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Functional and Regulatory Characteristics of IscR, a Global Regulator of Vibrio vulnificus
DC Field | Value | Language |
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dc.contributor.advisor | 최상호 | - |
dc.contributor.author | 임종규 | - |
dc.date.accessioned | 2017-07-13T08:20:29Z | - |
dc.date.available | 2017-07-13T08:20:29Z | - |
dc.date.issued | 2014-02 | - |
dc.identifier.other | 000000018376 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119461 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최상호. | - |
dc.description.abstract | Pathogenic bacteria have evolved global regulatory mechanisms to facilitate cooperation of the numerous virulence factors during pathogenesis. In the present study, a homologue of IscR, an Fe-S cluster-containing transcriptional regulator was identified from Vibrio vulnificus, a causative agent of food-borne diseases, and its role and regulatory characteristics were assessed. A mutant that exhibited less cytotoxic activity toward INT-407 human intestinal epithelial cells was screened from a random transposon mutant library of Vibrio vulnificus, and an open reading frame encoding an Fe-S cluster regulator, IscR, was identified using a transposon-tagging method. A mutational analysis demonstrated that IscR contributes to mouse mortality as well as cytotoxicity toward the INT-407 cells, indicating that IscR is essential for the pathogenesis of V. vulnificus. A whole genome microarray analysis revealed that IscR influenced the expression of 67 genes, 52 of which were up-regulated and 15 down-regulated. Among these, twelve genes most likely involved in motility and adhesion to host cells, hemolytic activity, and survival under oxidative stress of the pathogen during infection were selected and experimentally verified to be up-regulated by IscR. Accordingly, the disruption of iscR resulted in a significant reduction in motility and adhesion to the INT-407 cells, hemolytic activity, and resistance to reactive oxygen species (ROS) such as H2O2 and t-BOOH. Furthermore, the present study demonstrated that the iscR expression was induced by exposure of V. vulnificus to the INT-407 cells and the induction appeared to be mediated by ROS generated by the host cells during infection. Consequently, the combined results indicated that IscR is a global regulator contributing to the overall success in the pathogenesis of V. vulnificus by regulating the expression of various virulence and survival genes in addition to Fe-S cluster genes.
Furthermore, the regulatory mechanisms for the iscR expression of V. vulnificus were evaluated. The expression of iscR was found to be upregulated by a transcriptional regulator AphA, a homologue of the low cell density regulator AphA of the Vibrio species, in the exponential phase of growth. The promoter activity of iscR appeared to be activated and repressed by AphA and IscR, respectively. EMSA and DNase I protection assay showed that both AphA and IscR bind to the iscR regulatory region and the binding site for AphA overlapped with part of the binding site for IscR. Mutational analysis suggested that AphA upregulates the iscR expression only in the presence of functional IscR. An examination of the roles of AphA and the binding sites revealed that the binding of AphA would hinder the IscR-mediated repression of the iscR transcription. The combined results show that V. vulnificus AphA upregulates iscR expression by antagonizing its negative autoregulation. Furthermore, the disruption of aphA resulted in significantly reduced virulence in tissue cultures and in mice. Accordingly, AphA contributes the pathogenesis of V. vulnificus possibly by promoting the production of IscR, which activates the genes required for survival and virulence. The transcriptome analysis revealed that Vibrio vulnificus IscR upregulates a gene encoding a putative antioxidant, homologous to human peroxiredoxin 5. This gene was further identified as a peroxiredoxin-encoding gene of V. vulnificus and named as prx3. The prx3 mutant was hypersusceptable to killing by hydrogen peroxide and peroxynitrite, indicating that V. vulnificus Prx3 is required for survival under oxidative and nitrosative stress. In addition, mouse mortality test suggested that Prx3 is essential for the virulence of V. vulnificus. The expression of prx3 was increased upon iron starvation in IscR-dependent manner, implying that IscR-dependent sensing of the cellular Fe-S cluster status involves the regulation of prx3. Escherichia coli dual plasmid system assay showed that IscR3CA mutant (apo-form of IscR) also activates the prx3 expression, suggesting that Fe-S cluster of IscR is dispensible for the activation of prx3. qRT-PCR and primer extension analyses showed that the expression of prx3 in the iscR3CA mutant was more increased than that in the wild type. These results might be contributed to the increased level of IscR3CA in the iscR3CA mutant. A direct interaction between IscR3CA and the promoter region of prx3 was demonstrated by an EMSA, and a IscR3CA binding site, centered at 44 bp upstream of the transcription start site, was identified by a DNase I protection assay. The binding site for IscR3CA on the prx3 promoter matched the type 2 binding motif of Escherichia coli IscR, reinforcing that apo-IscR also activates the prx3 expression. Taken together, the expression of V. vulnificus Prx3, essential for the survival under conditions of oxidative and nitrosative stress and virulence in mice, is regulated by IscR. | - |
dc.description.tableofcontents | Abstract I
Contents V List of Figures X List of Tables XII Chapter I. Background 1 I-1. Vibrio vulnificus 2 I-2. Disease caused by V. vulnificus 4 I-3. Virulence factors and molecular pathogenesis of V. vulnificus 6 I-4. Objective of this study 15 Chapter II. IscR is a global regulator essential for the pathogenesis of Vibrio vulnificus and induced by host cells 17 II-1. Introduction 18 II-2. Materials and Methods 21 II-2-1. Bacterial strains, plasmids and culture conditions 21 II-2-2. Identification of V. vulnificus iscR and generation of iscR mutant 29 II-2-3. Complementation of the iscR mutant 30 II-2-4. Cytotoxicity and mouse mortality 30 II-2-5. Transcriptome analysis 31 II-2-6. Quantitative real-time PCR (qRT-PCR) 32 II-2-7. Purification of V. vulnificus iscR and electrophoretic mobility shift assay 37 II-2-8. Motility and adhesion assays 38 II-2-9. Hemolysis assay and survival under oxidative stress 38 II-2-10. Western blot analysis 39 II-2-11. Microarray data accession number 40 II-3. Results 41 II-3-1. Identification of V. vulnificus IscR 41 II-3-2. IscR is important for virulence 41 II-3-3. IscR-regulated genes involved in pathogenesis 45 II-3-4. Effects of the iscR mutation on the virulence-related phenotypes of V. vulnificus 57 II-3-5. Effects of host cells on IscR expression 64 II-4. Discussion 69 Chapter III. Low cell density regulator AphA upregulates the expression of Vibrio vulnficus iscR gene encoding the Fe-S cluster regulator IscR 73 III-1. Introduction 74 III-2. Materials and Methods 77 III-2-1. Bacterial strains, plasmids, and culture conditions 77 III-2-2. Generation of aphA and aphA iscR mutants 77 III-2-3. RNA purification and transcript analyses 78 III-2-4. Overexpression and purification of V. vulnificus AphA and IscR 79 III-2-5. Electrophoretic mobility shift assay (EMSA) and DNase I protection assay 79 III-2-6. E. coli dual plasmid system 80 III-2-7. Cytotoxicity assay 81 III-2-8. LD50 determination 82 III-3. Results 83 III-3-1. Effects of the cell growth and aphA mutation on the iscR expression 83 III-3-2. Effects of iscR or aphA mutation on activity of iscR promoter 86 III-3-3. IscR and AphA bind specifically to the iscR regulatory region 89 III-3-4. Identification of binding sites for IscR and AphA using DNase I protection analysis 91 III-3-5. AphA upregulates the iscR expression only in the presence of functional IscR 95 III-3-6. Examination of the roles of AphA and binding sites in the control of PiscR activity 97 III-3-7. AphA is important for virulence 100 III-4. Discussion 103 Chapter IV. Evidence that a Vibrio vulnificus peroxiredoxin gene, required for survival under oxidative and nitrosative stress and virulence, is regulated by Fe-S cluster regulator IscR 107 IV-1. Introduction 108 IV-2. Materials and Methods 111 IV-2-1. Bacterial strains, plasmids, and culture conditions 111 IV-2-2. Generation of prx3 mutant 111 IV-2-3. Site-specific mutagenesis of IscR 112 IV-2-4. Growth of V. vulnificus under oxidative and nitrosative stress 112 IV-2-5. Mouse mortality test 113 IV-2-6. Construction of a prx3-luxCDABE transcription fusion and measurement of cellular luminescence 113 IV-2-7. E. coli dual plasmid system 114 IV-2-8. RNA purification and analysis of prx3 transcripts 115 IV-2-9. Western blot analysis 116 IV-2-10. Overexpression and purification of V. vulnificus IscR3CA 116 IV-2-11. Electrophoretic mobility shift assay (EMSA) and DNase I protection assay 116 IV-3. Results 118 IV-3-1. Identification of V. vulnificus prx3 gene 118 IV-3-2. Effect of prx3 mutation on the growth of V. vulnificus under oxidative and nitrosative stress 120 IV-3-3. Effect of prx3 mutation on virulence of V. vulnificus in mice 123 IV-3-4. Transcription of prx3 is controlled by IscR and iron 125 IV-3-5. Transcription of prx3 in the presence of functional IscR or IscR3CA 127 IV-3-6. Effect of iscR or iscR3CA mutation on the expression of prx3 130 IV-3-7. IscR3CA binds specifically to the prx3 regulatory region 134 IV-3-8. Identification of the IscR3CA binding site using DNase I protection analysis 136 IV-4. Discussion 139 Chapter V. Conclusion 142 References 147 국문초록 167 | - |
dc.format | application/pdf | - |
dc.format.extent | 3696601 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Vibrio vulnificus | - |
dc.subject | Fe-S cluster | - |
dc.subject | IscR | - |
dc.subject | AphA | - |
dc.subject | Peroxiredoxin | - |
dc.subject.ddc | 630 | - |
dc.title | Functional and Regulatory Characteristics of IscR, a Global Regulator of Vibrio vulnificus | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.citation.pages | xii, 170 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2014-02 | - |
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