出版年:

研究生:

范忠錦

研究生(英文姓名):

Jong-Jin Fang

論文名稱:

創傷弧菌不產細胞溶解毒素突變株及不產細胞溶解毒素和蛋白雙突變株之分離和特性分析

英文論文名稱:

Isolation and characterization of Vibrio vulnificus mutants deficient in cytolysin and in both cytolysin and protease

指導教授:

何漣漪

指導教授(英文姓名):

Lein-I Hor

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46861076

學年度:

87

語文別:

中文

論文頁數:

84

關鍵詞:

創傷弧菌 ; 細胞溶解毒素突變株 ; 細胞溶解毒素和蛋白雙突變株 ; 同源性重組 ; 蛋白突變株 ; 動物毒性試驗 ; 細胞毒性試驗

英文關鍵詞:

Vibrio vulnificus ; cytolysin mutant ;
cytolysin-protease double mutant ;
homologous recombination ; protease mutant ;
animal virulence assay ; cytotoxicity assay

被引用次數:

3

[ 摘要 ]

創傷弧菌為一棲居於海水環境中的革蘭氏陰性桿菌，對人類會造成嚴重的傷口感染和致死性敗血症，但大多發生在有潛在性疾病的病人。創傷弧菌會分泌一種藉由破壞細胞膜而對許多細胞及紅血球具有毒性的細胞溶解毒素，此毒素也會造成血管通透性增加以及小鼠死亡。為了探討此細胞溶解毒素在創傷弧菌致病過程中所扮演的角色，我們利用DNA同源性互換的方法，分離出不產細胞溶解毒素單突變株及不產細胞溶解毒素和蛋白雙突變株，並進一步分析突變株的致病性。首先，利用已發表的細胞溶解毒素基因序列設計一DNA探針，藉以自本實驗室所構築的創傷弧菌基因庫中篩選出含此DNA片段的選殖株。其中一株具溶血活性，顯示該選殖株所含的重組質體帶有完整的細胞溶解毒素基因。將重組質體中的細胞溶解毒素基因，vvhA，其中一段0.8 kb的DNA以HpaI限制切除後，得到不表現溶血活性的缺陷基因。將含此缺失約3.6 kb的DNA片段移入一自殺型質體pCVD442內。此重組自殺型質體以接合生殖的方式分別送入創傷弧菌臨床株YJ016和其蛋白突變株CP080中，使重組質體上有缺失的細胞溶解毒素基因藉由前後二次同源性互換以取代細菌內完整的細胞溶解毒素基因，而得不產細胞溶解毒素單突變株及不產細胞溶解毒素和蛋白雙突變株。所得的突變株經由聚合連鎖反應、溶血試驗和南方雜交法加以確認。此兩突變株在營養培養基中的生長速率與野生株相似，且正如所預測的，細胞溶解毒素突變株的培養上清液不具溶血活性，而雙突變株的培養上清液不具蛋白和溶血活性。不產細胞溶解毒素單突變株或雙突變株的培養上清液雖然失去對HEp-2細胞的毒殺作用，但若以活菌感染細胞仍和野生株同樣造成細胞死亡，並且極少侵入細胞內。此外，各突變株對小鼠的半致死菌量，LD50，不論是由腹腔注射或是腸胃道感染小鼠，也與野生株沒有明顯的差異。因此，細胞溶解毒素可能不是主要的致死因子，也不是創傷弧菌造成細胞毒殺作用的唯一因素。細胞溶解毒素在細菌感染造成組織破壞作用的重要性則有待進一步的探討。

[ 英文摘要 ]

Vibrio vulnificus is a highly virulent estuarine bacterium that has been associated with serious wound infections and fatal septicemia, mostly to persons with underlying diseases. V. vulnificus strains produce an extracellular cytotoxin (cytolysin) that is known to be cytotoxic to a variety of cells, including the erythrocyte, by disrupting the cell membrane. The cytolysin also enhances vascular permeability and is lethal to mice. Cytolysin, therefore, is a potential virulence factor. The purpose of this study is to investigate the precise role of cytolysin in pathogenesis. We isolated two V. vulnificus mutants: one cytolysin-deficient (vvh-) and the other cytolysin-protease-double-deficient (vvh- vvp-), by allelic exchange. Clones containing the cytolysin gene (vvh) sequence were first screened from a V. vulnificus gene library by colony hybridization using a probe derived from the previously determined DNA sequence of this gene. One clone that exhibited hemolytic activity indicating the presence of an intact gene was identified. A 0.8 kb deletion by removing a HpaI fragment in the encoding sequence was introduced into the vvhA gene in the plasmid, resulting in a clone with non-hemolysis phenotype. A 3.6 kb fragment containing the deletion was then cloned into a suicide plasmid, pCVD442. This plasmid was transferred into a clinical V. vulnificus strain, YJ016, and an isogenic protease mutant, CP080, by conjugation. Cytolysin and cytolysin-protease double mutants were obtained by replacing the intact vvhA gene in the chromosome with the one with the deletion in pCVD442 via two sequential homologous recombination events. The deletion in the cytolysin gene in these mutants were confirmed by PCR, hemolytic assay, and Southern hybridization. The growth rates of these mutants in rich medium were similar to that of the parental strain YJ016. The vvh- mutant lacked cytolysin activity and the vvh- vvp- double mutant lacked both the cytolysin and the protease activities in the culture supernatant as expected. Although the culture supernatant of the vvh- or the vvh- vvp- mutant showed little cytotoxicity to the HEp-2 cells, these mutants were as cytotoxic as their parental strain when live bacteria at high MOI were used to infect the HEp-2 cells. In addition, the mutants as well as the parental strain showed little invasiveness to the HEp-2 cells. The virulence of both mutants, as represented by median lethal dose (LD50), was not significantly different from that of the parental strain. These results suggest that the cytolysin plays little role in the mouse virulence of V. vulnificus, and may not be the only factor causing cell death.