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Functional Analysis of Vibrio vulnificus SmcR, a Quorum-sensing Master Regulator, as a Novel Control Target : 식중독 패혈증 비브리오균 제어 표적으로서 quorum sensing 주 조절자인 SmcR의 기능 분석
DC Field | Value | Language |
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dc.contributor.advisor | 최상호 | - |
dc.contributor.author | 김승민 | - |
dc.date.accessioned | 2017-07-13T08:17:49Z | - |
dc.date.available | 2017-07-13T08:17:49Z | - |
dc.date.issued | 2012-08 | - |
dc.identifier.other | 000000002775 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119421 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2012. 8. 최상호. | - |
dc.description.abstract | Quorum sensing has been implicated as an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. Vibrio vulnificus is a model pathogen for studying many other foodborne pathogens because it causes life-threatening septicemia and gastroenteritis with various potential virulence factors controlled by quorum sensing. Recently, SmcR, a homologue of Vibrio harveyi LuxR, has been identified from V. vulnificus, and proposed as a quorum-sensing master regulator. In the present study, the roles of SmcR during an infectious process were examined in a series of experiments using biofilm cells, and comparing virulence of the smcR mutant with that of the parental wild type. When compared to the smcR mutant, the wild type showed a significant 2-fold increase in biofilm detachment rate in the extended time-course. To determine the genes involved in the biofilm detachment among SmcR-regulated genes, transcriptional profiles of the wild-type and smcR mutant biofilms were analyzed. The differentially expressed genes in the smcR mutant include genes that are known to stimulate biofilm dispersal in other bacteria. Interestingly, the smcR expression was induced upon exposure to intestinal epithelial cells. Thus, the ability of the wild type to detach cells from the biofilms was enhanced as biofilms were exposed to INT-407 human intestinal epithelial cells. On the other hand, the smcR mutant biofilms were not detached even upon exposure to INT-407 cells. In this regard, the defects in biofilm detachment in the smcR mutant resulted in a decrease in both intestinal colonization to new infection sites and histopathological damage in jejunum tissues from the mouse intestine after the intragastric administration of biofilm cells. Furthermore, the LD50 in ICR mice (specific-pathogen free) after intragastric infections of the smcR mutant was approximately 102 times higher than that of the parental wild type, suggesting that the smcR mutant biofilms were impaired in its ability to disperse and colonize new sites in the intestine in vivo. These differences between the wild-type and smcR mutant strain were not observed when planktonic cells were used. Therefore, these results indicate that upon entry into the host intestine, SmcR enables V. vulnificus biofilms to detach to find appropriate sites of infection and initiate a new infection cycle, demonstrating the importance of SmcR in V. vulnificus pathogenesis.
After the detachment of cells from the biofilms, flagellum-mediated motility is essential for dispersal of detached cells into the new colonization site. In the present study, the functions of FlhF and regulatory characteristics of the flhF expression of V. vulnificus were investigated. A deletion mutation of FlhF abolished motility, flagella formation, and flagellin synthesis, and introduction of flhF in trans complemented the defects. The flhF mutant revealed decreased expression of the class III and IV flagella genes, indicating that FlhF is a key regulator for the flagella biogenesis of V. vulnificus. The influence of global regulatory proteins on the expression of flhF was examined, and SmcR was found to downregulate the flhF expression at the transcriptional level. SmcR represses the flhF expression only in the stationary phase of growth and exerts its effects by directly binding to the flhF promoter region. Finally, an SmcR binding site, centered at 22.5-bp upstream of the transcription start site, was identified by a DNase I protection assay. The combined results demonstrate that a quorum sensing master regulator SmcR influences the motility and flagella biogenesis of V. vulnificus through modulating the expression of FlhF in a growth phase-dependent manner. During the initial stage of infection immediately after biofilm detachment, smcR expression is repressed because of low cell density, and expression of flhF is allowed, leading to flagella synthesis. The flagella prime V. vulnificus for initial colonization of host intestinal tissue, which is an important step required for the onset of its infectious cycle. In contrast, upon establishing preferred colonization niches with the increase in population density, the necessity of motility is superfluous, even detrimental, for a successful infection of hosts by the bacteria. In fact, flagellins of many enteropathogens have been well characterized as a major inducer as well as a target of host innate immune responses. Therefore, flagellar synthesis needs to be sophisticatedly regulated by quorum sensing regulatory pathways for optimal colonization and disease progression. These previous results led me to confirm that V. vulnificus quorum sensing is essential for the survival and pathogenesis of V. vulnificus. So identification and characterization of small molecules that inhibit quorum sensing are required for delineating novel strategies to control foodborne pathogens with low incidence of bacterial resistance. Therefore, a high throughput screening of small molecule libraries was performed to identify inhibitors of the V. vulnificus quorum sensing. Using a reporter strain PVVMO6_03194::luxAB whose activity entirely depends on SmcR, I identified a small molecule named U-262, 6-(phenylsulfonyl)nicotinonitrile. U-262 suppresses the activities of exoprotease and elastase without inhibiting bacterial growth itself. Attenuated cytotoxic activity, prolonged survival period and alleviated illness in mice were observed after treating V. vulnificus with the chemical. U-262 also decreased the luminescence of V. harveyi and the total protease activities of V. anguillarum which are regulated by quorum sensing, suggesting that it inhibits other quorum sensing of Vibrio spp.. Western blot analysis demonstrated that the chemical decreases the cellular level of SmcR in a dose dependent manner, indicating that the upper quorum-sensing signaling cascade is inhibited by the chemical. Meanwhile, E. coli dual plasmid system and EMSA showed that specific interactions between U-262 and SmcR resulted in the reduced DNA binding activity of SmcR, implying that U-262 affects both the cellular amount and the activity of SmcR. Taken together, these results suggested that U-262 is a novel anti-microbial agent inhibiting the quorum sensing of Vibrio spp. without the antibiotic resistance. It will be useful to protect food from the Vibrio spp. and help to enhance a public health. | - |
dc.description.tableofcontents | Abstract I
Contents VI List of Figures XI List of Tables XIV Chapter I. Introduction 1 I-1. Vibrio vulnificus 2 I-1-1. Virulence factors of V. vulnificus 4 I-1-2. Regulation of virulence in V. vulnificus 6 I-2. Quorum sensing 7 I-2-1. Quorum sensing in V. vulnificus 9 I-3. Previous study 11 I-4. Objective of this study 13 Chapter II. SmcR, a LuxR Homologue of Vibrio vulnificus, Controls Biofilm Dispersal 15 II-1. Introduction 16 II-2. Materials and Methods 19 II-2-1. Bacterial strains, plasmids and culture conditions 19 II-2-2. Biofilm formation assay 26 II-2-3. Sample preparation for RNA isolation 27 II-2-4. Transcriptome analysis 28 II-2-5. Quantitative RT-PCR 30 II-2-6. Competition assay 33 II-2-7. LD50 determination 34 II-2-8. Histopathological examination 34 II-2-9. Nucleotide sequence accession number 35 II-2-10. Data analyses 35 II-3. Results 36 II-3-1. Effect of smcR mutation on biofilm formation activity in V. vulnificus 36 II-3-2. Identification of the SmcR regulon by microarray expression profiling 38 II-3-3. Verification of microarray data 39 II-3-4. Purification of V. vulnificus RNA from within infected INT-407 cells and changes in the smcR expression 43 II-3-5. SmcR is essential for biofilm detachment toward epithelial cells in vitro 45 II-3-6. SmcR is required for intestinal colonization in vivo 48 II-3-7. Histopathology of mice infected with V. vulnificus 54 II-3-8. SmcR is important for virulence in mice 56 II-4. Discussion 60 Chapter III. Evidence that Vibrio vulnificus Flagellar Regulator FlhF is Regulated by Quorum Sensing Master Regulator SmcR 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 the V. vulnificus flhF deletion mutant 78 III-2-3. Motility assay and transmission electron microscopy 79 III-2-4. Preparation of protein samples and Western blot analysis 79 III-2-5. RNA purification and transcript analysis 80 III-2-6. Electrophoretic mobility-shift assay (EMSA) and DNase I footprinting 81 III-3. Results 82 III-3-1. Effects of an flhF mutation on the motility and flagella synthesis 82 III-3-2. Effect of an smcR mutation on the flhF transcription 87 III-3-3. Growth phase-dependent expression of flhF and its effect on motility 90 III-3-4. SmcR binds specifically to the flhF promoter region 94 III-3-5. Identification of the SmcR-binding site 96 III-4. Discussion 98 Chapter IV. Identification of a Small Molecule that Inhibits the Quorum Sensing of Vibrio vulnificus 102 IV-1. Introduction 103 IV-2. Materials and Methods 106 IV-2-1. Bacterial strains, plasmids, and culture conditions 106 IV-2-2. Selection of a gene directly regulated by SmcR 106 IV-2-3. Construction and development of high-throughput screening system 107 IV-2-4. Measurement of cellular luminescence 108 IV-2-5. Determination of β-galactosidase, total protease, and elastease activities 108 IV-2-6. Biofilm formation assay 110 IV-2-7. Cytotoxicity assay 111 IV-2-8. Morphological study 111 IV-2-9. Survival of V. vulnificus-infected mice 112 IV-2-10. Quantitative analysis of V. vulnificus in tissues 112 IV-2-11. Western immunoblotting 113 IV-2-12. Verification of hit chemicals using E. coli reporter system 113 IV-2-13. Electrophoretic mobility shift assay (EMSA) 114 IV-3. Results 115 IV-3-1. Selection of a gene from SmcR regulon for high-throughput screening 115 IV-3-2. High-throughput screening (HTS) system 119 IV-3-3. Effects of U-262 on virulence factors 122 IV-3-4. U-262 inhibits the cytotoxicity of V. vulnificus against human intestinal epithelial cells 127 IV-3-5. U-262 administration prolongs the survival period of V. vulnificus infected mice 132 IV-3-6. Effects of U-262 on quorum sensing of other Vibrio spp. 136 IV-3-7. Molecular mechanisms for inhibiting SmcR by U-262 138 IV-3-8. U-262 derivatives and their virulence 143 IV-4. Discussion 147 Chapter V. Conclusion 153 References 157 국문초록 174 | - |
dc.format | application/pdf | - |
dc.format.extent | 4919562 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Vibrio vulnificus | - |
dc.subject | quorum sensing | - |
dc.subject | SmcR | - |
dc.subject | biofilm | - |
dc.subject | FlhF | - |
dc.subject | quorum sensing inhibitor | - |
dc.subject.ddc | 630 | - |
dc.title | Functional Analysis of Vibrio vulnificus SmcR, a Quorum-sensing Master Regulator, as a Novel Control Target | - |
dc.title.alternative | 식중독 패혈증 비브리오균 제어 표적으로서 quorum sensing 주 조절자인 SmcR의 기능 분석 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Seung Min Kim | - |
dc.description.degree | Doctor | - |
dc.citation.pages | XIV, 178 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2012-08 | - |
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