臺灣博碩士論文加值系統

沙門氏菌為感染人類及動物之病原菌，主要透過沙門氏菌致病小島 （Salmonella pathogenicity island, SPI） 經轉錄及轉譯出致病蛋白，在感染初期，透過 SPI1 分泌蛋白質，使菌體入侵腸道上皮細胞。許多環境訊息及調控蛋白會調控 SPI1 上 hilA 基因的表現，其中蛋白酶扮演重要的角色。Lon 及 Clp family 都屬於細菌中 ATP 依賴蛋白酶（ATP-dependent protease）。已知 Lon 蛋白酶對 HilC、HilD 調控蛋白有負調控作用，使得SPI1基因表現量降低，且 ClpQY 蛋白酶已被證實與 Lon 蛋白酶在功能上有部份重疊。在本實驗中，將探討在不同環境條件下 ClpYQ （HslUV） 蛋白酶與沙門氏菌致病性之關係。將 SPI1 其中 4 個基因融合 lacZY 報導基因 （hilA:: lacZY、invF:: lacZY、sipA:: lacZY、sipC:: lacZY），利用噬菌體 P22 將基因片段帶入lon、clpY、clpQ 單一缺失或雙缺失突變株中。藉由不同氧氣含量及不同滲透壓（0%-3.5% NaCl）改變環境條件來培養菌體，經由 β-galactosidase assay，發現在氧氣限制及在 1% NaCl 條件下，各菌株在 SPI1 基因表現量達到最大值，在 clpQ 或 clpY 單一缺失突變株或是clpQ clpY 雙缺失突變株中，無法觀察到 SPI1 基因表現量和野生株有明顯差別；然而 lon 單一缺失突變株，其 SPI1 基因表現量比野生株高 3-5 倍；clpQ lon 雙缺失突變株，其 SPI1 基因表現又比 lon 單一缺失突變株高些，在 lon 缺失的背景下，會將 clpQ 基因缺失對於 SPI1 基因表現量的影響放大，推測 ClpYQ 蛋白酶在調控沙門氏菌致病力表現上，可能和 Lon 蛋白酶功能重複之地方。接著找出，lon 單一缺失突變株和 clpQ lon 雙缺失突變株表現量差異最大的環境條件，發現對於 invF 及 hilA 基因，在野生型及各個突變株中，其基因表現量受到環境因子的調控是不盡相同的，在供給氧氣及 1.5% NaCl 或 2% NaCl 條件下，發現 clpQ lon 雙缺失突變株表現量明顯高於 lon 單一缺失突變株；在 2% NaCl 時，lon 單一缺失突變株在SPI1 基因表現受到明顯抑制，比野生型還低。ClpQ 蛋白和 ClpY 蛋白在調控本實驗的四種基因表現量上，也扮演著不同的角色。在高氧氣環境培養條件下，氧氣含量會抑制整個 SPI1 基因的表現，lon 單一缺失突變株及 clpQ lon 雙缺失突變株對於氧氣含量敏感，由改變滲透壓實驗的數據發現，在供給氧氣條件下，lon 單一缺失突變株對滲透壓的改變較 clpQ lon 雙缺失突變株敏感許多，推測在環境不利於 SPI1 基因表現的環境下，不同於 Lon 蛋白酶的功能，ClpQY 蛋白酶扮演著一定的角色。

Salmonellae are pathogenic bacteria to both human and animals. Salmonella invades intestinal epithelial cells by using a type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle-like structure that injects effecter proteins directly into the cytosol of host cells. Salmonella pathogenicity is to maintain its survival and is able to replicate inside host cells. These virulence traits could cause systemic infections. SPI1 expression is influenced by a variety of environmental conditions and regulatory proteins, and the key regulator is the SPI1-encoded HilA, which activates their transcription directly or by increasing expression of another regulator, InvF. HilC and HilD accumulate in Lon-depleted cells and the enhancing effect on SPI1 expression. Lon and Clp family are ATP-dependent proteases shared of removing misfolded protein under stress conditions. In vivo proteolysis activities of Escherichia coli Lon and the ClpQY (HslUV) protease are also redundantly. In this study, we focused on the relationship between ClpQY (HslUV) protease and Salmonella SPI1 expression under different environmental conditions. The lon, clpQ and/or clpY single or double or triple mutation strains carrying hilA::Tn5lacZY, invF::Tn5lacZY, sipA::Tn5lacZY and sipC::Tn5lacZY fusions on the chromosome were constructed by bacteriophage P22 transductions. The resultant strains were incubated under different oxygen and osmolarity (0%-3.5% NaCl) environmental conditions. From β-galactosidase assay data, we found that bacteria with an optimal SPI1 gene expression were grown in oxygen limited and 1% NaCl condition. There is no significant difference in clpQ and clpY single or double mutants for SPI1 expression. However, the SPI1 gene expression in lon mutant is higher than wild type about 3 to 5 fold. The clpQ lon double mutant strain is slightly higher than lon single mutant in SPI1 gene expression. The influence of SPI1 gene expression in clpQ single mutant might be amplified in lon deletion background. ClpQY protease modulates Salmonella virulence; presumably that ClpQY shares redundant activities with Lon protease. Searching for the maximum difference in lon single mutant and clpQ lon double mutant, we found that the modulation by different environmental conditions are very diversely in invF and hilA gene expressions from wild type and all mutants. However, the SPI1 downstream gene expressions in clpQ lon double mutation strain are obvious higher than that in lon single mutant. We also notice that ClpQ and ClpY might play a different role in modulation the SPI1 gene expression. Afterwards, the SPI1 gene expression was repressed by incubation under high oxygen condition in wild type and all mutants.

壹、前言
一、沙門氏菌之重要性.......................................................................................... 1
二、細菌之熱休克蛋白 (heat shock proteins) 與病原菌致病力調控之相關研究 .................................................................................................................... 1
1. 細菌之熱休克反應....................................................................................... 2
2. 熱休克蛋白酶............................................................................................... 2
3. 熱休克蛋白酶與細菌致病性....................................................................... 3
3.1 Lon蛋白酶間接調控沙門氏菌入侵基因................................................. 3
3.2 ClpQY與Lon蛋白酶功能重疊................................................................. 4
4. Chaperone machinery DnaK/DnaJ與沙門氏菌致病力的關係..................... 5
三、沙門氏菌的致病力........................................................................................... 5
1. 沙門氏菌的致病機制................................................................................... 5
2. 沙門氏菌致病小島（Salmonella pathogenicity island）簡介.................... 6
3. 第三型分泌系統（type three secretion system）簡介................................. 7
4. 第三型分泌系統之組成分子....................................................................... 7
5. 第三型分泌系統之調控...................................................................... 8
6. 影響 SPI1 主要調控蛋白 HilA 基因之表現............................................ 9
四、沙門氏菌在哺乳類細胞內的作用................................................................. 10
1. 沙門氏菌與上皮細胞................................................................................... 10
2. 沙門氏菌與噬菌細胞................................................................................... 11
五、研究緣起與目的............................................................................................. 11

貳、材料與方法
一、實驗材料......................................................................................................... 13
(一)菌株與質體.................................................................................................. 13
(二)培養基.......................................................................................................... 13
(三)藥品與試劑.................................................................................................. 13
(四)實驗中使用之套組...................................................................................... 14
(五)儀器.............................................................................................................. 14
二、 實驗方法....................................................................................................... 14
(一)少量萃取細菌染色體DNA......................................................................... 15
(二)少量萃取質體DNA..................................................................................... 17
(三)大量萃取質體DNA..................................................................................... 17
(四)基因轉殖...................................................................................................... 17
(五)製備勝任細胞.............................................................................................. 18
(六)轉形作用...................................................................................................... 20
(七)噬菌體P22 轉導作用................................................................................. 22
(八) β-galactosidase assay in bacteria................................................................. 23

参、結果
一、突變菌株之建構............................................................................................. 26
二、在 1 % NaCl 培養液中，靜置培養對 SPI1 基因表現的影響 (氧氣限制) 27
三、在低滲透壓 (0% NaCl) 培養液中，靜置培養對 SPI1 基因表現的影響 (氧氣限制)...................................................................................................... 29
四、在高滲透壓 (2% NaCl) 培養液中，靜置培養對 SPI1 基因表現的影響 (氧氣限制)...................................................................................................... 30
五、在 1 % NaCl 培養液中，振盪培養對 SPI1 基因表現的影響 (高氧氣含量)................................................................................................................... 31
六、在低滲透壓 (0% NaCl) 培養液中，振盪培養對 SPI1 基因表現的影響 (高氧氣含量).................................................................................................. 32
七、在高滲透壓 (2% NaCl) 培養液中，振盪培養對 SPI1 基因表現的影響 (高氧氣含量).................................................................................................. 32
八、不同滲透壓，對於 sipA、sipC、invF 及 hilA 基因表現的影響............. 33
九、在振盪培養下，不同的滲透壓對於 invF 及 hilA 基因的表現............... 33
十、在 1% NaCl 下，不同的氧氣含量對於 invF 及 hilA 基因的表現.......... 35

肆、討論
一、氧氣限制條件下，沙門氏菌 SPI1 基因表現受滲透壓的分析.................. 37
二、供給氧氣條件下，沙門氏菌 SPI1 基因表現受滲透壓的分析................. 38
三、ClpQ 蛋白是否有參與沙門氏菌 SPI1 基因的調控................................... 40
四、ClpY 蛋白影響沙門氏菌 SPI1 基因表現之分析....................................... 42
五、不同滲透壓對於 invF 及 hilA 基因調控的分析....................................... 43
六、不同氧氣含量對於 invF 及 hilA 基因調控的分析.................................... 44
七、clpQ clpY lon 三缺失突變株的表現更為複雜.............................................. 45

伍、參考文獻........................................................................................................... 46

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