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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 廖淑貞 | |
dc.contributor.author | JIAN-FENG XIE | en |
dc.contributor.author | 謝建峰 | zh_TW |
dc.date.accessioned | 2021-06-17T06:27:51Z | - |
dc.date.available | 2021-08-30 | |
dc.date.copyright | 2018-08-30 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72188 | - |
dc.description.abstract | 奇異變形桿菌(Proteus mirabilis)屬於腸內菌科為健康人類腸道中的正常菌叢,常見為伺機性感染於尿導管植入之病患,是尿路感染的重要病原菌。細菌體內具有多種攝取環境中醣類的運輸系統,其一為phosphoenolpyruvate:sugar phosphotransferase system (PTS),此運輸系統由Enzyme I ( EI, encoded by ptsI )將磷酸烯醇式丙酮酸 ( phosphoenolpyruvate, PEP )上之一磷酸根 (phosphate, PO43-)轉移至HPr ( encoded by ptsH),而後再由HPr將該磷酸根轉移至不同的醣類特異性磷酸根攜帶酵素II ( phosphoryl carrier enzymes II, EII,包括EIIA,B,C )上,可攝取醣類同時將磷酸根由EIIB轉移至其上,最終磷酸化的醣類 (phosphoryl carbohydrate)進入代謝系統。過去已有研究指出PTS除了運輸醣類的功能外,尚扮演調控的腳色。除了carbohydrate PTS外,許多葛蘭氏陰性菌尚具有第二種PTS,被稱為氮相關PTS ( nitrogen-related PTS, PTSNtr)。PTSNtr 由EINtr (encoded by ptsP),NPr (encoded by ptsO)以及EIIANtr (encoded by ptsN)組成,其中ptsO與ptsN坐落於rpoN (54) 操縱子內。目前已有文獻指出ptsNNtr 參與許多調控機制。同時,EIIANtr在不同的磷酸化狀態下似乎具有不同之調控功能,而在棕色固氮菌中,以發現未磷酸化之EIIANtr可影響其細胞壁物質之合成。ptsN已於多種細菌被研究,諸如E. coli、K. pneumoniae,但尚未於Proteus mirabilis探討其調控角色。Mobley 及其團隊曾發現glutamine可增強P. mirabilis HI4320的表面移行能力;而Lee與其團隊中提出可藉由L-glutamine (L-gln)及-ketoglutarate (KG)影響大腸桿菌MG1655的EINtr而增加及減少去磷酸化 (dephosphorylated) EIIANtr之比例,並暗示PTSNtr可感應氮源可利用度。
本研究中,透過建構ptsN大量表現菌株,以觀察ptsN影響之表現型,並建構ptsN磷酸化位點突變大量表現菌株(ptsN(H72A))探討其中磷酸化狀態扮演之角色。結果顯示ptsN大量表現株在LB Lennox agar之表面移行能力較vector control顯著為低,而ptsN(H72A)大量表現株則恢復而與vector control 無差異;藉由添加L-gln與KG來觀察ptsN大量表現株減少之表面移行能力時是否藉由ptsN不同之磷酸化狀態來調控,發現不論添加與否,各菌株之間之表面移行能力並未出現新的變化,暗示此二物質在此條件下不能改變ptsN之磷酸化狀態,但野生株於L-gln中之rpoN表現降低。ptsN大量表現株之尿素酶(urease)活性較vector control顯著為低。ptsN大量表現株對於人類腎臟上皮細胞癌細胞 (A498)與移形上皮細胞癌(NTUB1)之附著力均分別較對照之野生株vector control略為提高;在移動相關基因之調控,qPCR結果顯示野生株於1.5%LB固態培養基時將顯著增加ptsNNtr表現;ptsN 大量表現株中glnA表現量明顯下降,暗示可能藉由抑制細胞內glutamine合成造成磷酸化EIIANtr增加進而影響表面移行能力。ptsN大量表現株與ptsN(H72A)大量表現株分別對於氧化壓力(30mM H2O2)耐受性均較vector control為高;在抗氧化壓力相關基因之調控中,30mM H2O2 環境下之野生型的ptsN表現增加,而 ptsN大量表現株中,sodB、soxR及rpoS表現增加,表示其可能藉由增加抗氧化相關基因表現而能提升對抗氧化壓力之能力。ptsN大量表現株與ptsN(H72A)大量表現株於含有5% NaCl之LB 液體培養基之耐受試驗均較vector control略為提高;在滲透壓相關基因之調控中,5% NaCl環境下並不影響ptsN基因表現,猜測是否藉由EIIANtr與KdpD之蛋白間作用 (protein-protein interaction, PPI)進而活化kdpFABC表現而影響細胞內離子濃度平衡。 | zh_TW |
dc.description.abstract | Proteus mirabilis belongs to Enterobacteriae as normal flora in health human and often diagnosed as an oppertunistic infection in patients with urinary-catheter implanted. The phosphoenolpyruvate:carbohydrate PTS, or sugar PTS, which transports and phosphorylates a carbohydrate into the cell also regulate the activities of a vast number of genes, and the parallel transfer cascade nitrogen PTS (PTSNtr), which does not transport carbohydrates but exerts many regulatory functions. The phosphate from phosphoenolpyruvate (PEP) was transferred through two general general phosphotransferase proteins: enzyme I (EI, encoded by ptsI or EINtr, encoded by ptsP in PTSNtr) and histidine protein (HPr, encoded by ptsH or NPr, encoded by ptsO in PTSNtr). In the sugar PTS, HPr subsequently phosphorylates the membrane-bound and substrate-specific transport protein enzyme II (EII) allowing uptake of the sugar. In PTSNtr, NPr phosphorylates EIIANtr(encoded by ptsN), which is not active in transport as the required domains are lacking. ptsO and ptsN were found located in the operon of rpoN (54). The phosphorylation state of EIIANtr may play distinct roles in various conditions as found the dephosphorylated form of EIIANtr affects the synthesis of cyst wall materials in A. vinelandii. The roles of ptsN has not been reported in Proteus mirabilis. Mobley et al suggested that the addition of L-glutamine(L-gln) may strengthen the swarming motility in P. mirabilis HI4320. Lee et al claimed that L-gln and-ketoglutarate (KG) in E. coli MG1655 affect the phosphorylation state of EIIANtr.
In this study, we investigated the phenotypes associated with ptsN and the role of phosphorylation states of ptsN by construction of overexpression of ptsN and non-phosphorylated EIIANtr (ptsN(H72A)) strains. The result showed that the overexpression of ptsN but not ptsN(H72A) delayed initiation time of swarming. -KG and L-gln were used to study whether the defect in swarming in the overexpression of ptsN was affected by the phosphorylation state of EIIANtr and the result showed that the compounds may not change the phosphorylation state of EIIANtr in this case but L-gln suppresses the mRNA expression level of rpoN in wild type. The overexpression of ptsN leads to lower urease activity and slighty increased the ability of cell adhesion. In the study of the regulation of motility-associated genes, the expression of ptsN significantly increased on solid surface and ptsN overexpression suppresed the expression of glnA which suggests that the suppression of intracellular glutamine concentration may reduce swarming motility by increasing the phosphorylated EIIANtr. The overexpression of ptsN and ptsN(H72A) increased the resistance of oxidative stress and high salt enviroment which may suggest that the interaction between EIIANtr and KdpD affects the homeostasis of intra-ion concentration by kdpFABC while the expression of ptsN elevated under 30mM H2O2. The expressoin of sodB, soxR and rpoS, which are associated with stress response, increased in the the overexpression of ptsN implied that ptsN improved the resistance under oxidative stress by upregulation the associated genes. | en |
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dc.description.tableofcontents | 口試委員審定書 I
中文摘要 II ABSTRACT III 誌謝 IV 圖目錄 IX 第一章 文獻回顧 1 第一節 奇異變形桿菌簡介 1 (1) P. mirabilis的基本介紹 1 (2) P. mirabilis的致病因子(附錄一) 1 生物膜(biofilm) 1 鞭毛 ( flagella ) 2 尿素酶(urease; urea amidohydrolase) 2 IgA蛋白酶(IgA-degrading protease) 3 溶血酶(haemolysin)[15] 3 胺基酸分解酶(deaminase)[15] 3 變形桿菌毒性凝集素( Proteus toxic agglutinin, Pta ) 3 (3) P. mirabilis的表面移形(swarming)能力(附錄二) 3 (4) P.mirabilis的抗藥性與感染症的治療 5 (5) P. mirabilis的感染與宿主的免疫關係 5 第二節 ptsN簡介 7 第三節 研究動機與目的 9 第四節 實驗設計 10 第二章 實驗材料與方法 11 第一節 實驗材料 11 (1) 實驗菌株、質體及引子 11 (2) 培養基、試劑、抗生素、儀器及網站 11 【培養基】 11 【試劑組 (kit)】 11 【抗生素】 12 【儀器】 12 【網站】 13 第二節 實驗方法 14 (1) 【建構P. mirabilis N2 pGEM vector control】(附錄六) 14 (2) 【建構ptsN overexpression】(附錄六) 15 (3) 【Site-direct mutagenesis: ptsNNtr(H72A) overexpression】(附錄十一) 16 (4) 【rpoN transcriptional reporter】(附錄七) 18 (5) 【定序及比對鑑定基因】 19 使用網站及軟體 19 分析方法 19 (6) 【聚合酶連鎖反應 (Polymerase chain reaction;PCR)】 20 (7) 【TA 選殖】 21 (8) 【質體DNA (plasmid DNA) 萃取】 22 (9) 【勝任細胞 (competent cell)製備】 23 A.電穿孔法 (electroporation) 勝任細胞 23 B.熱處理法 (heat shock) 勝任細胞 24 (10) 【轉型作用 (transformation)】 25 A.電穿孔法 (electroporation) 25 B.熱處理法 (heat shock) 25 (11) 【DNA純化】 26 A.電泳膠體純化 26 B.PCR產物純化 26 (12) 【DNA黏合作用 (ligation)】 27 (13) 【生長曲線 (growth curve) 測定】 28 (14) 【表面移行 (swarming) 試驗】 29 (15) 【泳動(Swimming)試驗 】 30 (16) 【生物膜 (biofilm) 生成能力】 31 (17) 【溶血素(haemolysin)活性測試】 32 (18) 【尿素酶(Urease)試驗】 33 (19) 【終點氧化壓力抵抗能力(Endpoint oxidative stress resistance)測試】 34 (20) 【細胞貼附 (cell adhesion)試驗】[53] 35 (21) 【Flagellin extraction】[54] 36 (22) 【不連續膠體電泳 (SDS-PAGE)】 37 (23) 【酸性及滲透壓耐受性測試】 39 (24) 【Reporter assay】[55] 40 (25) 【Catechol-2,3-dioxygenase (C23O) assay】 41 (26) 【RNA抽取 (RNA extraction)】 42 (27) 【RNA反轉錄 (reverse transcription)】 43 (28) 【同步定量PCR (real-time PCR)】 44 第三章 結果 45 第一節 ptsN序列分析 45 (1) ptsN基因位置分布及周邊基因(圖一) 45 (2) rpoN operon 驗證(附錄八)(圖二)(圖三) 45 (3) rpoN在P. mirabilis與E . coli之promoter序列分析(圖四) 48 第二節 建構P. mirabilis N2 之ptsN基因以pGEM大量表現菌株(附錄五)(圖五) 50 第三節 建構P. mirabilis N2 之位點突變ptsN基因(ptsN(H72A))並以pGEM大量表現菌株 …………………………………………………………………………………...51 (1) P. mirabilis N2 EIIANtr磷酸化位點預測(圖六) 51 (2) 建構含有pGEM-ptsN(H72A)之P. mirabilis N2 (附錄十一)(圖七) 51 第四節 ptsN大量表現菌株與ptsN(H72A)大量表現菌株之表現型分析 53 (1) ptsN大量表現株與ptsN(H72A)大量表現株生長與野生株vector control無差異(圖八) 53 (2) ptsN大量表現株在LB Lennox agar表面移型能力啟動時間點較野生株vector control為慢而ptsN(H72A)大量表現株則否(圖九) 53 (3) ptsN大量表現菌株在0.3% LB Lennox agar 泳動能力與野生株vector control無差異(圖十) 53 (4) ptsN 突變株形成生物膜 (biofilm)之能力與野生株相比無差異 (圖十一) 57 (5) ptsN 大量表現株之溶血素活性 (haemolysin) 與野生株vector control相比無差異而在第七小時略降 (圖十二) 57 (6) ptsN大量表現菌株尿素酶活性(urease)較野生株vector control顯著為低 (圖十三)..……………………………………………………………………………………60 (7) ptsN大量表現菌株貼附A498人類腎臟癌細胞的能力分別較野生株及野生株vector control略為提高 (圖十四) 60 (8) ptsN大量表現菌株表現鞭毛蛋白 (flagellin)與野生株vector control相比無可見差異(圖十五) 63 (9) ptsN大量表現菌株及 ptsN(H72A)大量表現株之氧化壓力耐受性 ( 30mM H2O2 ) 皆與野生株vector control相比顯著上升(圖十六) 64 (10) ptsN大量表現菌株酸性耐受性 ( pH5 ) 與野生株相比無差異(圖十七) 64 (11) ptsN 大量表現菌株尿素滲透壓耐受性 ( Urea ) 與野生株相比無差異(圖十八)…….………………………………………………………………………………….64 (12) ptsN大量表現菌株與ptsN(H72A)大量表現株高鹽滲透壓耐受性 ( 5% NaCl ) 與野生株相比略為提升(圖十九) 65 第四節 ptsN基因表現 69 (1) 野生株中ptsN mRNA表現量隨時間增加(圖二十一) 69 (2) rpoN promoter reporter中XylE活性隨時間增加(圖二十二) 70 (3) Transcriptome data顯示出crp突變株與rpoN突變株之ptsN現量上升及下降(圖二十三) 71 (4) L-gln 與a-KG 在P. mirabilis不影響ptsN大量表現菌株及ptsN(H72A)大量表現株於表面移行能力之差異(圖二十四) 72 (5) 1.5% LB agar中添加10 mM L-gln可於野生株中減少rpoN表現量而10mM -KG則不影響,而ptsN大量表現菌株及ptsN(H72A)大量表現株之rpoN表現量無差異(圖二十五)(圖二十六)(圖二十七) (圖二十八)(圖二十九) 74 (6) 分析ptsN大量表現菌株在1.5% LB agar中與表面移行相關之基因表現發現glnA明顯下降(圖三十)(附錄九) 80 (7) 野生株於30mM H2O2 LB broth中ptsN表現量較LB broth中為高,而ptsN大量表現菌提高抗氧化壓力相關基因中之sodB、soxR與rpoS(圖三十一)(圖三十二) ………………………………………………………………………………….81 (8) LB 含有5% NaCl不影響野生株之ptsN表現(圖三十三) (圖三十四)(附錄十)…….………………………………………………………………………………….83 (9) Urease activity之差異並非藉由影響ureC表現(圖三十五) 85 (10) 假設之ptsN調控關係圖(圖三十六) 86 第四章 結論 87 第五章 討論 88 第六章 表 90 表一、本研究中所使用之菌株及質體。 90 表二、實驗中所使用的引子 91 第七章 參考文獻 102 | |
dc.language.iso | zh-TW | |
dc.title | 探討尿道致病奇異變形桿菌ptsN(EIIANtr)所扮演的角色及其機制 | zh_TW |
dc.title | The roles of ptsN (EIIANtr) and underlying mechanisms in uropathogenic Proteus mirabilis | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧麗珍,邱浩傑,楊翠青 | |
dc.subject.keyword | 奇異變形桿菌,表面移行,抗氧化壓力, | zh_TW |
dc.subject.keyword | PTS,ptsN,oxidative stress resistance,swarming, | en |
dc.relation.page | 106 | |
dc.identifier.doi | 10.6342/NTU201803669 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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