臺灣博碩士論文加值系統

實驗室過去利用ceftriaxone 非抗藥菌株01-4 以multistep resistance selection 的方式，誘導出抗藥型菌株R200，經由RNA-sequencing 分析發現基因stm0084 的表現有顯著性改變；另外，我們也以protein fractionation 分析後發現STM0084 蛋白在ceftriaxone 抗藥型菌株R200 中的protein 表現量也有顯著增加，表示stm0084和ceftriaxone 的抗藥性可能有所關聯，因此我們以stm0084 作為本篇研究目標。
首先，利用one-step inactivation of chromosomal gene 方式將抗藥型菌株R200 的stm0084 基因剔除，並以E-test 測試MIC 值，結果發現stm0084 基因剔除後其其對ceftriaxne 藥物的MIC 值大幅下降約16 倍 (MIC 值為16 μg/ml)，且對於其他種類抗生素也有不同倍數的變化，由此說明stm0084 在抗ceftriaxone 及其他抗生素上的重要性。
雖然我們還無法確定STM0084 蛋白是以什麼樣的確切功能參與了ceftriaxone
抗藥性出現，但我們確認STM0084 蛋白是一個存在於間質區域的蛋白，且其存在
位置與ceftriaxone 抗藥性的出現是有相關性的，且STM0084 蛋白可被除了酸性環境之外的不同環境因子所誘發並具有sulfatase 的酵素活性，並可能以此參與其他的生理作用。
而從RNA-sequencing 和RT-PCR 的實驗結果發現R200(ΔcpxAR) 菌株中，其
stm0084 的mRNA 表現量相較於抗藥型菌株R200 都有下降趨勢，顯示STM0084
可能透過CpxAR 途徑的調控而與ceftriaxone 的抗藥性有所關聯。最後，透過reporter assay 與EMSA 實驗分析證實了stm0084 基因可以直接受CpxAR 二元訊息調控路徑的調控。
我們更進一步利用蛋白質圖譜及RT-PCR 分析發現，在過去研究中已知在鼠
傷寒沙門氏菌中會受CpxAR and BaeSR 二元訊息調控路徑所影響的抗ceftriaxone的外膜蛋白相關基因stm1530、stm3030、stm3031、ompD等，不管其mRNA 或蛋白質表現量在R200(Δstm0084) 菌株中都較R200 菌株有明顯差異。所以我們認為，STM0084 可能是經由CpxAR 二元訊息調控路徑的調控，進而影響了STM1530、STM3030、STM3031 與OmpD 等外膜蛋白的表現，從而對ceftriaxone 的抗藥性產生貢獻。

Significant increase in STM0084 protein level is present when the ceftriaxone-resistant Salmonella enterica serovar Typhimurium R200 strain is compared with the ceftriaxone-susceptible strain 01-4. Here, we examine the role of
STM0084 in ceftriaxone resistance.
A stm0084 gene deletion mutant, R200(Δstm0084), was generated and this strain showed 16-fold lower ceftriaxone resistance than R200, a fact suggesting an association between STM0084 and ceftriaxone resistance. Although the functions of the stm0084 gene remain unknown, we had confirmed the protein location of STM0084 is at periplasmic region and it can be induced by other environmental
factors besides acid condition and has sulfatase enzyme activity, thereby participating in other physiological response.
According to the RNA-sequencing and RT-PCR result, the stm0084 mRNA level was significantly decreased in both cpxAR gene deletion mutant, R200(ΔcpxAR) and baeSR gene deletion mutant, R200(ΔbaeSR). Finally, we find that the stm0084 gene
expression is directly regulated by CpxAR by a reporter assay and an EMSA assay.
Previously, the expression of STM3030, STM3031, STM1530, and OmpD, which are influenced by the CpxAR and BaeSR two-component system, play important roles in the ceftriaxone resistance of Salmonella enterica serovar Typhimurium.
Taken together, these result suggest that STM0084 is probably regulated by CpxAR but not BaeSR and influenced STM3030, STM3031, STM1530, and OmpD expression and that this contributed to S. enterica serovar Typhimurium ceftriaxone
resistance.

中文摘要 ..................................... 1
Abstract .................................. 2
第壹章、緒論…………………………………………………………………………..3
一、鼠傷寒沙門氏桿菌簡介……………………………………………………3
二、抗生素的作用機制………………………………………………………....4
三、羅氏芬 (Ceftriaxone, CRO )……………………………………………….4
四、細菌產生抗藥性之機制……………………………………………………5
五、藥物輸出幫浦 (drug efflux pump)……………………………….………...6
六、二元訊息傳遞系統 (Two Component-System).......7
七、孔洞蛋白 (Porin protein)...................7
八、硫酸酯酶蛋白………………………………………………………………8
九、酸性誘發硫酸酯酶蛋白 (Acid-Inducible Sulfatase) STM0084…..8
十、研究目的 ................................. ..9
第貳章、材料與方法.................................. 10
一、菌種與質體 (Bacterial strains and plasmids)........ 10
二、質體與引子序列 (Primers) ........................ 10
三、菌種儲存方法 ................................... 10
四、抗生素 (Antibiotics)................. 10
五、藥品、酵素及試劑組 ................................ 10
六、萃取核糖核酸 (Total RNA extraction).................... 11
七、反轉錄聚合酵素連合反應 (Reverse Transcription-PCR) ... 11
八、RNA 定序 ( RNA-sequencing; RNA-seq) ...... 12
九、聚合酵素鏈鎖反應 (Polymerase Chain Reaction, PCR) ...... 12
十、基因選殖 (DNA Cloning) ......... 13
十一、DNA 定序 (Sequencing) ......... 16
十二、One-step inactivation of chromosomal genes method ............................. 17
十三、抗生素敏感性試驗 (Antibiotics susceptibility test) .............................. 18
十四、生長曲線測定 (Growth curve determination) ........................................ 20
十五、細胞蛋白萃取 (Cell lysate extraction) ................................................... 20
十六、外膜蛋白萃取 (Outer membrane protein extraction) ............................. 21
十七、蛋白分層分離萃取法 (Protein fractionation) ........................................ 21
十八、蛋白質定量法 (Protein quantification) .................................................. 23
十九、蛋白質電泳及膠體製備 (SDS-PAGE, SDS Polyacrylamide Gel
Electrophoresis and Preparatio...............23
二十、西方點墨法 (Western blot) ..................... 24
二十一、組胺酸標記蛋白質的純化 (His-tag protein purification) ................. 25
二十二、電泳遷移率分析 (Electrophoretic Mobility Shift Assay, EMSA) ..... 26
二十三、鄰苯二酚加氧酵素報導基因分析 (Catechol-2,3-dioxygenase reporter assay)........................28
二十四、硫酸酯酶活性分析 (Sulfatase activity assay)........29
二十五、合成硫酸酯膽鹼 (Synthesis of choline-O-sulfate).....30
二十六、抗滲透壓壓力能力分析 (Osmoprotection assay)..........30
二十七、代謝碳、氮、硫元素生長能力分析 (Growth tests for carbon, nitrogen and sulphur source utilization).......30
第参章、實驗結果 .... 31
參之一、STM0084 新穎蛋白與鼠傷寒沙門氏桿菌的ceftriaxone 抗藥性的相關性
一、以RNA-sequencing 分析01-4、R200 的mRNA 表現量之差異 ............ 31
二、建構鼠傷寒沙門氏桿菌 stm0084 基因剔除菌株……………………..…31
三、stm0084 基因在鼠傷寒沙門氏菌中對ceftriaxone 抗藥性扮演重要角色312
四、STM0084 蛋白座落於鼠傷寒沙門氏桿菌中的periplasmic 區域……….32
五、大量表現STM0084 在01-4 及R200(Δstm0084)探討對抗藥性之影響 ..33
六、STM0084 protein 表現的位置及穩定性與ceftriaxone 抗藥性的相關性354
七、STM0084 protein 表現位置的不同與ceftriaxone 抗藥性出現的相關性 34
八、以Reverse-Transcription PCR 分析01-4、R200 與R200(Δstm0084) 的 mRNA 表現量之差異 ............... 35
九、新穎蛋白STM0084 與外膜蛋白組成的相關性 .......... 36
參之二、STM0084新穎蛋白與鼠傷寒沙門氏桿菌的二元訊息傳遞路徑的相關性
十、以RNA-sequencying 和RT-PCR 分析BaeSR 與CpxAR 二元訊息傳遞路
徑對stm0084 基因表現的影響…………………………………………..……38
十一、以Catechol-2,3-dioxygenase reporter assay 分析BaeSR 與CpxAR 二元訊息傳遞路徑對stm0084 的影響 .......... 38
十二、以EMSA 證明stm0084 的啟動子區域會受CpxR 蛋白所結合……...39
十三、以RT-PCR 分析Ceftriaxone 藥物對CpxAR 二元訊息傳遞路徑及
stm0084 基因表現的影響……………………………………………………....40
參之三、STM0084新穎蛋白的sulfatase功能在鼠傷寒沙門氏桿菌所扮演的角色
十四、以酵素動力學測試不同strain 中STM0084 新穎蛋白是否具有受酸性
生長環境所誘發的sulfatase 酵素活性………………………………………..41
十五、觀察STM0084 新穎蛋白是否具有choline sulfatase 酵素活性…….....42
參之四、STM0084 新穎蛋白在鼠傷寒沙門氏桿菌中與其他種類抗生素的相關性
十六、剔除stm0084 基因造成部份β-lactam 類抗生素的敏感性增加…….....45
第肆章、總結與討論 ........................... 46
第伍章、圖表 ................................ 50
參考文獻 ................................... 86

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