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系統識別號 U0026-0812200911170285
論文名稱(中文) ‧以蛋白質體分析法偵測創傷弧菌無細胞毒殺性突變株內表現量降低之蛋白質 ‧創傷弧菌TolC 缺失突變株之補償作用
論文名稱(英文) ‧Detection of a protein down-regulated in a non-cytotoxic Vibrio vulnificus mutant by proteomic analysis ‧Complementation of a TolC-deficient V. vulnificus strain
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 92
學期 2
出版年 93
研究生(中文) 陳吟竹
研究生(英文) Yin-Chu Chen
電子信箱 YinChu1229@msn.com
學號 s4691403
學位類別 碩士
語文別 中文
論文頁數 88頁
口試委員 指導教授-何漣漪
口試委員-吳俊忠
口試委員-賴信志
中文關鍵字 補償作用  蛋白質體  細胞毒殺性  創傷弧菌  外膜蛋白質 
英文關鍵字 complementation  TolC  proteomic analysis  Vibrio vulnificus  cytotoxicity 
學科別分類
中文摘要   創傷弧菌為棲息於海水環境中的革蘭氏陰性細菌,為人類的伺機性致病菌。創傷弧菌感染人後,會造成嚴重的組織壞死與敗血症,特別是具有潛在性疾病的人更為危險族群。創傷弧菌會產生許多細胞外產物,如:莢膜、siderophores、蛋白酶、細胞溶解毒素以及磷脂酶。但根據本實驗室之前的研究顯示,不產生蛋白酶、細胞溶解毒素以及磷脂酶的突變株,仍然對HEp-2 細胞具有毒殺能力且對小鼠的致病能力與野生型菌株並無差異,因此我們認為這三者並非創傷弧菌主要的毒力因子。然而,我們在篩選細胞溶解毒素以及磷脂酶雙缺失突變株的過程中,意外的分離到一株不具有細胞毒殺能力的雙缺失突變株,NY303,不僅喪失了細胞毒殺能力,對小鼠的毒力也明顯的下降。因此我們推論,NY303 可能在基因上有所變異使其喪失了細胞毒殺能力;且此與細胞毒殺能力相關的因子,可能是一個極為重要的毒力因子。在本實驗中,我們嘗試以蛋白質體學方法去找尋NY303 所缺失或受到影響的蛋白質。我們以一維(SDS-PAGE)以及二維電泳(2-DE)分析NY303 以及另一株同為細胞溶解毒素與磷脂酶雙缺失突變株,但仍具有細胞毒殺能力的菌株NY303-2,在全細胞溶解性蛋白質、細胞外分泌型、外膜以及細胞膜間質的蛋白質表現量的差異。當我們以一維電泳分析時,我們發現NY303 在細胞膜間質以及外膜蛋白質各有一條約35 kDa 大小的色帶的缺失,以LCQ/MS 鑑定此色帶所含的蛋白質並以RNA 溝槽墨點雜交試驗進行確認。我們發現其中只有Vv2338 確實僅在NY303-2 會轉錄出RNA。而我們以核酸引子將NY303 的Vv2338 的基因放大並以電泳分析後發現,NY303 的Vv2338 基因並未有缺損。此外,在細胞膜間質以及細胞外分泌型蛋白質的二維電泳圖中,我們可以發現NY303-2 與NY303 有許多蛋白質點的表現量有所不同。顯示NY303 失去細胞毒殺能力,可能是由於某個扮演調控功能的因子發生變異而引起的一連串變化,而非單一基因與其產物的缺損。

  另一方面,由於TolC 外膜蛋白質參與許多分子的運輸,包括小分子的抗生素到大分子的毒素,一向被認為與細菌的抗藥性以及致病機制有關。在我們實驗室先前的研究中,我們發現創傷弧菌的TolC 缺失突變株對膽鹽和紅黴素的感受性增加,且失去對HEp-2 細胞株的毒殺能力以及對小鼠的致病能力。在確認過TolC 的缺失並不會影響創傷弧菌的蛋白酶與細胞溶解毒素的分泌後,我們推測TolC 的缺失影響了一個未知的細胞毒素的活性或運送。為了確認我們所觀察到的現象確實是由於TolC 缺失所引起,需要選殖完整的基因送進缺失突變株內以進行補償實驗加以佐證。我們發現,以高複製數的載體於E. coli 中不易選殖到正確的tolC 基因,選殖到的tolC 基因則帶有一個以上的點突變,因此,創傷弧菌的TolC 過度表現可能對E. coli 是有害的。而我們以低複製數的載體進行選殖時,得到一個菌株YC019 可以在含有膽鹽的LB 培養液中生長,但生長情形不如野生型菌株。我們將生長於0.02%含膽鹽LB 培養液的細菌塗佈在TCBS 培養基上,篩選在含膽鹽LB 培養液中生長情形與野生型菌株相似的菌株,並得到YC025。此菌株對膽鹽和紅黴素的感受性可恢復到與野生型菌株相同,但其對於HEp-2 細胞仍不具有毒殺能力。我們發現YC025 比YC019 多了一個silence mutation 和一個胺基酸的缺失。我們推論這個胺基酸的缺失可能改變了TolC的結構使其對紅黴素的感受性回復,然而TolC 缺失所影響的細胞毒素並沒有因此回復,以致於YC025 仍不具有細胞毒殺能力。關於TolC 對細胞毒素的影響有待進一步的實驗證實。
英文摘要 Vibrio vulnificus, a gram-negative halophilic marine bacterium, is an opportunistic human pathogen. It causes severe wound infection and septicemia, particularly in those with underlying diseases. V. vulnificus produce a number of potential virulence factors including capsular polysaccharide, siderophores, protease, cytolysin and phospholipase. Our previous study showed that disruption of the genes encoding protease, cytolysin and phospholipase did not influence the virulence in mice or cytotoxicity to HEp-2 cells. Hence we concluded that these three factors are not the major virulence factors in V. vulnificus. However, a non-cytotoxic mutant, NY303, isolated fortuitously from a phospholipase-cytolysin double mutant was shown to be much less virulent in mice. This data suggested that cytotoxicity may be an important virulence factors. We suspected that NY303 might have lost the activity of an unidentified cytotoxin. In this study, we tried to identify the proteins that are lost in NY303 by proteomic analysis. The total soluble proteins, outer membrane proteins, secreted extracellular proteins and periplasmic proteins of NY303 and its isogenic, cytotoxic strain, NY303-2, were extracted and separated by one-dimensional (SDS-PAGE) or two-dimensional electrophoresis (2-DE). When we separated the periplasmic and outer membrane proteins by SDS-PAGE, one band about 35 kDa was found lost in NY303 in each fraction. These bands were subjected to LCQ/MS for protein identities and the transcriptions of genes encoding these proteins were examined by RNA slot blot hybridization. Only with VV2338, the open-reading-frame of one of the identified proteins, the transcript was more abundant in NY303-2 than NY303. However, VV2338 in NY303 can be amplified without any deletion, indicating that the lost of VV2338 protein in NY303 was not caused by a deletion in the gene. Meanwhile, some protein spots differentially expressed in NY303-2 and NY303 were identified on 2-DE gels of the periplasmic and extracellular fraction, suggesting that the lost of cytotoxicity in NY303 might be due to the dysfunction of a regulatory factor.

On the other hand, TolC and its homologues are outer membrane proteins essential for the transport of a variety of molecules, including the antibiotics and toxins, across the cell envelope. In our previous study, we found that a TolC-deficient V. vulnificus mutant, MW021, was more sensitive to bile and erythromycin, non-cytotoxic to HEp-2 cells, and much less virulent in mice compared to the parental strain, YJ016. Since the disruption of tolC did not influence the secretion of protease and cytolysin, we suspected that the activity or transport of an unknown cytotoxin was affected by TolC dysfunction. To confirm that the phenotypes we observed were associated with the disruption of tolC, we performed a complementary experiment. We found it’s very difficult to clone the wild-type V. vulnificus tolC gene in Escherichia coli with a high copy number vector, and we always obtained at least one point mutation in the coding sequence of cloned tolC. These suggested that overexpression of V. vulnificus tolC might be harmful for E. coli. When we used the low copy number vector, partial complementation with delayed growth in the 0.02% bile-containing LB medium was observed in one strain, YC019, which contained one point mutation 19-bp upstream of the cloned tolC open reading frame and one silent mutation in the coding sequence. We plated the bacteria grown in the bile-containing LB medium on TCBS (Thiosulfate-Citrate-Bile salt-Sucrose) agar and screened for those that grew as well as YJ016 in bile-containing LB medium. One strain, YC025, was obtained and its phenotypes, including cytotoxicity to HEp-2 cells and susceptibility to erythromycin were similar to YJ016. We found that the tolC gene on the plasmid contained in YC025 had one additional silent mutation and one amino acid deletion. We suspected that the deletion of the amino acid might have altered the TolC structure that resulted in restoration of the phenotypes. Whether TolC has an effect on the activity of an unknown cytotoxin or RTX toxin awaits further investigation.

論文目次 壹、中文摘要 i
貳、英文摘要 iii
叁、致謝 v
肆、目錄 vi
伍、表目錄 ix
陸、圖目錄 ix
柒、符號及縮寫 x
捌、緒論 1
玖、材料與方法 8
I.實驗菌株與質體及保存方法 8
1.細菌菌種與質體 8
2.菌種的培養與保存方法 8
II.實驗細胞株的培養、保存 8
1.細胞培養 8
2.細胞保存 9
III.核酸技術 9
1.小量純化質體DNA的方法 9
2.商業化套組萃取質體DNA 9
3.大量純化質體DNA 10
4.商業化套組萃取染色體DNA 11
5.限制酶切割質體DNA 11
6.DNA片段之分離 11
7.商業化套組回收DNA片段 12
8.DNA片段之粘端補齊反應 12
9.DNA片段去磷酸化反應 12
10.DNA接合反應 13
11.聚合酶連鎖反應 13
12.PCR放大片段之選殖 (TA cloning) 13
13.質體轉移方法 14
13.1.熱休克轉形作用 14
13.2.電擊轉形作用 15
13.3.細菌接合生殖 15
14.DNA定序 16
IV.菌株的特性分析 16
1.測定細菌生長曲線 16
2.細菌對膽鹽的感受性試驗 16
3.細菌對紅黴素的感受性試驗 16
4.細胞毒殺性試驗 17
V.蛋白質體學方法 18
1.蛋白質樣品的製備 18
1.1.萃取蛋白質 18
1.1.1.全細胞溶解性蛋白質 18
1.1.2.細胞外分泌型蛋白質 18
1.1.3.外膜蛋白質 18
1.1.4.細胞膜間質蛋白質 19
1.2.蛋白質沉澱 19
1.2.1.方法一 19
1.2.2.方法二 20
1.3.蛋白質回溶 20
1.4.蛋白質定量 20
2.一維電泳 21
2.1.硫酸十二酯納聚丙烯胺膠體電泳 21
2.2.蛋白質染色:Coomassie blue staining 22
3.二維電泳 22
3.1.第一維:等電點聚焦 22
3.2.第二維:硫酸十二酯納聚丙烯胺膠體電泳 22
3.3.蛋白質染色:銀染(silver staining) 23
4.膠片保存 23
5.二維電泳膠影像處理 24
6.二維電泳膠影像分析比對 24
7.蛋白質鑑定 24
VI.RNA溝槽墨點試驗 25
1.RNA的製備 25
2.RNA溝槽墨點法的前處理 25
3.DNA探針的製備 26
4.雜交反應 26
拾、結果 27
I.以蛋白質譜分析尋找NY303菌株中表現量降低之基因 27
1.NY303與NY303-2蛋白質圖譜之比較 27
2.NY303中表現量降低之蛋白質的鑑定 28
3.所鑑定出之蛋白質其對應的基因其mRNA表現量的測定 28
II.TolC補償株之構築及特性分析 29
1.以高複製數質體選殖tolC基因 29
2.過度表現tolC基因對E. coli的影響 30
3.以低複製數質體pJRD215選殖tolC基因 30
4.TolC補償株的表現型(phenotypes) 31
a) YC019對膽鹽的感受性 31
b) 以0.02% bile stress篩選TolC功能恢復的菌株 31
c) 對紅黴素的感受性 31
d) 對表皮細胞的細胞毒殺性 32
e) YC019與YC025的tolC轉錄作用 32
5.YC025的tolC基因定序 32
6.以轉形作用將pYC025傳送入E. coli 33
拾壹、討論 34
I.NY303蛋白質缺失及表現量改變的探討 34
II.TolC補償株的構築與特性分析 38
拾貳、參考文獻 43
拾叁、圖表 50
拾肆、附錄 70
I.Original data of Mass spectrometry (periplasmic fraction) 70
II.Original data of Mass spectrometry (outer membrane fraction) 85
III.Crystal structure of E. coli TolC 90
拾伍、自述 91
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