攝鐵調控蛋白及鐵離子對於腸炎弧菌胞外蛋白酶調控之研究

Regulation of extracellular protease in Vibrio parahaemolyticus by Iron and Fur

趙芳筠(Fang-Yun Chao)

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(2012年)

https://doi.org/10.6342/NTU.2012.10116

摘要

腸炎弧菌（Vibrio parahaemolyticus）胞外蛋白酶為潛在之致病因子，其致病能力可能源自於菌體攝取外界養分而造成，其中，攝鐵能力即在細胞存活及生長過程扮演重要且必須之角色。腸炎弧菌膠原蛋白酶 PrtV（VPA0459）基因的上游區域有兩處可能之攝鐵調控蛋白結合位（Fur box），絲胺酸蛋白酶 PrtA（VPA0227）及小 RNA RyhB 上游具一處可能之結合位，故本研究利用一株腸炎弧菌缺乏 tdh 及 trh 之台灣臨床分離菌株， V. parahaemolyticus NO.93 為研究材料，分別建構 fur 突變株及 ryhB過量表現菌株，擬探討胞外蛋白酶 PrtV 及 PrtA 此兩蛋白酶基因之調控是否與鐵離子、攝鐵調控蛋白（Fur），以及 RyhB密切相關。由 FURTA 實驗結果可得知， RyhB 基因上游啟動子區域具有與 Fur 結合之能力。 Q-PCR 結果顯示， Fur 負向調控 prtV 及 prtA 轉錄，但由 FURTA 結果可推測此調控並非由於 Fur 直接結合於此兩蛋白酶上游啟動子區而抑制；而 RyhB 則正向調控 prtV 及 prtA 轉錄。由西方墨點法結果也顯示， PrtV 及 PrtA 皆能夠受 Fur 及鐵離子負向調控，於 RyhB 過量表現菌株也可偵測到較野生株高量之 PrtA 表現，因此推測 Fur 調控 PrtA 表現是經由 RyhB；而 PrtV 雖然能夠受 Fur 及鐵離子的負向調控，但於 RyhB 過量表現菌株中無論在胞內或胞外並無法偵測到較野生株高量之 PrtV 表現，顯示 RyhB 對 PrtV 的調控並不影響其分泌途徑。本篇研究證實腸炎弧菌胞外蛋白酶 PrtV 及 PrtA 會受到 Fur-Fe2+ 及 RyhB所調控，此發現有助於未來探討此系統對於腸炎弧菌致病機制之研究。

關鍵字

腸炎弧菌 ； 胞外蛋白酶攝鐵調控蛋白 ； RyhB

並列摘要

Vibrio parahaemolyticus causes acute gastroenteritis in humans and it is one of the most important food-borne pathogens in many countries. The putative Fur (ferric uptake regulator) boxes, each containing 19-bp highly conserved sequence, were found in the upstream of promoter regions of prtV encoding metalloprotease (VPA0459), prtA encoding alkaline serine protease (VPA0227) and small RNA RyhB (between VP0105 and VP0106) genes. To clarify whether the production of PrtV and PrtA are regulated by iron, Fur and RyhB, the fur deletion mutant and ryhB overexpression derivatives were generated. The results of FURTA indicated that Fur can bind to a putative Fur box sequence on ryhB and it is functional as recognized by E. coli Fur protein. The Q-PCR showed that the prtV and prtA genes were negatively regulated by Fur protein, but they were positively regulated by small RNA RyhB. Western blot analysis demonstrated that V. parahaemolyticus senses iron availability via Fur to regulate the production of PrtV and PrtA. While the extracellular PrtA was increased in ryhB overexpression strain, the production of PrtV was not elevated neither in the fraction of extracellular nor of intracellular, these results indicated that RyhB did not regulate the secretion of PrtV and PrtA. These observations demonstrate that in V. parahaemolyticus the Fur/RyhB regulatory pathway provides a mechanism to regulate the expression of extracellular protease PrtV and PrtA.

並列關鍵字

Vibrio parahaemolyticus ； extracellular protease ； ferric uptake regulator ； Fur ； RyhB

參考文獻

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