攝鐵調控蛋白及 VPA0458 調控腸炎弧菌膠原蛋白酶 PrtV

Abstract

腸炎弧菌是一種海洋弧菌，為亞洲地區常見的食品病原菌，人類常因食用含有腸炎弧菌的海鮮而造成腹瀉、嘔吐等腸胃疾病。它可分泌許多種胞外蛋白酶，本次研究觀察的 PrtV 為腸炎弧菌之胞外金屬蛋白酶，同時也是一種膠原蛋白酶。本研究以腸炎弧菌 no.93 作為實驗菌株，探討膠原蛋白酶 PrtV 之基因 prtV (VPA0459) 與其周圍鄰近基因之轉錄情形，並闡明攝鐵調控蛋白 Fur (Ferric uptake regulator, VPI_2457) 對其基因轉錄的影響。首先利用反轉錄聚合酶連鎖反應 (RT-PCR) 確認 prtV 周圍基因VPA0456～VPA0460 之 RNA 轉錄狀況，並且利用 ARF-TSS 方法鑑定出 prtV 的轉錄起始點位於其轉譯起始點上游第 32 個核苷酸的位置；vpa0458 的轉錄起始點位於其轉譯起始點上游第 167個核苷酸的位置。前人利用即時定量聚合酶連鎖反應 (qRT-PCR) 檢測發現 prtV 上游之反向基因 vpa0458 及轉錄抑制子 fur 對 prtV 具有負調控的現象，且在 prtV-vpa0458 基因間區域 (intergenic region) 發現兩段序列可能為 Fur 的結合區 (Fur box 1 及 Fur box 2)，在本研究中發現了第三段可能為Fur 的結合區 (Fur box 3)，該序列與弧菌屬保守 Fur 結合序列 (5’–AATGANAATNATTNTCATT–3’) 比較，具有 80% 相同度。文獻指出 Fur 的調控功能會受到細菌生長環境中亞鐵離子 (Fe2+) 濃度的影響，利用 qRT-PCR 觀察腸炎弧菌在不同鐵離子環境中基因表現量的變化，結果顯示低鐵環境下，fur 表現量顯著降低、vpa0458 及 prtV 表現量顯著增加，推測亞鐵離子會促進 fur 表現並抑制 vpa0458 及 prtV 表現；比較不同突變株中的 prtV 表現量，∆VPA0458、∆Fur 及 ∆VPA0458∆Fur 相較於野生株的 prtV 表現量都有顯著提升，推測 prtV 會受到 fur 及 vpa0458 的抑制。為更進一步確認 fur 與 prtV 的調控關係，建構重組蛋白 His6-Fur 並進行大量純化，以之與含有 prtV 上游 Fur box 之 DIG 探針進行電泳遷移率實驗 (EMSA)，證實 Fur 能夠結合在 prtV 上游預測到的三個 Fur box。在前人研究中以 Fur(partially frame shift) 進行電泳遷移率實驗發現 Fur 疑似能夠結合在 Fur box 1。在本研究中證實腸炎弧菌之 Fur 除了能夠結合於 Fur box 1，也能結合在 Fur box 2 以及本研究發現之 Fur box 3。

Vibrio parahaemolyticus is a commom marine pathogen of humans in Asia and is a causative agent of seafood-associated food poisoning. This organism can cause gastroenteritis such as diarrhea and vomiting. It can secret many extracellular proteases. The extracellular metalloprotease PrtV of Vibrio parahaemolyticus no.93 is also a collagenase. In this study, we validated the transcription status of prtV (VPA0459) and its adjacent genes: VPA0456VPA0460 and investgated the expression of prtV to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. We used RT-PCR to prove VPA0456~VPA0458 and VPA0459~VPA0460 are not operons. By using ARF-TSS assay, we found the transcription start site of prtV was 32 nucleotides upstream of the translation start site, and the transcription start site of vpa0458 was 167 nucleotides upstream of the translation start site. Previously, transcription regulator Fur and hypothetical protein VPA0458 were found to down regulate the expression of prtV by qRT-PCR. Furthermore, there were found two predicted Fur boxes, named Fur box 1 and Fur box 2, in prtV-vpa0458 intergenetic region. In this study, we found the third Fur box (Fur box 3) shows 80% similarity compared to Vibrios conserved Fur binding sequence (5’–AATGANAATNATTNTCATT–3’). Also, it was well proved that the concentrantion of Fe2+ in cell culture will affect fur expression. Thus, we added dip into medium to chelate the Fe2+ and observed the difference of gene expression level. The result of qRT-PCR shows the gene expression level of prtV and vpa0458 were significantly promoted in iron-limited cultures, while fur was repressed. We also compared prtV expression level in different strains. In ∆VPA0458, ∆Fur and ∆VPA0458∆Fur, prtV expression level was significantly higher than in WT, thus we thought prtV can be negative regulated by fur and vpa0458. Fur box 1 was prevouly found that Fur(partially frame shift) might interact on this region by EMSA analysis. In this study, we reconstructed the recombinant protein Fur (His6-Fur) and validated the interaction of His6-Fur with not only predicted Fur box 1, but Fur box 2 and new-found Fur box 3. This study demonstrates that Fur regulates PrtV at the transcription level by binding on the Fur box.