腸炎弧菌 prtV基因表現受 Fur及 VPA0458的調控

Regulation of Vibrio parahaemolyticus prtV gene expression by Fur and VPA0458

吳培萱(Pei-Syuan Wu)

指導教授 ： 李佳音

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603262

摘要

腸炎弧菌的胞外蛋白酶為造成人類腹瀉或腸胃炎之潛在致病因子，本次研究觀察胞外蛋白酶基因prtV 上游基因對它的調控，PrtV除了是金屬蛋白酶 (Metalloprotease) 也是一種膠原蛋白酶 (Collagenase)，目前發現大部分會產生膠原蛋白酶的細菌皆為致病菌。故本次研究以腸炎弧菌 (Vibrio parahaemolyticus no.93) 為研究菌株，探討腸炎弧菌的金屬蛋白酶PrtV (VPA0459) 基因表現的的調控，本次探討的調控因子有兩項，其中一項為Fur (Ferric uptake regulator; VP0833) 蛋白質對prtV基因轉錄的影響，另一項則為prtV上游基因 (Hypothetical protein, VPA0458) 是否參與prtV基因表現的調控。首先利用即時定量聚合連鎖反應 (qRT-PCR) 檢測發現調控因子Fur和VPA0458 對prtV基因具有負調控情形。再利用報導基因pLuxAB螢光表現分析，同樣發現在細胞內fur和vpa0458 因子對prtV上游啟動子有負調控結果。在其他的文獻顯示弧菌中Fur調控會受到Fe2+濃度影響，因此也探討不同二價鐵離子濃度的情況下Fur的調控情形，以qRT-PCR分析結果顯示在低鐵環境下相較於高鐵生長條件下prtV基因表現下降，因此推測Fur在高鐵環境下會抑制prtV基因表現。prtV上游基因序列有兩段高保守19-bp 序列可能為Fur box binding site，分別命名為Fur box 1以及Fur box 2，電泳延滯(EMSA)和足跡實驗 (Footprinting)實驗證實Fur蛋白質直接結合在prtV啟動子之Fur box 1。

關鍵字

腸炎弧菌 ； 膠原蛋白? ； 調控子

並列摘要

The extracellular proteases of Vibrio parahaemolyticus is a potential virulence factor that causes diarrhea or gastroenteritis. In this studies, we discussed the upstream regulation of extracellular proteases prtV gene. PrtV protein is not only a metalloprotease but also collagenase. A variety of bacteria have been known as collagenase producers. Majority of them are pathogenic strains, applying the enzyme as the virulence factor. In this study, we used Vibrio parahaemolyticus no.93 strain to investigate gene expression of PrtV (VPA0459). We were interested in prtV gene expression of two regulators between Fur (Ferric uptake regulator; VP0833) and VPA0458 (hypothetical protein). We designed and performed a series of in vivo and in vitro tests on prtV gene expression. In vivo test, Fur and VPA0458 have negative regulation in qRT-PCR analysis. In vitro test, Fur and VPA0458 have negative regulation in LuxAB activity analysis. In other literature shows that importance of Fe2+ in Vibrio. Therefore, we describe how changed in iron levels influence gene expression through Fur-mediated regulation of prtV. Our result reveals that prtV gene expression was higher in iron-limited cultures than in iron-replete cultures in qRT-PCR analysis. We hypothesized that Fur repressed prtV under iron-replete condition. There are two putative Fur box binding sites containing highly conserved 19-bp sequence on the upstream of prtV gene. They are named Fur box 1 and Fur box 2. We also found that prtV gene expression is subject to negative regulation through the binding of Fur on the Fur box 1 with electrophoretic mobility shift assay and Footprinting assay.

並列關鍵字

Vibrio parahaemolyticus ； collagenase ； regulator

參考文獻

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