腸炎弧菌胞外蛋白酶受Fur調控及RyhB特性分析

Abstract

腸炎弧菌是台灣及亞洲國家重要的食品病原菌，人類常因為生食被汙染的海鮮引起感染導致腹瀉或腸胃炎。本研究證實腸炎弧菌Fur攝鐵調控蛋白對於腸炎弧菌胞外蛋白酶prtA(VPA0227)及prtV(VPA0459)基因的調控機制，是藉由Fur直接結合在此兩個基因的啟動子區域導致prtA及prtV基因表現受到抑制。小片段核醣核酸 RyhB參與細胞中鐵離子平衡之調控，迄今僅在霍亂弧菌和大腸桿菌中研究較透徹，腸炎弧菌目前尚未報導。本研究證實腸炎弧菌RyhB扮演調控基因的角色。以生物資訊學方法分析預測腸炎弧菌RyhB序列，與NCBI 資料庫中弧菌屬及大腸桿菌RyhB序列比對相似性。在ryhB基因的上游區域比對到一段含有19-bp保守序列可能的Fur box，利用電泳遷移率(EMSA)方法證實Fur攝鐵調控蛋白可以結合於該預測之Fur box序列，推測RyhB受到Fur及Fe2+的負向直接調控。同時建構腸炎弧菌的ryhB過量表現菌株，以即時定量聚合酶鏈鎖反應方法證實RyhB可以正向調控基因的表現，包含lafK (sigma54依賴調節因子)、flaD (鞭毛)、fliS (鞭毛蛋白)、flaK (極性鞭毛蛋白)、rpoS (RNA聚合酶-sigma38)、fhuE (ferric coprogen和ferric-rhodotorulic acid的外膜受體)；RyhB亦可以負向調控部分基因的表現，包含sodB (超氧歧化酶)、sdhCDAB (琥珀酸脫氫酶)、iscS (半胱胺酸去硫酶)、cheY (趨化性蛋白)、motB及motA (鞭毛驅動蛋白)。以北方氏雜交法亦證實RyhB可以調控上述基因表現，除了flaD、fliS、flaK 結果訊號微弱較難判讀。本研究結果闡明腸炎弧菌RyhB具有調控趨化性、移動性及與鐵離子相關基因的功能。

Vibrio paraheamolyticus is a major food-borne pathogen in many countries that causes diarrhea through the consumption of raw or undercooked seafood. These results indicated that PrtA (VPA0227) and PrtV (VPA0459) are repressed directly by Fur and iron in V. parahaemolyticus No.93. A small untranslated RNA, RyhB, was discovered in Escherichia coli and Vibrio cholerae that were involved in the regulation of iron metabolism. In this study, we identify and characterize a small RNA RyhB ( gene code VP0105-VP0107 intergenic ) in V. parahaemolyticus No.93 to clarify its regulation roles. Bioinformatics analysis appeared a putative RyhB sequence in V. paraheamolyticus that has the similarity with other small non-coding RNA RyhB in NCBI database. Interestedly, a putative fur box containing 19-bp conserved sequence was found in the upstream of ryhB gene from V. parahaemolyticus. We used EMSA to demonstrate the interaction of Fur protein and ryhB promoter. We show that RyhB is functional and is down regulated by Fur. The transcription level of twelve RyhB-regulated target genes were examined in RyhB overexpression strain by using RT-PCR and Northern Blot hybridization. LafK (sigma54-dependent regulator), FlaD (flagellin), FlaK (polar flagellar protein), RpoS (RNA polymerase-sigma38) and FhuE (Outer membrane receptor for ferric coprogen and ferric-rhodotorulic acid) were positively regulated by RyhB, while SodB (superoxide dismutase), SdhC (succinate dehydrogenase), IscS (cysteine desulfurase), CheY (chemotaxis vi protein), MotB and MotA (flagellar motor protein) were negatively regulated by RyhB. In this report, we identify and characterize a small RNA RyhB in V. paraheamolyticus and show that it is similar in sequence to that of V. cholerae and is functional to regulate the chemotaxis, motility, metabolism and iron-related genes.