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Genomic analysis of cronobacter sakazakii and molecular study of hfq and CSK29544_02616 genes as virulence factors : Cronobacter sakazakii균의 유전체 분석과 병원성 인자 hfq와 CSK29544_02616 의 분자적 연구
DC Field | Value | Language |
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dc.contributor.advisor | 유상렬 | - |
dc.contributor.author | 김성옥 | - |
dc.date.accessioned | 2017-07-13T08:25:19Z | - |
dc.date.available | 2017-07-13T08:25:19Z | - |
dc.date.issued | 2016-08 | - |
dc.identifier.other | 000000137239 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119532 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2016. 8. 유상렬. | - |
dc.description.abstract | Cronobacter sakazakii is considered to be an opportunistic pathogen causing life-threatening diseases, including necrotizing enterocolitis, septicemia, and meningitis particularly to infants with high fatality. However, the mechanisms of its pathogenicity and virulence-associated factors remain largely unknown. To understand them, complete genome of C. sakazakii ATCC 29544, a type strain, was sequenced and analyzed using bioinformatics. The complete genome of C. sakazakii ATCC 29544 is composed of a circular chromosome (Genbank accession No. CP011047) and three plasmids, including pCSK29544_p1 (Genbank accession No. CP011048), pCSK29544_p2 (Genbank accession No. CP011049), and pCSK29544_p3 (Genbank accession No. CP011050). It has been known that C.sakazakii infection mainly occurs via contaminated reconstituted infant formula milk. In this regard, I tried to identify several gene clusters, predicted to be responsible for the survival of C.sakazakii in harsh environments, including dried infant formula. Three gene clusters were found on chromosome, which may confer advantages on C.sakazakii survival against extremely dried conditions. These gene clusters include biosynthesis of capsular proteins (CSK29544_00281-00284) as protectants from osmotic shock, cellulose (CSK29544_01124-01127) to form biofilms, and nanKTAR (CSK29544_00587-590) to utilize sialic acid for energy production. Moreover, the strain ATCC 29544 may have arsenic resistance activity for survival in formula milk powder (CSK29544_3p0051-0055), on the plasmid 3p. These gene clusters, in part, may contribute to C.sakazakii survival under dried infant formula.
On human infection, C. sakazakii has invasins, OmpA (CSK29544_03699) for BMEC adhesion and the ibeB-homologous cusC (pCSK29544_3p0028) for the penetration of BMECs. For survival within the host, the strain ATCC 29544 has the privileged iron acquisition system, including siderophore biosynthesis system (iucABCD/iutA | - |
dc.description.abstract | CSK29544_1p0024- CSK29544_1p 0028) and an ABC-type iron transport system (eitCBAD | - |
dc.description.abstract | CSK29544_1p0056- CSK29544_1p 0059). The analysis of complete genome sequences would shed light on the understanding of virulence mechanisms by C. sakazakii.
As hfq has been reported as a critical virulence factor in many pathogenic bacteria, it was chosen to see its effects on C. sakazakii virulence. Hfq is a global regulator conversed in many pathogens, but its contributions to virulence vary from one species to another. Therefore, the role of Hfq in C. sakazakii virulence was investigated for the first time. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion into animal cells and survival within host cells, and exhibited low resistance to hydrogen peroxide. Remarkably, the loss of hfq led to hyper-motility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyper-flagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagella biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes. To identify new virulence factors required for invasiveness of C. sakazakii ATCC 29544, transposon-mediated random mutant library was constructed and screened for detection of mutants with reduced invasion ability into human intestine epithelial Caco-2 cells compared with wild type. One of the mutants showing the most reduced invasion ability but a comparable growth with WT was found to have transposon insertion in the CSK29544_02616, which is highly conserved in only Cronobacter species and encodes an unidentified protein. The deletion mutant of CSK29544_02616 was constructed and various virulence-associated traits, including invasion to intestinal Caco-2 cell, in vivo animal study, survival/phagocytosis within/into cultured-macrophages, motility, and biofilm formation, was characterized. The lack of CSK29544_02616 showed attenuations in invasiveness, in vivo colonization and dissemination, phagocytosis and survival into/within macrophage-like cells. Based on the observations, I questioned the function of CSK29544_02616 of C.sakazakii ATCC 29544 in terms of virulence. As it is an unidentified protein, I determined to perform ligand fishing assay to screen players which help the function of CSK29544_02616 to be unveiled. I found that LpxA, the first enzyme involved in lipid A biosynthesis, was shown to specifically interact with CSK29544_02616. The amount of lipid A-core was decreased in CSK29544_02616 mutation, whereas that of phospholipids was increased. This altered ratio between Lipid A and phospholipids in outer membrane influenced outer membrane assembly and changed cell surface to be more hydrophobic, resulting in increased cell autoaggregation and biofilm formation. Interestingly, the mutant was non-motile although it had flagella and torque generation, indicating loss of CSK295444_02616 caused paralyzed flagella. Taken together, CSK29544_02616 plays critical roles in the virulence of C. sakazakii by modulating LPS production. CSK29544_02616 would be a good target for biocontrol of C. sakazakii in the way that the deletion of CSK29544_02616 does not affect C. sakazakii growth, but influence its virulence as an antibacterial agent. In conclusion, understanding the pathogenesis of C. sakazakii ATCC 29544 by both genomic analysis and molecular studies of virulence factors would be the cornerstone for the development of a new strategy to control C. sakazakii pathogenesis. | - |
dc.description.tableofcontents | Chapter I. Introduction 1
I-1. Cronobacter sakazakii 2 I-1-1. Clinical manifestations of C. sakazakii infections 4 I-1-2. Environmental reservior and mode of transmission 5 I-2. Pathogenicity and virulence factors of C. sakazakii 7 I-2-1. Invasins 7 I-2-2. Survival within host immune cells 8 I-2-3. Stress tolerance 9 I-3. Objectives of this study 10 Chapter II. Complete genome sequence analysis of Cronobacter sakazakii 12 II-1. Introduction 13 II-2. Materials and Methods 16 II-2-1. Bacterial strains, plasmids and growth conditions 16 II-2-2. Genome sequencing and assembly 16 II-2-3. Genome annotation 17 II-2-4. Bioinformatics 17 II-3. Results and Discussion 19 II-3-1. Genome properties of C. sakazakii ATCC 29544T 19 II-3-2. Pathogenicity and virulence factors 27 II-3-3. Comparative genome analysis 31 II-3-4. Comparative phylogenetic tree analysis 32 Chapter III. The hfq, a RNA chaperone, plays important roles in virulence and stress adaptation in Cronobacter sakazakii ATCC 29544 36 III-1. Introduction 37 III-2. Materials and Methods 41 III-2-1. Bacterial strains, plasmids, and culture conditions. 41 III-2-2. Construction of a hfq deletion mutant using the Lambda-Red recombination method 41 III-2-3. Complementation study 42 III-2-4. Gentamicin protection (invasion) assay 43 III-2-5. Motility assay 44 III-2-6. Bacterial survival assay in animal cells 44 III-2-7. Bacterial resistance against hydrogen peroxide 45 III-2-8. qRT-PCR (quantitative real time) 46 III-2-9. Animal study in vivo 46 III-2-10. TEM (transmission electron microscopy) analysis 47 III-2-11. Western blot analysis 47 III-2-12. Primer extension analysis 48 III-2-13. Statitical analysis 50 III-3. Results 58 III-3-1. Construction and growth characteristics of the hfq mutant in C. sakazakii 58 III-3-2. The loss of hfq attenuates colonization by C. sakazakii in rat pups 63 III-3-3. Hfq is involved in the invasion of C. sakazakii into human epithelial cells 67 III-3-4. Hfq is required for oxidative-stress resistance in C. sakazakii 70 III-3-5. Loss of the hfq gene reduces intracellular survival of C. sakazakii in macrophage-like cells 74 III-3-6. A C. sakazakii hfq strain is hypermotile due to higher expression of flagella 77 III-4. Discussion 83 Chapter IV. CSK29544_02616, a newly identified protein, influences C. sakazakii virulence by modulating LPS production 91 IV-1. Introduction 92 IV-2. Materials and Methods 96 IV-2-1. Strains, plasmids, and culture conditions 96 IV-2-2. Construction of random transposon mutant libraries and screening 96 IV-2-3. Determination of the transposon insertion sites 96 IV-2-4. Generation of ΔCSK29544_02616 deletion mutant by using site-specific mutagenesis 97 IV-2-5. Construction of plasmids 98 IV-2-6. Invasion assay 98 IV-2-7. Survival assay 99 IV-2-8. In vivo animal study 100 IV-2-9. Suspension-clearing and microscopic analysis 101 IV-2-10. Overexpression and purification of the tagged-proteins 101 IV-2-11. Ligand fishing assay 102 IV-2-12. LC/MS/MS analysis 103 IV-2-13. Database search 104 IV-2-14. BACTH (Bacterial adenylate cyclase based two-hybrid system) assay 104 IV-2-15. β-galactosidase assay 106 IV-2-16. In vitro GST pull-down assay 106 IV-2-17. Western blot analysis 107 IV-2-18. LpxA enzyme assay 108 IV-2-19. LPS extraction 112 IV-2-20. Outer membrane fraction 114 IV-2-21. Phospholipid quantitation 115 IV-2-22. Hydrophobicity assay 116 IV-2-23. Biofilm assay 117 IV-2-24. RNA isolation and sequencing 117 IV-3. Results 128 IV-3-1. A gene discovered in an invasion-attenuated mutant 128 IV-3-2. The CSK29544_02616 gene is required for C .sakazakii invasion 131 IV-3-3. The CSK29544_02616 gene contributes to C .sakazakii virulence 133 IV-3-4. CSK29544_02616 directly interacts with LpxA 137 IV-3-5. CSK29544_02616 helps LpxA enzyme activity in a synergistic manner 146 IV-3-6. CSK29544_02616 is involved in modulating the ratio of lipid A and phospholipid 156 IV-3-7. The altered balance between Lipid A and phospholipids in CSK29544_02616 mutation influenced outer membrane assembly 158 IV-3-8. Reduction of LPS production in ΔCSK29544_02616 increased cell surface hydrophobicity and biofilm formation 163 IV-3-9. CSK29544_02616 abrogated flagella-mediated motility 166 IV-4. Discussion 170 References 178 국문 초록 215 | - |
dc.format | application/pdf | - |
dc.format.extent | 3704598 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Cronobacter sakazakii | - |
dc.subject | Complete genome sequences | - |
dc.subject | Pathogenesis | - |
dc.subject | hfq | - |
dc.subject | CSK29544_02616 | - |
dc.subject | LpxA | - |
dc.subject.ddc | 630 | - |
dc.title | Genomic analysis of cronobacter sakazakii and molecular study of hfq and CSK29544_02616 genes as virulence factors | - |
dc.title.alternative | Cronobacter sakazakii균의 유전체 분석과 병원성 인자 hfq와 CSK29544_02616 의 분자적 연구 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Kim, Seongok | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 219 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2016-08 | - |
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