桿菌的先天免疫反應。
我們發現出血性大腸桿菌會感染並殺死線蟲，也發現相同於此致病菌感染哺乳動物之特徵如細菌寄殖於腸道中且引發粘著/塗抹病變(A/E lesions)現象，在此病菌感染的線蟲腸道中亦會出現。兩種演化高保留度之先天免疫的訊息路徑，p38 MAP kinase 和DAF-2 insulin-like的訊息路徑，在線蟲遭受出血大腸桿菌感染時也會被活化並調控宿主對此病菌的感受性。我們利用甲基磺酸乙酯(EMS)這種突變劑去篩選對於出血性大腸桿菌寄殖於宿主所參與的宿主因子。在所篩選的124株線蟲突變株當中，其中一株突變株，wf059，發現其對於出血大腸桿菌是有抗性的，並且經遺傳學方法分析，得知其突變區可能位於染色體I上。既然先天免疫在許多生物體中是相當保守性的反應，我們希望藉由瞭解線蟲對於出血性大腸桿菌感染的免疫防禦系統之分子基礎，可為免疫系統抵禦出血性大腸桿菌之研究，帶來新的曙光。

Abstract
Escherichia coli O157:H7 is a member of pathogenic E. coli, known as enterohemorrhaghic E. coli (EHEC), can colonize the gastrointestinal tract and cause life-threatening infections worldwide. However, current treatment for the EHEC infection is still limited to supportive care. Although several animal models have been proposed for studying the EHEC infection, the lack of naturally infected and genetic tractable animal model hinders the study of EHEC infection with holistic and unbiased genetic screening approaches in vivo. Here we applied the genetic tractable animal, Caenorhabditis elegans—which may encounter EHEC in its niche naturally as a surrogate host for studying the host innate immunity to this pathogen.
Our results showed that E. coli O157:H7, infected and killed C. elegans. Colonization and induction of the characteristic attaching and effacing (A/E) lesions by E. coli O157:H7 were concomitantly demonstrated in the intact intestinal epithelium of C. elegans in vivo. The C. elegans p38 MAP kinase and the DAF-2 insulin-like signaling pathways, two evolutionally conserved innate immune signaling pathways, were activated and mediated in the regulation of host susceptibility to EHEC infection. We also applied the EMS-mediated genetic screen for the host factors involved in E. coli O157:H7 colonization in C. elegans. Among the 124 candidates, one of the allele, wf059, with a mutation been mapped to the chromosome I, showed resistant to E. coli O157:H7.Given that the innate immune responses are highly conserved among different organisms, understanding the molecular basis of the immune defense systems in C. elegans should shed light onto some aspects of immunity against EHEC infection.

Reference
1 Brenner, S. NATURE’S GIFT TO SCIENCE. (2002).
2 Brenner, S. In the Beginning Was the Worm.. Genetics 182, 413-415 (2009).
3 Tan, M. W., RAHME, L. G., STERNBERG, J. A., TOMPKINS, R. G. & AUSUBEL, F. M. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence. (1999).
4 Tan, M. W. & Ausubel, F. M. Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. (2000).
5 Darby, C. Interactions with microbial pathogens. WormBook : the online review of C. elegans biology (2005 ).
6 Waterfield, N. R., Wren, B. W. & H., F.-C. R. Invertebrates as a source of emerging human pathogens. Nat Rev (2004).
7 Pennington, H. Escherichia coli O157. The Lancet 376, 1428-1435 (2010).
8 Mohawk, K. L. & O'Brien, A. D. Mouse models of Escherichia coli O157:H7 infection and shiga toxin injection. J Biomed Biotechnol 2011, 258185 (2011).
9 Panda, A. et al. Escherichia coli O157:H7 infection in Dutch belted and New Zealand white rabbits. Comp Med 60, 31-37 (2010).
10 Golan, L., Gonen, E., Yagel, S., Rosenshine, I. & Shpigel, N. Y. Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models. Dis Model Mech 4, 86-94 (2011).
11 Donnenberg, M. S. et al. The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model. J Clin Invest 92, 1418-1424 (1993).
12 Chase-Topping, M., Gally, D., Low, C., Matthews, L. & Woolhouse, M. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157. Nature Reviews Microbiology 6, 904-912 (2008).
13 Renter, D. G., Sargeant, J. M., Oberst, R. D. & Samadpour, M. Diversity, frequency, and persistence of Escherichia coli O157 strains from range cattle environments. Appl Environ Microbiol 69, 542-547 (2003).
14 Kenney, S. J., Anderson, G. L., Williams, P. L., Millner, P. D. & Beuchat, L. R. Persistence of Escherichia coli O157:H7, Salmonella Newport, and Salmonella Poona in the gut of a free-living nematode, Caenorhabditis elegans, and transmission to progeny and uninfected nematodes. International journal of food microbiology 101, 227-236 (2005).
15 Anderson, G. L., Kenney, S. J., Millner, P. D., Beuchat, L. R. & Williams, P. L. Shedding of foodborne pathogens by Caenorhabditis elegans in compost-amended and unamended soil. Food microbiology 23, 146-153 (2006).
16 Anyanful, A. et al. Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene. Molecular microbiology 57, 988-1007 (2005).
17 Lee, Y. et al. The role of disulfide bond isomerase A (DsbA) of Escherichia coli O157:H7 in biofilm formation and virulence. FEMS microbiology letters 278, 213-222 (2008).
18 Farfan, M. J. & Torres, A. G. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains. Infection and immunity 80, 903-913 (2012).
19 Naylor, S. W. et al. Escherichia coli O157 : H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon. Microbiology 151, 2773-2781 (2005).
20 Hamada, D., Hamaguchi, M., Suzuki, K. N., Sakata, I. & Yanagihara, I. Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: a case for EspB as an intrinsically less-ordered effector. The FEBS journal 277, 2409-2415 (2010).
21 Campellone, K. G. Cytoskeleton-modulating effectors of enteropathogenic and enterohaemorrhagic Escherichia coli: Tir, EspFU and actin pedestal assembly. The FEBS journal 277, 2390-2402 (2010).
22 Tobe, T. Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: role of EspL2 in adherence and an alternative pathway for modulating cytoskeleton through Annexin A2 function. The FEBS journal 277, 2403-2408 (2010).
23 Campellone, K. Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7. Current opinion in microbiology 6, 82-90 (2003).
24 Strockbine, N. A. et al. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect Immun 53, 135-140 (1986).
25 Perna, N. T. et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409, 529-533 (2001).
26 Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974).
27 Valdivia, R. H. & Falkow, S. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol Microbiol 22, 367-378 (1996).
28 Chen, C. S. et al. WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like Signaling Network Involved in Pore-Forming Toxin Cellular Defenses in Caenorhabditis elegans. PLoS One 5 (2010).
29 Bellier, A., Chen, C. S., Kao, C. Y., Cinar, H. N. & Aroian, R. V. Hypoxia and the hypoxic response pathway protect against pore-forming toxins in C. elegans. PLoS pathogens 5, e1000689 (2009).
30 Larry J. Bischof, Danielle L. Huffman & Aroian, a. R. V. Assays for Toxicity Studies in C. elegans With Bt Crystal Proteins. Methods in Molecular Biology 351, 139-154 (2006).
31 Irazoqui, J. E. et al. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathog 6, e1000982(2010).
32 Bischof, L. J. et al. Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLoS pathogens 4, e1000176 (2008).
33 Davis, M. W. et al. Rapid single nucleotide polymorphism mapping in C. elegans. BMC genomics 6, 118, doi:10.1186/1471-2164-6-118 (2005).
34 Zhang, Y., Lu, H. & Bargmann, C. I. Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438, 179-184 (2005).
35 McGhee, J. D. The C. elegans intestine. WormBook, 1-36 (2007).
36 Sato, K. et al. Caenorhabditis elegans SNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells. Mol Biol Cell 22, 2579-2587 (2011).
37 Lee, J., Kang, J., Shin, D. & Yu, J. R. Lats kinase is involved in the intestinal apical membrane integrity in the nematode Caenorhabditis elegans. Development 136, 2705-2715 2009).
38 Beanan, M. J. & Strome, S. Characterization of a germ-line proliferation mutation in C. elegans. Development 116, 755-766 (1992).
39 Syntichaki, P., Troulinaki, K. & Tavernarakis, N. eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445, 922-926 (2007).
40 Tohyama, D., Yamaguchi, A. & Yamashita, T. Inhibition of a eukaryotic initiation factor (eIF2Bdelta/F11A3.2) during adulthood extends lifespan in Caenorhabditis elegans. FASEB J 22, 4327-4337 (2008).
41 TeKippe, M. & Aballay, A. C. elegans Germline-Deficient Mutants Respond to Pathogen Infection Using Shared and Distinct Mechanisms. PLoS One 5(2010).
42 Alper, S. et al. The Caenorhabditis elegans germ line regulates distinct signaling pathways to control lifespan and innate immunity. J Biol Chem 285, 1822-1828 (2010).
43 Huffman, D. L. et al. Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins. Proceedings of the National Academy of Sciences of the United States of America 101, 10995-11000 (2004).
44 Ewbank, J. J. & Zugasti, O. C. elegans: model host and tool for antimicrobial drug discovery. Dis Model Mech 4, 300-304 (2011).
45 Irazoqui, J. E., Urbach, J. M. & Ausubel, F. M. Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates. Nature reviews. Immunology 10, 47-58 (2010).
46 Aroian, R. & van der Goot, F. G. Pore-forming toxins and cellular non-immune defenses (CNIDs). Current opinion in microbiology 10, 57-61, (2007).
47 Kim, D. H. et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 297, 623-626, (2002).
48 Garsin, D. A. et al. Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens. Science 300, 1921, (2003).
49 Henderson, S. T. & Johnson, T. E. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr Biol 11, 1975-1980, (2001).
50 Cheng-Yuan Kao, Ferdinand C O Los & Aroian, R. V. Nervous about immunity: neuronal signals control innate immune system. Nature immunology 9 (2008).
51 Alejandro Aballay, Peter Yorgey & Ausubel, F. M. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Current Biology 10, 1539–1542 (2000).
52 Garsin, D. A. et al. A simple model host for identifying Gram-positive virulence factors. Proceedings of the National Academy of Sciences of the United States of America 98, 10892-10897 (2001).
53 Styer, K. L. et al. Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors. EMBO reports 6, 992-997 (2005).
54 Laura E. Fuhrman, Kevin V. Shianna & Aballay, A. High-Throughput Isolation and Mapping of C. elegans Mutants Susceptible to Pathogen Infection. PloS one 3 (2008).
55 Stephen R. Wicks, Raymond T. Yeh, Warren R. Gish, Robert H. Waterston & Plasterk, R. H. A. Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nature genetics 28 (2001).
56 Katie L. Styer et al. Innate Immunity in Caenorhabditis elegans Is Regulated by Neurons Expressing NPR-1/GPCR. SCIENCE 322 (2008).
57 Aballay, A. Neural regulation of immunity: Role of NPR-1 in pathogen avoidance and regulation of innate immunity. Cell Cycle 8, 966-969 (2009).
58 Schulenburg, H. & Ewbank, J. J. The genetics of pathogen avoidance in Caenorhabditis elegans. Molecular microbiology 66, 563-570 (2007).
59 Kirthi C. Reddy, Erik C. Andersen, Leonid Kruglyak & Kim, D. H. A Polymorphism in npr-1 Is a Behavioral Determinant of Pathogen Susceptibility in C. elegans. SCIENCE 323 (2009).
60 Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974).
61 Wells, M. B., Snyder, M. J., Custer, L. M. & Csankovszki, G. Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes. Molecular and cellular biology 32, 1710-1719 (2012).