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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂 | zh_TW |
| dc.contributor.advisor | Jin-Town Wang | en |
| dc.contributor.author | 陳曉蝶 | zh_TW |
| dc.contributor.author | Hsiao-Tieh Chen | en |
| dc.date.accessioned | 2023-09-24T16:08:40Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-23 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-09 | - |
| dc.identifier.citation | 1. Ashayeri-Panah, M., M.M. Feizabadi, and F. Eftekhar, Correlation of Multi-drug Resistance, Integron and blaESBL Gene Carriage With Genetic Fingerprints of Extended-Spectrum beta-Lactamase Producing Klebsiella pneumoniae. Jundishapur J Microbiol, 2014. 7(2): p. e8747.
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Borlee, The NarX-NarL two-component system regulates biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei. Sci Rep, 2022. 12(1): p. 203. 25. Fang, C.T., et al., A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med, 2004. 199(5): p. 697-705. 26. Blumenkrantz, N. and G. Asboe-Hansen, New method for quantitative determination of uronic acids. Anal Biochem, 1973. 54(2): p. 484-9. 27. Freeman, Z.N., S. Dorus, and N.R. Waterfield, The KdpD/KdpE two-component system: integrating K(+) homeostasis and virulence. PLoS Pathog, 2013. 9(3): p. e1003201. 28. Dhiman, R.K., et al., Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis. PLoS One, 2019. 14(4): p. e0214958. 29. Ho, J.Y., et al., Functions of some capsular polysaccharide biosynthetic genes in Klebsiella pneumoniae NTUH K-2044. PLoS One, 2011. 6(7): p. e21664. 30. 莊依萍, The cps region responsible for capsular serotype K1 in klebsiella pneumonia causing human primary pyogenic liver abscess., in Department of microbiology. 2005: National Taiwan University. 31. Mehra, S., et al., The DosR Regulon Modulates Adaptive Immunity and Is Essential for Mycobacterium tuberculosis Persistence. Am J Respir Crit Care Med, 2015. 191(10): p. 1185-96. 32. Tazzyman, S., et al., Macrophage-mediated response to hypoxia in disease. Hypoxia (Auckl), 2014. 2: p. 185-96. 33. Stuehr, D.J. and M.A. Marletta, Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A, 1985. 82(22): p. 7738-42. 34. Martin, B., et al., Expression and maintenance of ComD-ComE, the two-component signal-transduction system that controls competence of Streptococcus pneumoniae. Mol Microbiol, 2010. 75(6): p. 1513-28. 35. Ruanto, P., et al., Activation by NarL at the Escherichia coli ogt promoter. Biochem J, 2020. 477(15): p. 2807-20. 36. Goh, E.B., et al., Hierarchical control of anaerobic gene expression in Escherichia coli K-12: the nitrate-responsive NarX-NarL regulatory system represses synthesis of the fumarate-responsive DcuS-DcuR regulatory system. J Bacteriol, 2005. 187(14): p. 4890-9. 37. Chuang, Y.P., et al., Genetic determinants of capsular serotype K1 of Klebsiella pneumoniae causing primary pyogenic liver abscess. J Infect Dis, 2006. 193(5): p. 645-54. 38. Berg, B.L. and V. Stewart, Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12. Genetics, 1990. 125(4): p. 691-702. 39. Lin, T.H., et al., FNR-Dependent RmpA and RmpA2 Regulation of Capsule Polysaccharide Biosynthesis in Klebsiella pneumoniae. Front Microbiol, 2019. 10: p. 2436. 40. Cortes, G., et al., Molecular analysis of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia. Infect Immun, 2002. 70(5): p. 2583-90. 41. Schembri, M.A., et al., Capsule and fimbria interaction in Klebsiella pneumoniae. Infect Immun, 2005. 73(8): p. 4626-33. 42. Sahly, H., et al., Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae. Infect Immun, 2000. 68(12): p. 6744-9. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90236 | - |
| dc.description.abstract | 本實驗室先前研究發現在分枝結核桿菌中，過度表現雙分子調控系統（TCS）的缺氧反應基因devS及devR會抑制NLRP3的活化，而克雷伯氏肺炎桿菌的narX及narL和前兩者皆有高達32%的序列相似度。已有研究指出NarX-NarL 雙分子調控系統在E. coli中可以調控硝酸鹽之呼吸作用，但此系統與細菌致病力的關係尚未受到討論。本研究目的為瞭解NarX-NarL 雙分子調控系統在克雷伯氏肺炎桿菌感染時的重要性，並探討其相關下游傳導路徑。我們以酵素結合免疫吸附分析法測量無標記基因剔除株∆narX 感染THP-1巨噬細胞時IL-1β的釋放量，結果顯示野生株及∆narX之間並無顯著差異，表示narX不太參與活化發炎小體的路徑。並且，剔除narX也不會影響生物膜的生成。然而，我們觀察到在厭氧環境的minimal agar上， ∆narX生長不論有無硝酸鹽的刺激，生長情形都相似，但視野生株的生長情形在添加硝酸鹽後有明顯好轉。基因微陣列分析結果發現narX在有氧環境下似乎對整體細胞的total RNA表現影響不大。接著，in vitro的實驗顯示∆narX對THP-1巨噬細胞的胞噬作用具更高的抵抗力，而從比色法再加上轉錄活性稍微較高的莢膜多醣基因群來推知，此現象可能是由較厚的莢膜多醣所造成。綜合上述，此研究再次證明narX能夠調節硝酸鹽呼吸作用的相關基因，且可能參與莢膜多醣的合成，影響感染時宿主胞噬作用的效力。 | zh_TW |
| dc.description.abstract | Klebsiella pneumoniae genes: narX and narL exhibit 32% sequence homology with hypoxia-induced two component system genes: devS and devR in Mycobacterium tuberculosis, whose over-expressions were shown to surpress NLRP3 inflammasome activation from our previous study. The molecular function of NarX-NarL two component system was known as the nitrate respiration modulator in E. coli. However, the role it plays in bacterial pathogenicity remains unknown. This study aims to understand the importance of NarX-NarL system during Klebsiella pneumoniae infection. ELISA was performed to record IL-1β release upon THP-1 macrophage infection by narX deletion mutant, ∆narX, which was constructed through unmarked deletion technique. The result showed no significant change in IL-1β release compared to the wild type strain, indicating narX played a minor role in inflammasome activation. Insignificant change in biofilm formation was also observed upon deletion of narX. However, although cell growth assay revealed that the growth of ∆narX remained the same with or without nitrate induction anaerobically, wild type strain’s growth was improved upon nitrate supplementation. DNA array analysis demonstrated that narX had less impact on the overall RNA expression profile while growing aerobically. Further in vitro experiment showed that ∆narX displayed increased resistance to phagocytosis by THP-1 macrophage. This might be explained by higher capsule polysaccharide (CPS) production as was determined with a colormeteric assay and with a mild elevation in transcriptional activity within the CPS loci. Collectively, these data confirmed the role of narX in regulating nitrate respiration genes and capsule polysaccharide production, and demonstrated its relevance to phagocytic escape during infection. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-24T16:08:40Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-24T16:08:40Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 I
誌謝 II 中文摘要 III Abstract IV 目錄 VI 表目錄 IX 圖目錄 X 第一章、緒論 1 1.1 克雷伯氏肺炎桿菌（Klebsiella pneumoniae） 1 1.2 發炎小體（Inflammasome） 2 1.3 克雷伯氏肺炎桿菌與發炎小體（K. pneumoniae and inflammasome） 2 1.4 雙分子調節系統（Two-component system） 3 1.5 NarX-NarL雙分子調控系統 ( NarX-NarL two-component system) 4 1.6 NarX-NarL 系統及生物膜之形成（NarX-NarL system and biofilm formation） 5 1.7 研究目標 （Study aim） 7 第二章、材料與方法 8 2.1 菌株 （Bacterial strain） 8 2.2 細胞株（Cell line） 8 2.3 建構∆narX之基因剔除株（Construction of deletion mutant ∆narX） 8 2.4 建構∆narX之基因補回株（Construction of ∆narX complementation strains） 9 2.5 巨噬細胞感染 （Macrophage infection） 10 2.6 酵素免疫吸附分析法 （Enzyme-linked immunosorbent assay; ELISA） 11 2.7 乳酸脫氫酶細胞毒性測試 （LDH cytotoxicity assay） 11 2.8 生物膜形成測試（Biofilm assay） 11 2.9 藥物感受性測試（Antimicrobial susceptibility test） 11 2.10 萃取克雷伯氏肺炎桿菌之RNA（RNA extraction of K. pneumoniae） 12 2.11 DNA微陣列分析（DNA microarray） 12 2.12 細菌生長測試（Bacterial growth assay） 16 2.13 胞噬作用檢測（Macrophage phagocytosis assay） 17 2.14 即時聚合酶連鎖反應（Real-time quantitative PCR） 17 2.15 莢膜多醣之定量（Capsular polysaccharide qauntification） 18 2.16 統計分析（Statistical analysis） 19 第三章、結果 20 3.1 基因序列對比（BLAST） 20 3.2 ∆narX基因剔除確認 （∆narX confirmation） 20 3.3 ∆narX活化發炎小體之情形 （Effect of ∆narX infection on inflammasome） 20 3.4 ∆narX生物膜之生成 （Biofilm formation of ∆narX） 21 3.5 藥物感受性測試（Antimicrobial susceptibility test） 21 3.6 基因微陣列分析 （DNA microarray） 21 3.7 NTUH-K2044和∆narX補回株之回補確認（complementation strain confirmation） 23 3.7 ∆narX的生長情形（Growth of ∆narX） 23 3.8 對胞噬作用的影響（Effect on phagocytosis） 23 3.9 莢膜多醣的生成改變（Capsular polysaccharide production change） 24 第四章、討論 26 參考文獻 31 表 一、本論文使用之引子 37 表 二、基因微陣列表現量顯著改變之基因 39 圖 一、以NCBI blast NTUH-K2044 genome序列之結果 40 圖 二、pKO3-narX-FR-km 41 圖 三、無標記基因剔除（unmarked deletion）流程圖 42 圖 四、NTUH-K2044之突變株∆narX基因剔除結果 43 圖 五、∆narX感染THP-1的細胞死亡率及IL-1β 分泌量 44 圖 六、生物膜之生成 45 圖 七、藥物感受性測試 46 圖 八、基因微陣列分析NTUH-K2044及∆narX中RNA之表現量流程圖 47 圖 九、NTUH-K2044與∆narX的基因微陣列分析 48 圖 十、NTUH-K2044和∆narX補回株之回補結果 49 圖 十一、∆narX生長情形 50 圖 十二、胞噬作用測試 51 圖 十三、莢膜多醣定量 52 圖 十四、莢膜基因群mRNA表現量 53 附錄 54 附錄 一、E.coli中NarX-NarL相關基因的分佈和功能之示意圖 54 附錄 二、pJET1.2/blunt map 55 附錄 三、pCR®-Blunt II-TOPO® vector map 56 附錄 四、初步結果：∆narX感染THP-1的細胞死亡率及IL-1β 分泌量 57 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 胞噬作用 | zh_TW |
| dc.subject | 克雷伯氏肺炎桿菌 | zh_TW |
| dc.subject | NarX-NarL雙分子調節系統 | zh_TW |
| dc.subject | 發炎小體 | zh_TW |
| dc.subject | IL-1β | zh_TW |
| dc.subject | inflammasome | en |
| dc.subject | NarX-NarL two-component system | en |
| dc.subject | Klebsiella pneumoniae | en |
| dc.subject | phagocytosis | en |
| dc.subject | IL-1β | en |
| dc.title | 探討克雷伯氏肺炎桿菌NarX-NarL雙分子調控系統之功能 | zh_TW |
| dc.title | Functional characterization of two component system NarX-NarL in Klebsiella pneumoniae | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 賴信志;董馨蓮;張書蓉 | zh_TW |
| dc.contributor.oralexamcommittee | Hsin-Chih Lai;Xin-Lian Dong;Shu-Jung Chang | en |
| dc.subject.keyword | 克雷伯氏肺炎桿菌,NarX-NarL雙分子調節系統,發炎小體,IL-1β,胞噬作用, | zh_TW |
| dc.subject.keyword | Klebsiella pneumoniae,NarX-NarL two-component system,inflammasome,IL-1β,phagocytosis, | en |
| dc.relation.page | 58 | - |
| dc.identifier.doi | 10.6342/NTU202303665 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2023-08-10 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| 顯示於系所單位： | 微生物學科所 | |
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