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研究生： 王信沺
Wang, Sin-Tian
論文名稱： 腸出血性大腸桿菌之OmpR蛋白協調毒力因子表達於秀麗隱桿線蟲之消化道中
OmpR coordinates the expression of virulence factors of enterohemorrhagic Escherichia coli in the alimentary tract of Caenorhabditis elegans
指導教授： 陳昌熙
Chen, Chang-Shi
學位類別： 博士
Doctor
系所名稱： 醫學院 - 基礎醫學研究所
Institute of Basic Medical Sciences
論文出版年： 2020
畢業學年度： 109
語文別： 英文
論文頁數： 87
中文關鍵詞： 腸出血性大腸桿菌 、反應調控蛋白(OmpR) 、感應蛋白(EnvZ) 、第三型分泌系統 (T3SS) 、類志賀毒素 、秀麗隱桿線蟲
外文關鍵詞： Enterohemorrhagic Escherichia coli (EHEC), OmpR, EnvZ, Type III secretion system (T3SS), Shiga-like toxins (Stxs), Caenorhabditis elegans (C. elegans)
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    • 腸出血性大腸桿菌 (Enterohemorrhagic E. coli) 為腸道病原菌之一，會藉由第三型分泌系統 (T3SS) 與類志賀毒素 (Shiga-like toxins) 等基因表達，幫助細菌寄殖於腸道中，並造成病患產生嚴重腹瀉、出血性結腸炎以及溶血性尿毒症。然而，當腸出血性大腸桿菌進入消化道中，細菌會感受環境中的酸鹼值與滲透壓的改變，並同時協同調控應變基因與毒力因子的表達，但是對其調控機轉仍具有許多未解之謎。許多第三型分泌系統相關蛋白的表達，除了受到腸道細胞消除基因組調控子Ler (locus of enterocyte effacement-encoded regulator) 的控制，也於不同環境因子刺激下，受到非腸道細胞消除基因組調控子 (non-LEE-encoded regulators) 的調控。本論文，我們發現OmpR蛋白除了參與大腸桿菌對於滲透壓和酸鹼值的適應性外，也參與腸出血性大腸桿菌感染秀麗隱桿線蟲的致病機轉。我們的結果顯示，OmpR蛋白會直接地結合至ler基因的啟動子與stx1基因的啟動子，進而於轉錄層級中調節-ler、escV、escC、eae、espA第三型分泌系統相關基因等與類志賀毒素1 (Stx1) 的表達。除此之外，我們也證實腸出血性大腸桿菌於活體感染時，Ler基因的表達除了受秀麗隱桿線蟲的腸道環境影響外，也受OmpR蛋白調節Ler基因的表達。總結以上結果，我們發現OmpR蛋白為腸出血性大腸桿菌重要的調控子，可在體內胃腸道感染期間協調毒力因子的表達。

    Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen colonizes in the intestine, causes severe diarrhea, hemorrhagic colitis, and sometimes leads to hemolytic uremic syndrome in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC senses and responds to the changes of pH and osmolarity when entering the alimentary tract and concomitantly coordinate the expression of the virulence genes remained elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, mainly the Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation for osmolarity and pH alterations in E. coli, is required for EHEC infection in Caenorhabditis elegans. Our data also showed that the OmpR protein directly binds the ler promoter and the promoter of Shiga-like toxin 1 (stx1) gene to transcriptionally regulate the expression of T3SS and Stx1 toxin. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and under regulation by OmpR in vivo. Taken together, we unveil that OmpR is an important regulator to coordinate the expression of virulence factors in EHEC during gastrointestinal infection in vivo

    目錄 口試合格證明書 I 中文摘要 II 英文摘要 III 致謝 IV 目錄 V Introduction 1 Enterohemorrhagic Escherichia coli 1 Type III secretion system (T3SS) 1 Shiga-like toxin 3 Two component system 4 Caenorhabditis elegans 5 Intestine of Caenorhabditis elegans 6 Bacterial infection by using C. elegans model 6 Specific Aim 8 Materials and methods 9 Bacterial and nematode strains 9 Construction of ompR transposon mutants 9 Construction of ompR and envZ deletion mutants 10 Construction of plasmids 11 Construction of dual fluorescence transcriptional reporter plasmid 11 The survival assay of C. elegans 12 Bacterial colonization in C. elegans intestine 12 The microvillar actin rearrangement in C. elegans 13 In silico analysis by PRODORIC 13 Protein purification 14 Western blotting of phosphorylated OmpR 14 Electrophoretic mobility shift assay (EMSA) 15 In vivo Reporter assay in C. elegans 16 In vitro reporter assay in LB broth 16 RNA extraction and qRT-PCR 17 Scanning electron microscopy (SEM) 17 Immunofluorescence stain 18 Results 19 ompR is required for the pathogenicity of EHEC in C. elegans 19 The EnvZ/OmpR two-component system is required for intestinal infection of EHEC in C. elegans 21 OmpR directly binds to the promoter regions of the ler and stx1A genes 23 OmpR is required for the expression of ler and stx1A in the host alimentary tract in vivo 24 OmpR is required for the expression of EHEC virulence genes 25 OmpR regulates pathogenesis of EHEC against human cells 26 Conclusion 28 Discussion 29 References 32 Table 40 Table 1. Nematode and bacterial strains 40 Table 2. Plasmids 41 Table 3. Primers used in cloning, mutant construction, and qRT–PCR 42 Table 4. The potential OmpR-binding sites in the EHEC virulence genes.. 44 Figure 45 Figure 1. Reduced toxicity of ompR mutants toward to C. elegans N2 strain. 45 Figure 2. The ompR mutants display attenuated-phenotype to colonize intestine of C. elegans. 46 Figure 3. The ompR mutants attenuated the ability of actin rearrangement in C. elegans.. 48 Figure 4. The ompR is also required in clinical EHEC isolates. 49 Figure 5. The ompR deletion mutants in other EHEC strains affected the ability of colonization in intestine of C. elegans.. 50 Figure 6. The function of ompR plays an important role in EHEC-induced actin rearrangement in C. elegans. 52 Figure 7. The mutants disrupted envZ-ompR signaling affecte EHEC toxicity in C. elegans. 53 Figure 8. The function of envZ-ompR signaling regulates the capacity of colonization in intestine of C. elegans. 54 Figure 9. The envZ-ompR signaling involves in the mechanism of EHEC-induced actin rearrangement in C. elegans. 56 Figure 10. The mimic-phosphorylated OmpR mutant, OmpR (D55E) rescues the toxicity of EHEC toward C. elegans. 57 Figure 11. The phosphorylated OmpR, OmpR (D55E) restored the capacity of colonization in intestine of C. elegans. 58 Figure 12. The phosphorylated OmpR mutant, OmpR (D55E) plays important role in EHEC-induced microvillar actin rearrangement in C. elegans. 60 Figure 13. The OmpR is solo phosphorylated at the Aspartate55 residue.. 61 Figure 14. Recombinant OmpR proteins purified by nickel column chromatography. 62 Figure 15. Recombinant proteins, OmpR directly bind to the ler promoter. 63 Figure 16. The OmpR proteins also directly bind to the promoter region of stx1A gene. 64 Figure 17. The phospho-mimic OmpR exhibits high affinity to binding the promoter region of ler gene.. 65 Figure 18. The ler promoter region (-5 to -255) is important for OmpR binding the biotin-labeling DNA. 68 Figure 19 Establishment of the in vivo reporter system of EHEC in the intestine of C. elegans. 69 Figure 20. The expression of ler was regulated by OmpR in vivo. 71 Figure 21. OmpR was required for stx1 expression in EHEC after mitomycin C treatment. 72 Figure 22. OmpR upregulated the expression of T3SS genes. 73 Figure 23. OmpR upregulated the expression of Shiga-like toxin 1. 74 Figure 24. OmpR is involved in EHEC-induced microvilli effacement in human cells. 75 Figure 25. OmpR is involved in pathogenesis of EHEC-induced microvilli effacement in human cells. 77 Figure 26. Schematic illustrating the relationship between EnvZ/OmpR-mediated pH and osmotic pressure sensing in the alimentary tract and coordination of the expression of EHEC virulence genes in vivo. 78 Appendix 79 Figure S1. The flowchart is a diagram that the intensity of fluorescence proteins in the intestine of C. elegans is quantified by the analysis software, Image Pro Plus Analysis Software. 79 Figure S2. The flowchart is a diagram that the areas of microvilli actin on the surface of Caco2 are quantified by Image J system.. 81 Figure S3. The virulent determine genes in EHEC were identified from EHEC Tn5 transposon mutant library by using EHEC-C. elegans platform.. 82 Table S6: Transposon screening candidate genes 83

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