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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
| dc.contributor.author | Miao-Hsia Lin | en |
| dc.contributor.author | 林妙霞 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:28:39Z | - |
| dc.date.copyright | 2010-02-04 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-01-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22799 | - |
| dc.description.abstract | 克雷伯氏肺炎桿菌NTUH-K2044引起之肝膿瘍及其併發轉移性病灶是種台灣近年來新興的感染性疾病，例如腦膜炎、眼內炎。這種人類細菌性病原外層包覆著多醣類物質成為物理性屏障，這種醣鞘結構稱為莢膜多醣體，是一種重要的致病因子，用來躲避人體免疫系統的攻擊。目前已知酪胺酸去磷酸脢與酪胺酸磷酸化激脢參與在莢膜多醣體生合成的調控之中，其他的調控蛋白並不清楚。為了了解酪胺酸磷酸化系統與細菌莢膜多醣體生合成的關聯性抑或是更進一步對於未來藥物的設計開發，我們必須找到更多參與調控的蛋白質。這篇論文主要利用散彈隨機 (shotgun approach) 質譜分析方式分析克雷伯氏肺炎桿菌NTUH-K2044 的磷酸化蛋白質體，在更進一步去探討磷酸化蛋白與莢膜多醣體生成的關係。此研究在菌株細胞內一共找到117條含有磷酸化的胜肽，其中包含了93個精確的磷酸化氨基酸位置，分佈於81個磷酸化蛋白中。
在這81個磷酸化蛋白之中，有三個酪胺酸磷酸化蛋白坐落於莢膜多醣體合成有關的基因組位於cps (capsule polysaccharide synthesis) 基因座上，這三個蛋白可能參與莢膜生成的調控之中，分別為酪胺酸磷酸化激脢 (Wzc)、磷酸甘露糖變位酶 (ManB) 和磷酸十一葵烯醇轉醣基酶 (WcaJ)。由於酪胺酸磷酸化激脢的調控機制較為清楚，因此，我們將目標放在磷酸甘露糖變位酶和磷酸十一葵烯醇轉醣基酶，並建立了點突變株分別為ManBY26F和WcaJY5F。由莢膜醣的定量及老鼠的腹腔注射實驗發現WcaJY5F此突變株不僅生成較少的莢膜多醣體，其致死力也較野生型菌株K2044低兩百倍。然而，ManBY26F此突變株多醣生成量不變，致死力也只差了六倍。我們的研究發現磷酸化十一葵烯醇轉醣基酶的第五個酪胺酸磷酸化參與了莢膜多醣體生合成的調控，且此酪胺酸的磷酸化與否與致病力息息相關。此外，已成功表現出Wzc、Wzb與ManB重組蛋白，未來將可進一步探討磷酸化與蛋白酵素活性的關係；其中在Wzc重組蛋白N端同時加上His以及HA胜肽標記 (His-HAWzc445-717)，可應用於免疫蛋白沉澱的實驗，企圖尋找相互作用的蛋白，期望建立整體訊息傳導網絡系統。若能對酪胺酸磷酸化系統對於莢膜生合成的影響有更多的了解，有機會發展出更多適合的治療與防預的方法，本研究提供了一個線索發展未來相關研究。 | zh_TW |
| dc.description.abstract | Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease often complicates with metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found protein tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we have analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins.
Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein tyrosine kinase (Wzc), phosphomannomutase (ManB) and undecaprenolphosphate glycosyltransferase (WcaJ), were found to distribute in the cps locus and thus were speculated to involve in the converging signal transduction of capsule biosynthesis. Subsequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. And as a result, the capsular polysaccharides on WcaJ mutant (WcaJY5F) was dramatically reduced quantitatively and the LD50 increased by 200 fold in mouse peritonitis model comparing to the wild-type strain. While the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity and the LD50 increased by merely 6 fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. Furthermore, the recombinant protein: Wzc, Wzb and ManB have been successfully generated; these proteins would be used to clarify the relationship between tyrosine phosphorylation and enzyme activity. In immunoprecipitation experiment, the N-terminal His and HA double tagged Wzc recombinant protein (His-HAWzc445-717) is used to find out the undiscovered interaction proteins. As a result, the signaling cascade will be well-established. Based on the further understanding of the precise roles of tyrosine phosphorylation system in bacterial CPS biosynthesis, more precise therapeutical and preventice methods must be developed. Our investigation provides a clue for further research on drug discovery in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:28:39Z (GMT). No. of bitstreams: 1 ntu-99-D94b46010-1.pdf: 9688586 bytes, checksum: 80cb54d9a23f6652da0440a1e3cbf8e7 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | Preface .............................................................................................................................. i
中文摘要 .......................................................................................................................... ii Abstract .......................................................................................................................... iv List of Figures .............................................................................................................. viii List of Tables .................................................................................................................. ix List of Appendixes ........................................................................................................... x Abbreviations ................................................................................................................. xi Introduction ..................................................................................................................... 1 1. Klebsiella pneumoniae .............................................................................................. 1 2. Bacterial exopolysaccharides .................................................................................... 3 3. Phosphorylation in bacteria ...................................................................................... 5 Materials and Methods ................................................................................................. 13 1. Bacterial growth and lysate preparation ................................................................. 13 2. Western blotting ...................................................................................................... 13 3. Protein digestion ..................................................................................................... 14 4. Phosphopeptide enrichment .................................................................................... 14 5. Affinity purification of pTyr proteome subset ........................................................ 15 6. LC-MS/MS analysis ............................................................................................... 16 7. Data analysis ........................................................................................................... 16 8. Bioinformatics analysis ........................................................................................... 17 9. Generation of site-specific mutants ........................................................................ 18 10. Extraction and quantification of CPS ................................................................... 19 11. Hypermucosity String test .................................................................................... 19 12. Precipitation speed test ......................................................................................... 20 13. Sugar composition analysis .................................................................................. 20 14. Resistance to serum killing ................................................................................... 21 15. Mouse lethality assay ............................................................................................ 21 16. Cloning and purification of Wzc cytoplasmic domain ......................................... 21 17. Cloning and purification of Wzb .......................................................................... 23 18. Over-expression and purification of ManB .......................................................... 23 19. Cloning and over-expression of WcaJ .................................................................. 24 20. ManB phosphorylation and dephosphorylation .................................................... 25 Results ............................................................................................................................. 26 1. Phosphoproteome of mid-log phase K. pneumoniae .............................................. 26 2. Phosphotyrosine proteome subset of K. pneumoniae ............................................. 28 3. Generation and characterization of site-directed mutants ....................................... 29 4. Overexpression and purification of Wzb, Wzc cytoplasmic domain, ManB and WcaJ. ........................................................................................................................... 30 5. ManB phosphorylation and dephosphorylation ...................................................... 31 Discussion ....................................................................................................................... 32 Figure .............................................................................................................................. 39 Table ............................................................................................................................... 48 Reference ........................................................................................................................ 53 Appendix ........................................................................................................................ 62 | |
| dc.language.iso | en | |
| dc.subject | 磷酸甘露糖變位酶 | zh_TW |
| dc.subject | 酪胺酸磷酸化激脢 | zh_TW |
| dc.subject | 酪胺酸磷酸化 | zh_TW |
| dc.subject | 克雷伯氏肺炎桿菌NTUH-K2044 | zh_TW |
| dc.subject | 肝膿瘍 | zh_TW |
| dc.subject | 莢膜多醣體生合成 | zh_TW |
| dc.subject | 磷酸十一葵烯醇轉醣基酶 | zh_TW |
| dc.subject | capsular polysaccharide biosynthesis | en |
| dc.subject | undecaprenolphosphate glycosyltransferase | en |
| dc.subject | phosphomannomutase | en |
| dc.subject | tyrosine kinase | en |
| dc.subject | tyrosine phosphorylation | en |
| dc.subject | Klebsiella pneumoniae NTUH-K2044 | en |
| dc.subject | liver abscess | en |
| dc.title | 以磷酸化蛋白質體學方法探討克雷伯氏肺炎桿菌中蛋白質酪胺酸磷酸化與致病機轉 | zh_TW |
| dc.title | Phosphoproteomics of Klebsiella pneumoniae NTUHK-K2044 reveals a tight link between tyrosine phosphorylation and virulence | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王錦堂(Jin-Town Wang),陳水田(Shui-Tein Chen),邱繼輝(Kay-Hooi Khoo),蔡明道(Ming-Daw Tsai) | |
| dc.subject.keyword | 克雷伯氏肺炎桿菌NTUH-K2044,肝膿瘍,莢膜多醣體生合成,酪胺酸磷酸化,酪胺酸磷酸化激脢,磷酸甘露糖變位酶,磷酸十一葵烯醇轉醣基酶, | zh_TW |
| dc.subject.keyword | Klebsiella pneumoniae NTUH-K2044,liver abscess,capsular polysaccharide biosynthesis,tyrosine phosphorylation,tyrosine kinase,phosphomannomutase,undecaprenolphosphate glycosyltransferase, | en |
| dc.relation.page | 110 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-01-28 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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