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系統識別號 U0026-1602201623012700
論文名稱(中文) 創傷弧菌對小鼠毒力之廣泛調控子Lrp所調控基因之鑑識
論文名稱(英文) Identification of the target genes of Lrp, a global regulator involved in the virulence of Vibrio vulnificus in mice
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 104
學期 1
出版年 105
研究生(中文) 翁肇徽
研究生(英文) Chao-Hui Weng
學號 s46021058
學位類別 碩士
語文別 中文
論文頁數 65頁
口試委員 指導教授-何漣漪
口試委員-陳振暐
口試委員-橋本昌征
口試委員-陳盈璁
中文關鍵字 創傷弧菌  鐵攝取能力  趨化力 
英文關鍵字 Vibrio vulnificus  Lrp  RNA-seq  chemotaxis  iron-acquisition  vulnibactin 
學科別分類
中文摘要 創傷弧菌(Vibio vulnificus) 屬於革蘭氏陰性病原菌,藉由傷口或食入汙染的海鮮感染人類,會造成敗血症和壞死性筋膜炎。我們實驗室先前的研究發現廣泛調控子Lrp會調控一株臨床株,YJ016,在含0.3%瓊脂之LB培養盤上的移動性和對小鼠的毒力。進一步分離Δlrp突變株YH01,發現YH01在小鼠感染部位的存活能力較野生株YJ016差;且YH01在小鼠血清的生長也較YJ016差,但當在小鼠血清中外加鐵來源,如血紅蛋白(hemoglobin)、氯化血紅素(hemin) 或檸檬酸鐵銨(ferric ammonium citrate),YH01的生長能力就會恢復。本篇研究首先確認了Lrp在第一和第二生物型創傷弧菌對小鼠之毒力皆扮演重要角色。另外,我發現雖然將野生株YJ016中合成細胞溶解素(vvhA) 和磷脂酶(vpl) 之基因雙雙剔除後,並未造成對小鼠之毒力下降的情況,但在帶有自然發生之lrp點突變之突變株中將此兩基因剔除,則此菌株之毒力便下降至與突變株YH01相當的程度,表示細胞溶解素和磷脂酶對毒力之影響需在Lrp有缺陷的情況下才會顯現,而帶有此點突變之Lrp可能還具有功能。接著,為了瞭解Lrp如何調控創傷弧菌對小鼠之毒力,我比較了野生株YJ016和突變株YH01培養在80%小鼠血清中2小時之全轉錄體,發現在突變株YH01中有597個基因的表現量下降2倍以上,而有103個基因的表現量上升2倍以上,當中有許多參與在和新陳代謝相關的反應。最特別的是,參與在細菌趨化功能以及vulnibactin生合成和鐵攝取系統中之基因,有相當高的比例在突變株YH01中的表現量顯著下降。突變株YH01中和鞭毛合成相關之基因的表現量改變不大,且其鞭毛長度、寬度和在液態基質中之游動功能皆是正常的,因此,Lrp很可能是透過調控細菌趨化功能,而非鞭毛之生合成或功能影響創傷弧菌在含0.3%瓊脂之LB培養盤上的移動性,但創傷弧菌之趨化功能是否會影響對小鼠的毒力,目前還不清楚。另一方面,我也發現突變株YH01的vulnibactin產量顯著降低,而將細菌的vulnibactin合成酶基因剔除後,其在小鼠血清中之生長明顯變差。因此推測,失去Lrp的突變株YH01很可能由於與鐵攝取和vulnibactin生合成有關基因的表現量降低,進而影響其自宿主獲取鐵的能力,導致其在小鼠體內的生長變差,而減弱其對小鼠的毒力。未來預期可以在突變株YH01表現量顯著改變的其它基因中,找到更多與細菌毒力相關的基因。
英文摘要 Vibrio vulnificus, a gram-negative bacterial pathogen, can cause necrotizing fasciitis and fulminant septicemia via wound or food-borne infection. Our laboratory has previously found that the leucine-responsive transcriptional regulator (Lrp), a global regulator, is involved in the regulation of migration on soft agar and virulence in the mouse in a clinical strain YJ016. In this study, I first demonstrated that Lrp played an important role in the virulence for mouse not only in biotype 1 but also biotype 2 V. vulnificus strains. It has been shown previously that deletion of the genes encoding cytolysin (vvhA) and phospholipase (vpl) in strain YJ016 did not affect the virulence in mice. In this study, I showed that deletion of these two genes in a mutant containing a spontaneous mutation in lrp, which showed partially reduced virulence compared to the Lrp-deficient mutant YH01, further reduced the virulence to the level of mutant YH01. These indicate that the importance of vvhA and/or vpl is obvious only in the presence of the mutated Lrp, and such Lrp mutant may retain partial function. To understand how Lrp regulates the virulence of V. vulnificus, I identified the target genes of Lrp by comparing the transcriptomes of mutant YH01 incubated in 80% mouse serum for 2 hours with that of the wild-type strain YJ016. It showed that 597 genes were down-regulated and 103 genes were up-regulated by ≥ 2 folds in mutant YH01, and many of them were predicted to be involved in the metabolic processes. Particularly, the transcriptional levels of genes involved in bacterial chemotaxis, vulnibactin biosynthesis and iron uptake were down-regulated in the mutant. The transcription of most genes involved in flagellum biosynthesis was not affected in mutant YH01, the flagellum of the mutant appeared normal, and this mutant could swim freely in liquid medium. Therefore, the defect of mutant YH01 in migration on soft agar might be due to reduced chemotaxis ability, but not the biosynthesis or function of flagellum. On the other hand, I found that the production of vulnibactin was reduced in mutant YH01, and a mutant deficient in vulnibactin synthetase grew much poorer in mouse serum compared to the wild-type strain. These results suggest that mutant YH01, with reduced transcription of the genes involved in vulnibactin biosynthesis and iron uptake, might be defective in iron-acquisition, which lead to poor growth and, consequently, reduced virulence of the mutant in the mouse. We expect to identify more virulence-associated genes among those with altered expression levels in mutant YH01 in the future.
論文目次 中文摘要…............................................................................................................. I
Extended Abstract .................................................................................................. II
誌謝...................................................................................................................... VII
目錄...................................................................................................................... IX
表目錄.................................................................................................................. XII
圖目錄...................................................................................................................XIII
符號及縮寫.......................................................................................................... XIV
緒論....................................................................................................................... 1
材料與方法............................................................................................................ 9
I. 實驗菌株、質體及菌種保存.......................................................................... 9
1. 實驗菌株與質體........................................................................................ 9
2. 實驗菌種的培養與保存.............................................................................9
II. 實驗細胞株與實驗動物................................................................................ 9
1. 細胞株培養................................................................................................ 9
2. 細胞株保存方法...................................................................................... 10
3. 實驗動物..................................................................................................10
III. 核酸及分子生物學技術............................................................................. 10
1. 商業化套組純化質體DNA.................................................................... 10
2. 聚合酶連鎖反應( polymerase chain reaction, PCR).................................11
3. 核酸引子之製備.....................................................................................11
4. DNA電泳分析........................................................................................11
5. 商業化套組回收DNA片段....................................................................12
6. 限制酶切割DNA....................................................................................12
7. DNA片段去磷酸化反應.........................................................................12
8. DNA黏合反應........................................................................................13
9. 質體轉移方法.........................................................................................13
10. 商業化套組萃取細菌RNA.....................................................................15
11. 反轉錄作用(reverse transcription) ...........................................................15
12. 即時偵測聚合酶連鎖反應(real-time PCR) .............................................16
13. RNA-seq定序分析..................................................................................17
IV. 創傷弧菌突變株之構築.............................................................................17
1. 以自然轉形法分離突變株........................................................................17
2. 以對偶基因置換法(allelic exchange) 分離突變株....................................18
V. 細菌特性分析..............................................................................................19
1. 細菌移動能力(Migration assay) ................................................................19
2. 細胞毒性分析(Cytotoxicity assay) ............................................................19
3. 細菌在小鼠血清中之生長........................................................................20
4. 細菌對小鼠之半致死劑量測定(LD50) ..................................................... 20
VI. 蛋白質分析................................................................................................21
1. 商業化套組萃取全細胞蛋白質................................................................ 21
2. 蛋白質樣品之定量.................................................................................. 21
3. 蛋白質電泳分析.......................................................................................22
VII. Vulnibactin產量之檢測.............................................................................23
VIII. 統計分析方法......................................................................................... 23
結果..................................................................................................................... 24
I. Lrp在不同創傷弧菌菌株中於調控小鼠毒力所扮演的角色..........................24
1. 分離自不同創傷弧菌菌株之Δlrp突變株的特性分析..............................24
1-1. 細菌移動性/趨化性(Migration/Chemotaxis ) ..................................... 24
1-2. 細胞毒性(Cytotoxicity) ……............................................................... 24
1-3. 對小鼠之毒力 …………………………….................................................... 25
II. Vpl和VvhA在帶有NY303 lrp上的點突變之菌株中對小鼠毒力所扮演的角色................................................................................................................. 25
1. 帶有ΔvvhAΔvpl以及NY303 lrp上的點突變之突變株的特性分析……... 25
1-1. 對小鼠之毒力 …………………………….................................................... 25
1-2. 細菌移動性/趨化性(Migration/Chemotaxis ) ..................................... 26
1-3. 細胞毒性(Cytotoxicity) ……................................................................ 26
III. Lrp所調控下游基因之鑑識........................................................................ 26
1. 野生株在不同培養基中lrp之表現量....................................................... 26
2. 野生株在80 %小鼠血清中Lrp之蛋白質產量..........................................26
3. 野生株和Δlrp突變株在80 %小鼠血清中其鐵獲取能力相關基因之表現量............................................................................................................... 27
4. 比較野生株和Δlrp突變株以RNA-seq分析之全轉錄體(Transcriptome) ..........................................................................................27
IV. Lrp對參與趨化功能之基因的調控..............................................................28
1. 野生株和Δlrp突變株細菌之鞭毛功能.....................................................28
2. 野生株和Δlrp突變株中參與趨化功能之基因的mRNA表現量 ..............29
3. ΔcheY突變株之趨化功能...........................................................................29
V. Lrp對鐵獲取能力相關基因之調控...............................................................30
1. 野生株和Δlrp突變株鐵獲取能力相關基因之表現量...............................30
2. Vulnibactin生合成在創傷弧菌於小鼠血清中之生長所扮演的角色...........30
3. Δlrp突變株之Vulnibactin產量..................................................................31
討論...................................................................................................................... 32
參考文獻............................................................................................................... 39
圖表集................................................................................................................... 45
自述....................................................................................................................... 65
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