出版年:

研究生:

林美薇

研究生(英文姓名):

Mai-Wei Lin

論文名稱:

TolC外膜蛋白在創傷弧菌毒力所扮演的角色

英文論文名稱:

Role of TolC in virulence of Vibrio vulnificus

指導教授:

何漣漪

指導教授(英文姓名):

Lien-I Hor

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物及免疫學研究所

學年度:

90

語文別:

中文

論文頁數:

82

關鍵詞:

毒力 ; 創傷弧菌

英文關鍵詞:

TolC ; vibrio vulnificus

被引用次數:

0

[ 摘要 ]

創傷弧菌為一棲居於海水環境的革蘭氏陰性菌，對人類而言，它屬於伺機性病原，在一些免疫有缺陷的病人造成相當嚴重的傷口感染及引發致死性敗血症。創傷弧菌產生很多細胞外產物，如：莢膜、siderophores、蛋白酶、細胞溶解毒素及磷脂酶，均被認為與其毒力有關。但根據我們實驗室之前的研究顯示，蛋白酶、細胞溶解毒素及磷脂酶並非重要的毒力因子，因為觀察不產生蛋白酶、細胞溶解毒素及磷脂酶的三重突變株，發現它們仍然對HEp-2細胞具有毒殺作用且對小鼠的半致死劑量與野生株沒有明顯差異。然而，從實驗室之前偶然分離到一株對HEp-2細胞幾乎沒有毒性的細胞溶解毒素及磷脂酶雙突變株 (NY303)，發現其對小鼠的毒力比野生株要來得低，顯示創傷弧菌的細胞毒殺性可能是一個重要的毒力因素，我們推測NY303突變株可能含有一個未知的突變而導致細菌失去細胞毒殺作用。我們曾在大腸桿菌菌株S17-1lpir構築此菌的基因庫，並擬由當中篩選出能夠回復NY303之細胞毒性的DNA片段，以進一步找出此未知之細胞毒素。然而，我們尚未得到足夠數目的轉形株以進行這項實驗。另一方面，有文獻指出，霍亂弧菌之RTX蛋白毒素會造成HEp-2細胞型態上的改變且可能與El Tor霍亂弧菌的竄起有關，我們發現創傷弧菌也具有rtx gene cluster。並由染色體上含有rtx gene cluster的區域設計五對核酸引子，利用聚合酶連鎖反應檢視NY303的rtx gene cluster是否含有deletion。結果發現，由NY303 rtx gene cluster放大出的PCR產物的長度與野生株並沒有明顯差異，但我們並不能排除NY303之RTX發生點突變的可能。同時，有研究指出霍亂弧菌的TolC外膜蛋白質參與RtxA的運輸過程且與膽鹽的耐受性有關，我們利用tolC突變株 (MW021) 的構築以了解TolC在創傷弧菌的致病過程可能扮演的角色，發現MW021對於膽鹽及紅黴素敏感性大於野生株。觀察活菌對HEp-2細胞之毒性試驗時，我們發現MW021對HEp-2細胞幾乎不具毒性且對小鼠的毒力也比野生株要來得低。這些結果顯示，TolC可能藉由影響一個未知細胞毒素的活性或分泌來參與創傷弧菌的致病過程。此外，我們懷疑tolC可能為創傷弧菌之必要基因，因為我們很難分離到tolC有缺失的菌株。為了釐清tolC是否為創傷弧菌之必要基因，我們在分離tolC突變株過程中，in trans提供一個完整的tolC基因，以避免染色體上的tolC當置換成缺失tolC基因而造成的細胞死亡。結果顯示，我們並沒有得到更多染色體上tolC已缺失的菌株。有關於tolC是否為創傷弧菌之必要基因，有待更進一步證實。

[ 英文摘要 ]

Vibrio vulnificus, a gram-negative marine bacterium, is an opportunistic pathogen that causes severe wound infection and septicemia in immunocompromised patients. V. vulnificus strains produce a number of potential virulence factors including capsular polysaccharide, siderophores, protease, cytolysin and phospholipase. Our previous data showed that the protease, cytolysin and phospholipase are not the major virulence factors, because disruption of all the three genes encoding these factors did not reduce the virulence in mice or cytotoxicity to HEp-2 cells. However, a non-cytotoxic mutant, NY303, isolated fortuitously from a phospholipase-cytolysin double-deficient mutant was shown to be much less virulent in mice, indicating that cytotoxicity is an important virulence factor. We suspected that NY303 might have lost the activity of an unidentified cytotoxin. We have tried to construct a library in E. coli strain S17-1lpir and will screen for clones that complement the defect of NY303 in cytotoxicity. However, we have not had sufficient numbers of clones in this library for doing this complementation experiment. On the other hand, a homologue of V. cholerae rtx gene cluster, which has been shown to cause the morphological change of cultured cells and seemed to serve as a key role in the emergence of the El Tor strain, was identified in V. vulnificus. We designed primers to amplify the RTX gene cluster in the wild type strain and NY303. Although there was no distinct difference in the length of the PCR products between these two strains, we could not rule out the possibility of point mutation in the rtx gene cluster in NY303. Meanwhile, tolC, an outer membrane protein involved in the export of diverse molecules ranging from large protein toxin to antibiotics, was shown to be required for the secretion of RTX toxin and colonization of V. cholerae. A tolC homologue was also identified in V. vulnificus. To determine the role of tolC in the virulence of V. vulnificus, a tolC-deficient mutant, MW021, was isolated by allelic exchange. MW021 was more sensitive to bile and erythomycin compared with the parental strain. In addition, MW021, was non-cytotoxic to the HEp-2 cells and much less virulent in mice, suggesting that tolC may be involved in the pathogenesis of V. vulnificus, probably by secreting an unidentified cytolysin. The tolC gene was suspected to be an essential gene because it was extremely difficult to obtain a tolC-deficient mutant. To test this, a plasmid carrying tolC was introduced into MW021 and the frequency of isolating the ΔtolC mutant was determined. No increase in the isolation frequency ofΔtolC mutant was observed in the presence of tolC in trans. Whether tolC is essential for V. vulnificus viability awaits further investigation.