出版年:

2001

研究生:

邵長平

研究生(英文姓名):

Chung-Ping, Shao

論文名稱:

創傷弧菌蛋白之表現調控機制及其在致病過程中之角色

英文論文名稱:

Regulation of Vibrio vulnificus metalloprotease (Vvp) expression and role of Vvp in pathogenesis

指導教授:

何漣漪

指導教授(英文姓名):

Lien-I, Hor

學位類別:

博士

校院名稱:

國立成功大學　

系所名稱:

基礎醫學研究所

學號:

S58861082

學年度:

89

語文別:

中文

論文頁數:

117

關鍵詞:

創傷弧菌 ; 蛋白 ; 細胞融解毒素

英文關鍵詞:

Vibrio vulnificus ; metalloprotease ;
quorum sensing ; LuxR ; SmcR ; cytolysin ;
Vvp

被引用次數:

0

[ 摘要 ]

創傷弧菌是一種存在於海水環境中的革蘭氏陰性菌，在人類可造成嚴重的組織壞死及敗血症。此菌可以產生多種與致病有關的毒力因子，其中蛋白（Vvp）由於對實驗動物具有多種生物作用因而被認為是重要的毒力因子。本研究的目的在了解vvp基因表現的調控，以及分離並分析無法產生此酵素的突變株，以探討Vvp在致病機轉中所扮演的角色。據我們的研究顯示，Vvp受到quorum-sensing系統所調控，且由一屬於V. harveyi LuxR轉錄活化子家族的一員，稱為SmcR，在此菌生長進入穩定期時活化vvp基因的表現。此SmcR蛋白也參與細胞溶解毒素基因表現的負調控，而此細胞溶解毒素也被認為是重要的毒力因子。我們利用DNA電泳位移改變測定法發現SmcR在vvp啟動子區域的DNA結合序列有二：一為自-35區域算起涵蓋整個上游達39氮鹼基對的區域，稱為B box；另一則位於B box上游85氮鹼基對，其涵蓋範圍有45氮鹼基對，稱為A box。另一方面我們以 in vivo allelic exchange 的方式分離到不產Vvp的突變株（PD突變株，CP104）。 此突變株在老鼠的腹腔感染模式中其致死力和野生株一致，但是在灌食途徑上卻比野生株高十倍。 此外，PD突變株由腹腔侵襲至血流、在小鼠血液中的生長，以及促進鐵離子之攝取等方面的能力均和野生株相當。因此，Vvp對小鼠並非是主要的毒力因子。此外PD突變株的培養上清液中其細胞溶解毒素的活性要比野生株高出一倍而且作用亦能持續一段時期。對此，我們進一步利用另一PD突變株（CP080）、不產細胞溶解毒素的CD突變株，以及不產Vvp以及細胞溶解毒素的DD突變株等菌株，研究Vvp和細胞溶解毒素的作用，並針對小鼠致死力，組織損害以及對培養上皮細胞的毒性等三點做分析。除了CP080在灌食模式上其毒力（包括組織損害方面）要比野生株及其餘突變株高之外，這些菌株不論是以皮下注射或是灌食的方式感染小鼠其致死力均和野生株相當。另外，在對上皮細菌的毒性分析上，我們認為細胞溶解毒素是目前創傷弧菌唯一分泌性的細胞毒素，然而不產細胞溶解毒素之突變株其菌體本身仍然有極強的細胞毒性。由此可知，在此菌中存有另一個細胞毒素。綜合以上結果，雖然細胞溶解毒素在造成小鼠消化道的組織破壞方面似乎是較Vvp重要，但是Vvp或是細胞溶解毒素均非導致此菌對小鼠有致死力的主要原因。

[ 英文摘要 ]

Vibrio vulnificus, a gram-negative marine bacterium, is an opportunistic pathogen that causes severe wound infection and septicemia in humans. Among the potential virulence factors proposed, the purified secretory protease (Vvp) exhibited a variety of biological activities that are possibly detrimental to the animals. To study the role of Vvp in pathogenesis, we investigated the regulation of vvp gene and, in addition, constructed and characterized two isogenic protease-deficient (PD) mutants. The expression of Vvp was regulated by a quorum-sensing mechanism. A positive regulator SmcR, a member of the Vibrio harveyi LuxR family, activated the transcription of vvp when the bacterial culture entered the stationary phase. In addition, SmcR negatively regulated the cytolysin, which is another potential virulence factor of V. vulnificus. Two SmcR-binding DNA sequences in the vvp promoter region were identified by gel mobility shift assay with the purified his6-tagged SmcR. One, which covers a region of 39 bp, is located around the -35 promoter sequence (B box), and the other (A box), which covers a region of 45 bp is located 85 bp upstream of the B box. The PD mutant CP104, which was isolated by in vivo allelic exchange, was as virulent as its parental strain in mice challenged intraperitoneally, but was about 10-fold more virulent than its parental strain in mice challenged via the oral route. Furthermore, CP104 was as efficient as its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin as iron sources. These data together demonstrate that Vvp is not a major virulence factor. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twice as that of the wild-type strain and maintained for a much longer period. The actions of Vvp and cytolysin were further analyzed with another PD mutant CP080, a cytolysin-deficient (CD) mutant, and a Vvp-cytolysin-double deficient (DD) mutant for 1) virulence in mice; 2) ability to cause tissue damage; and 3) cytotoxicity to cultured epithelial cells. The virulence of these mutants, except for CP080, in mice infected by subcutaneous injection or force feeding was comparable to that of the wild type strain. CP080 was the most virulent one when given via the oral route and severely damaged the alimentary tract among the bacteria analyzed. Although the cytolysin was the only detectable secretory cytotoxin to the HEp-2 cells, the mutants devoid of cytolysin damaged the HEp-2 cells by direct cell-to-cell contact to similar extent as the wild-type strain did. This result suggests the presence of another cytotoxin that awaits identification. In conclusion, although the cytolysin, rather than Vvp, causes damage in the alimentary tract, neither Vvp nor cytolysin was essential for virulence of V. vulnificus in mice.