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Identification of the roles of EIIANtr in amino sugar homeostasis and virulence regulation in salmonella enterica serovar typhimurium : Salmonella enterica serovar typhimurium에서 EIIANtr의 아미노당 항상성과 병원성에 대한 역할 규명
DC Field | Value | Language |
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dc.contributor.advisor | 유상렬 | - |
dc.contributor.author | 유웅재 | - |
dc.date.accessioned | 2017-07-13T08:25:42Z | - |
dc.date.available | 2017-07-13T08:25:42Z | - |
dc.date.issued | 2017-02 | - |
dc.identifier.other | 000000140867 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119538 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 유상렬. | - |
dc.description.abstract | Salmonella enterica serovar Typhimurium is a rod-shaped, flagellated, facultative anaerobic, and Gram-negative bacterium. Salmonella Typhimurium is one of the major food-borne pathogens which causes a wide variety of diseases from gastroenteritis in millions of people worldwide to severe systemic infection in humans and animals. Various virulence factors and their regulation mechanisms have been identified in Salmonella, but the functions and mammalian targets of only a few have been known.
The term phosphotransferase system (PTS) describes a system in which enzymes transfer phosphate moieties derived from phosphoenolpyruvate (PEP) from one enzyme to the other in a sequential order. In general, two different branches of the PTS exist | - |
dc.description.abstract | the sugar-PTS, responsible for the phosphorylation and uptake of sugars into the cell and the nitrogen-metabolic PTS or PTSNtr, which fulfils exclusively regulatory functions.
Many Proteobacteria possess the paralogous nitrogen-metabolic phosphotransferase system (PTSNtr) which consists of enzyme INtr (encoded by ptsP), NPr (encoded by ptsO), and enzyme IIANtr (encoded by ptsN). Due to the location of ptsO and ptsN downstream of rpoN in the same operon, this system is postulated to be involved in nitrogen metabolism. Since a specific substrate transferred by PTSNtr is yet to be determined, this system is supposed to function mainly in a regulatory capacity. In order to define the primary role of PTSNtr in Salmonella Typhimurium, ligand fishing was performed with EIIANtr as a bait and revealed that D-glucosamine-6-phosphate synthase (GlmS) directly interacted with EIIANtr. GlmS, which converts D-fructose-6-phosphate (Fru6P) into D-glucosamine-6-phosphate (GlcN6P), is a key enzyme producing amino sugars through glutamine hydrolysis. Amino sugar is an essential structural building block for bacterial peptidoglycan and lipopolysaccharide (LPS). I further verified that EIIANtr interacted with GlmS in a heterotrimeric formation and inhibited GlmS activity in a phosphorylation-state-dependent manner. EIIANtr was dephosphorylated in response to excessive nitrogen sources and was rapidly degraded by Lon protease upon amino sugar depletion. The regulation of GlmS activity by EIIANtr and the modulation of glmS translation by RapZ (RNase adaptor protein encoded immediately downstream of ptsN) suggest that the genes comprising the rpoN operon play a key role in maintaining amino sugar homeostasis in response to nitrogen availability and the amino sugar concentration in the bacterial cytoplasm. In order to understand roles of nitrogen-metabolic PTS in Salmonella Typhimurium, whole transcriptome was also compared between the wild-type and a mutant strain lacking ptsN by RNA sequencing (RNA-Seq). Genome-wide transcriptomic analysis revealed that 3.5% of the whole annotated genes was up or down regulated by ptsN by three-fold or more. Genes differentially regulated by ptsN could be grouped into 5 categories based on their predicted functions including carbohydrate transport and metabolism, amino acid transport and metabolism, energy production and conversion, transcriptional regulation, and cell wall/membrane/envelope biogenesis. Among them, the expression of genes involved in 1,2-propanediol (1,2-PDL) utilization and Ado-B12 synthesis was significantly decreased in the ΔptsN mutant strain, and this phenomenon was complemented with the addition of glutathione (GSH) into the growth medium. Therefore the quantity of GSH was compared between wild-type and ΔptsN mutant strain, and confirmed that GSH was about 3 folds lower in ΔptsN mutant strain than wild-type. Decreased expression level of genes involved in 1,2-PDL utilization and Ado-B12 synthesis in ΔptsN mutant strain results in the lower production of propionate in the presence of 1,2-PDL compared to wild-type. Interestingly, the invasion ability of ΔptsN mutant strain was about 5-fold higher than that of wild-type in the presence of 1,2-PDL. Concentration of 1,2-PDL can be high in the intestine because it can be produced by the fermentation of the common plant sugars L-rhamnose and L-fucose. L-fucose is also found in the glycoconjugates of intestinal cells, where it is involved in host-pathogen interaction. As previously reported, propionyl-CoA, the intermediate of 1,2-PDL metabolism, reduces the protein stability of HilD, a master regulator of Salmonella pathogenicity island-1 (SPI-1), leading to attenuation in Salmonella virulence. Interestingly, EIIANtr protein level was increased by over 3 folds in the presence of 1,2-PDL. Based on these results, it is suggested that EIIANt can modulate Salmonella fitness and virulence via 1,2-PDL metabolism. In conclusion, it was discovered that discovered the new roles of EIIANtr, one of the components of nitrogen-metabolic PTS, in the regulation of amino sugar homeostasis and 1,2-PDL utilization pathway in Salmonella enterica serovar Typhimurium. GlmS is the only enzyme synthesizing amino sugar required for the peptidoglycan and LPS biosynthesis, and 1,2-PDL is a good carbon and energy source for Salmonella Typhimurium to compete with other intestinal microbiota. In this study, EIIANtr controlled GlmS enzyme activity and the expression of cob-pdu operon in response to environmental cues including amino sugar and 1,2-PDL, and the expression of EIIANtr was also regulated either post-translationally or translationally in response to the same environmental cues such as amino sugar and 1,2-PDL. It means that EIIANtr is a key player modulating Salmonella fitness and virulence through the interaction with its host environment, and I suggest the new possibility to reduce Salmonella disease by controlling the activity or the expression of EIIANtr. | - |
dc.description.tableofcontents | Chapter I. Introduction 1
I-1. Salmonella enterica serobar Typhimurium 2 I-2. Salmonella pathogenesis and its virulence mechanisms 7 I-3. Objectives of this study 13 Chapter II. Fine-tuning of amino sugar homeostasis by EIIANtr in Salmonella Typhimurium. 15 II-1. Intdoruction 16 II-2. Materials and Methods 22 II-2-1. Bacterial strains, plasmids, and culture conditions 22 II-2-2. Plasmid construction 23 II-2-3. Ligand fishing to search for EIIANtr-His6 protein targets 25 II-2-4. Bacterial two-hybrid system for studying protein-protein interactions 26 II-2-5. β-Galactosidase assay 28 II-2-6. Immunoprecipitation of EIIANtr using His6-Glms in vivo. 28 II-2-7. In vitro phosphorylation assay 29 II-2-8. Purification of His6-tagged proteins 30 II-2-9. In vitro protein-protein interactions between phosphorylated/unphosphorylated EIIANtr and GlmS 31 II-2-10. Size exclusion chromatography with multi-angle light scattering 32 II-2-11. Glucosamine-6-phosphate synthase activity assay 33 II-2-12. RNA isolation and quantitative real-time RT-PCR 33 II-2-13. SDS-PAGE and Western blotting analysis 34 II-2-14. Analysis of protein stability 35 II-3. Results 44 II-3-1. Specific interaction between Enzyme IIANtr and glucosamine-6-phosphate synthase in Salmonella Typhimurium 44 II-3-2. Phosphorylation status of EIIANtr influences its binding affinity with GlmS. 49 II-3-3. EIIANtr inhibits GlmS activity 62 II-3-4. EIIANtr affects the GlmS-mediated bacterial growth rate 68 II-3-5. EIIANtr and GlmS interact to form a heterotrimeric complex 74 II-3-6. EIIANtr stability is modulated by Lon in response to amino sugar availability. 79 II-4. Discussion 90 Chapter III. Transcriptomic analysis of Salmonella enterica serovar Typhimurium lacking ptsN 100 III-1. Introduction 101 III-2. Materials and Methods 105 III-2-1. Bacterial strains, media, and culture conditions 105 III-2-2. Construction of strains and plasmids 105 III-2-3. RNA isolation and sequencing 106 III-2-4. Heat-map generation 107 III-3. Results and Discussion 117 III-3-1. Comprehensive understanding of EIIANtr roles in Salmonella using transcriptome analysis 114 III-3-2. EIIANtr activated expression of genes involved in vitamin B12 synthesis and 1,2-PDL utilization 118 III-3-3. EIIANtr negatively controlled expression of SPI-1 and SPI-4 important for Salmonella invasion 122 Chapter IV. EIIANtr regulates Salmonella virulence via 1,2-propanediol utilization pathway. 130 IV-1. Introduction 131 IV-2. Materials and Methods 137 IV-2-1. Bacterial strains, media, and culture conditions 137 IV-2-2. Construction of strains and plasmids 137 IV-2-3. Quantitative real-time RT-PCR 139 IV-2-4. Propanediol utilization assay 140 IV-2-5. β-Galactosidase assay 140 IV-2-6. Western blotting analysis 141 IV-2-7. Analysis of EIIANtr and HilD stability 141 IV-2-8. Gentamicin protection assay 142 IV-2-9. Glutathione (GSH) assay 143 IV-3. Results 148 IV-3-1. EIIANtr positively regulates the expression of genes involved in Ado-B12 synthesis and 1,2-PDL utilization 148 IV-3-2. Dissecting EIIANtr roles in transcriptional regulation of pdu operon and SPI-1 and -4 153 IV-3-3. Translational levels of EIIANtr is up-regulated in response to 1,2-PDL 160 IV-3-4. EIIANtr positively regulates the expression of genes involved in 1,2-PDL utilization pathway by controlling glutathione (GSH) production 163 IV-4. Discussion 167 References 173 국문 초록 207 | - |
dc.format | application/pdf | - |
dc.format.extent | 6563261 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | 1 | - |
dc.subject | 2-propanediol utilization (pdu) | - |
dc.subject | Glucosamine-6-phosphate synthase (GlmS) | - |
dc.subject | Nitrogen-metabolic phosphotransferase system (PTSNtr) | - |
dc.subject | RNA-sequencing (RNA-seq) | - |
dc.subject | Salmonella pathogenicity island-1 (SPI-1) | - |
dc.subject | Salmonella pathogenicity island-4 (SPI-4) | - |
dc.subject | Salmonella Typhimurium | - |
dc.subject.ddc | 630 | - |
dc.title | Identification of the roles of EIIANtr in amino sugar homeostasis and virulence regulation in salmonella enterica serovar typhimurium | - |
dc.title.alternative | Salmonella enterica serovar typhimurium에서 EIIANtr의 아미노당 항상성과 병원성에 대한 역할 규명 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Woongjae Yoo | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 230 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2017-02 | - |
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