創傷弧菌是一海洋中的革蘭氏陰性菌，若經由傷口感染嚴重會導致敗血病及休克，死亡率高達50%以上，當食入不潔的海鮮食物則會造成急性下痢。先前實驗室利用跳躍基因（mini-Tn5）建立一個創傷弧菌突變株基因庫，並以血清抗性試驗篩選出一株對血清高感受性的變異株，利用基因選殖技術分析此段基因，且經由比對後得知其與σ54-dependent response regulator有相當高的相似性，並將其命名為polR，並且在polR序列前隔一個核苷酸的位置發現另一個ORF(open reading frame)，其對histidine sensor kinase有相當高的相似性，將其命名為polS。之前實驗室的學長利用磷酸化實驗及單點突變試驗證明polS本身會自我磷酸化並且把磷酸根傳遞給下游的polR，即表示PolS-PolR之間會形成一雙因子調控系統。在本實驗中我們利用EMSA證明σ54與polS promoter之間會互相作用，並以單點突變及 promoter fusion assay實驗證明polS前端-76 bp的GG-N11-GC具有promotor功能，與之前所有文獻指出的σ54 binding site是GG-N10-GC不同。之後我們加入polymyxin B、macrophage及PMN刺激polS promotor，皆有出現誘導的現象。除此之外，我們篩選出一polS isogenic mutant並與之前實驗室構築好的polR、σ54 isogenic mutants一起與野生株比較其表現型和致病力的改變，在表現型實驗中顯示σ54 isogenic mutant失去運動性及生物膜形成能力﹔而致病力方面顯示σ54 isogenic mutant對人類血清感受性降低且毒性不論在健康老鼠或是在給予鐵劑的老鼠，其毒性皆明顯下降，由以上結果皆指出σ54在創傷弧菌中的致病力上扮演了一重要角色。我們發現σ54在創傷弧菌中是屬於持續表現的狀態，利用promotor預測軟體預估σ54還會調控哪些下游基因，發現σ54在創傷弧菌中可以調控與鞭毛運動、碳和氮的代謝及其他雙因子調控系統等相關基因，之後利用穿透式電子顯微鏡觀察菌體鞭毛的有無，可以明顯看出σ54突變株鞭毛形成能力喪失，並且下游σ54調控的regulator與致病力有關，因此σ54調控下游的鞭毛、雙因子調控系統在創傷弧菌中是一重要的毒力因子。

Vibrio vulnificus is a marine gram-negative bacteria. It can cause serious wound infections, septicemia and diarrhea in human. Mutagenesis with the mini-Tn5 has been successfully utilized in V. vulnificus in our lab. We isolated a serum susceptible mutant (polR) and also found an ORF (polS) which is located immediately upstream of polR. PolS shows sequence homologous to sensor protein and PolR homologous to the σ54-dependent response regulators of two component signal transductional system (TCSTS). In this study, we demonstrated the interaction of σ54 protein and polS promotor region by EMSA. A typical consensus sequence of σ54 dependent promotor is GG-N10-GC. Although no such a typical consensus sequence was found in promoter region of polS, there is a GG-N11-GC sequence locating about 76 bp upstream region of polS. Site-directed mutagenesis and reporter fusion experiments demonstrated that the GG-N11-GC motif has a promoter function. Promoter fusion experiments indicated that polS promoter could be markedly induced during exposure of bacteria to polymyxin B, polymorphonuclear leukocyte (PMN) and macrophage in stationary phase. In addition, the polS, polR and σ54 isogenic mutants were constructed by homologous recombination, respectively. Phenotypical analysis revealed that σ54 isogenic mutant is non-motile and has the lowest biofilm formation ability compared to those of wild type, polS and polR isogenic strains. The infection experiments demonstrated virulence attenuation when σ54 isogenic mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice. Expresssion of σ54 transcription activator is constitutive. Observation with the TEM clearly showed that wild type V. vulnificus retained a polar flagellum but that the σ54 isogenic mutant had completely lost its flagellum. These data indicateσ54 dependent flagellum and two component sustems are important virulenlce factors in V. vulnificus.

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陳昱仲. (2004) 創傷弧菌致病因子之研究。中興大學食品科學系研究所博士論文。

袁道成.(2005) 創傷弧菌感測及反應雙因子調控系統對血清、抗菌蛋白及嗜中性白血球抗性之探討。國立成功大學生物化學研究所碩士論文。