出版年:

2000

研究生:

李寧宜

研究生(英文姓名):

Li, Ningyi

論文名稱:

創傷弧菌磷脂之定序及磷脂缺失突變株之分離和特性分析

英文論文名稱:

Nucleotide sequencing of phospholipase gene in Vibrio vulnificus and isolation and characterization of mutants deficient in phospholipase

指導教授:

何漣漪

指導教授(英文姓名):

Hor, Lein-I

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:

s46871110

學年度:

88

語文別:

中文

論文頁數:

71

關鍵詞:

創傷弧菌 ; 磷脂 ; 基因定序 ; 磷脂缺失突變株

英文關鍵詞:

Vibrio vulnificus ; phospholipase ; Nucleotide sequencing ;
mutants deficient in phospholipase

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，但大多發生於有潛在性疾病的病人。創傷弧菌會產生許多種細胞外產物，包括莢膜、蛋白、細胞溶解毒素和磷脂，均被認為
可能是毒力因子。本研究使用遺傳學方法，探討創傷弧菌的磷脂在致病過程中所扮演的角色。從創傷弧菌基因庫中先後共篩選出9株產磷脂選殖株。從中得到一個含有磷脂活性的4 kb的DNA插入片段，經由DNA定
序，解讀出兩個開放閱讀區域。其中長度為1254 bp開放閱讀區域，可轉譯出417個胺基酸，且此胺基酸序列與腸炎弧菌的不耐熱溶血素胺基酸序列有75％的相似性。此基因命名為創傷弧菌磷脂（Vpl）。根據由
DNA序列推衍出的胺基酸序列推算，成熟型的Vpl其分子量約47 kDa，而等電點為5.83。另一個開放閱讀區域則與V. metschnikovii的alklaine serine protease的D
NA序列有76％的相似性，而破壞這個開放閱讀區域，並不會影響到vpl的表現。Vpl在E. coli選殖株中，被分泌到細胞間質中。以E. coli選殖株的細胞間質蛋白進行蛋白質電泳及磷脂活性分析，可以
確定Vpl的分子量約為46 kDa，與由vpl DNA序列推測的結果吻合。以RNA槽溝墨點法分析，得知vpl基因在創傷弧菌中持續性地表現。接著以遺傳學法分離到各種不產生磷脂的創傷弧菌突變株：不產生磷
脂單突變株（LD）、不產生磷脂及蛋白雙突變株（LPD）、不產生磷脂及細胞溶解毒素雙突變株（LCD），和不產生磷脂、蛋白及細胞溶解毒素三重突變株（TD）。所有突變株在營養培養基中的生長速度與
野生株相似。LPD突變株的培養上清液由於含有較大量的細胞溶解毒素，對HEp-2細胞的毒殺作用最強，其餘各突變株的培養上清液幾乎不具細胞毒殺作用。然而，觀察活菌對HEp-2細胞的細胞毒殺作用時，則發現除
了LCD突變株（NY303）外，其餘各突變株對細胞都有很強的毒性，顯示有其他的因素參與在活菌的細胞毒殺作用中。重新分離另一株LCD突變株（NY303-2），卻發現此突變株與野生株或其他突變株一樣，對細
胞也有很強的毒性。由於NY303以重組質體使其恢復產Vpl的能力後，仍然不具細胞毒性，因此，我們推測以突變株所以失去細胞毒性的原因，很可能是在其他基因上產生了突變。此外，不產磷脂單突變株及不產磷脂

[ 英文摘要 ]

Vibrio vulnificus, a halophilic, gram-negative bacterium, is associated with serious wound infections and fatal septicemia in humans, particularly in those with underlying diseases. V. vulnificus strains produce a number of extracellular products including capsular polysaccharide, protease, cytolysin, and phospholipase (PL) , all of which have been proposed as possible virulence factors. The aim of this study was to determine the role of PL in the pathogenesis of V. vulnificus by genetic approach. PL-producing recombinant clones, which produced a clear zone of lecithin degradation on an egg yolk agar plate, were screened from a V. vulnificus gene library. A 4.0 kb DNA fragment conferring thePL activity was isolated and its nucleotide sequence determined. Two open reading frames (ORFs) were identified. One was a 1254 bp segment encoding a polypeptide of 417 amino acids (aa) that shared 82% sequence homology with the thermolabile haemolysin of V. parahaemolyticus. The molecular weight and PI of the mature form of this polypeptide predicted from the deduced aa sequence were 47 kDa and 5.83, respectively. The other ORF shared 76% DNA sequence homology with the alkline serine protease of V. metschnikovii. Disruption of this ORF by removal of a sequence in it did not affect the expression of the PL activity. PL activity was located in the periplasm of a recombinant E. coli strain. Polyacrylamide gel electrophoresis of the periplasmic fraction of this strain combined with egg yolk agarose gel overlay revealed a phospholipase with an apparent molecular weight of 46 kDa, which was consistent with that predicted from the deduced aa sequence of polypeptide. This locus was, therefore, designated as the vpl (V. vulnificus phospholipase gene). PL expression was found to be constitutive in V. vulnificus when examined by RNA slot blot test. The following phospholipase-deficient mutants, which contained an in-frame internal deletion in vpl, were isolated by allelic exchange technique: a phospholipase-deficient (LD) mutant, a protease-phospholipase double-deficient (LPD) mutant, acytolysin-phospholipase double-deficient (LCD) mutant, and a protease-cytolysin-phospholipase triple-deficient (TD) mutant. The growth rate of these mutants in rich medium was similar to that of the parental strain. The culture supernatant of LPD mutant showed strong cytotoxicity against HEp-2 cells due to high level of the cytolysin, while none of the culture supernatant of the other mutants wasytotoxic. However, when HEp-2 cells were infected with washed bacteria, all but LCD mutant exhibited strong cytotoxicity like the wild-type strain, suggesting that there were unidentified factors involved in cytotoxicity. Intriguingly, a newly isolated LCD mutant, in contrast to the previous one, showed strong cytotoxicity as the other mutants. Complementation of NY303 with a recombinant plasmid, from which Vpl was expressed, did not restore cytotoxicity of this mutant. Therefore, lack of cyto
toxicity of this mutant might be caused by a mutation in some other gene, in stead of in vpl. The virulence of LD and TD mutants in mice, as represented by median lethal dose (LD50), was not significantly different from that of the wild-type strain. These results suggested that the phospholipase pl