腸炎弧菌Cpx雙分子系統與調控 磷脂醯絲胺酸脫羧酶基因之研究

Abstract

Cpx雙分子調控系統由感應器CpxA組胺酸激酶 (Histidine kinase)，及反應調控因子CpxR共同組成，參與細菌多種生理作用的調控。磷脂醯乙醇胺 (phosphatidylethanolamine, PE) 為腸炎弧菌細胞膜的主要組成分，負責維持細菌細胞膜的功能以及完整性。磷脂醯絲胺酸脫羧酶 (phosphatidylserine decarboxylase, Psd) 是磷脂醯乙醇胺生合成路徑中最末端的重要酵素，將磷脂醯絲胺酸 (phosphatidylserine) 脫羧反應後形成磷脂醯乙醇胺。本研究以腸炎弧菌 (Vibrio parahaemolyticus no. 93, VP93) 作為實驗菌株，建構cpxA及cpxRA刪除突變株與實驗室前人建構之cpxR刪除突變株，測試比較野生株及突變株對於環境壓力的耐受性。實驗結果顯示，CpxR促進腸炎弧菌對抗低溫、極端酸鹼值、高滲透壓及抗氧化壓力反應。接著利用生物資訊分析方法預測psd的啟動子區域，發現在腸炎弧菌psd上游之啟動子區域及其上游基因末端分別具有一段與CpxR保守性結合序列相似之核苷酸序列。在psd的啟動子區域利用ARF-TSS的方法鑑定出psd的轉錄起始點是位於其轉譯起始點上游第48個核苷酸位置。由於本研究室發現VP93之CpxR與psd有負調控相關，本論文繼續針對CpxA及CpxRA進行研究，探討cpxA及cpxRA刪除突變株對於psd的調控情形，並以CpxR蛋白質進行電泳遷移率實驗 (EMSA)，實驗結果顯示CpxR結合於cpxR上游區域，但不結合於cpxP及psd上游區域。EMSA證明psd與CpxR的調控關係並非直接由CpxR結合於psd上游區域而行使負調控關係，而是間接受CpxR的調控。進一步探討感應器CpxA組胺酸激酶的自磷酸化對於psd的調控情形，建構cpxA的自磷酸化H258A點突變株及其回補株。將CpxA自磷酸化位點為胺基酸序列258位置的Histidine (cac) 進行點突變為Alanine (gcc)，結果確認若CpxA無法自磷酸化，會使得下游CpxR無法活化，進而造成下游psd基因無法被抑制而大量表現。總結以上結果，本研究首次證實腸炎弧菌中CpxRA雙分子系統對於環境壓力的反應以及其對於磷脂質代謝途徑的探討。

The CpxRA two component system comprised of the histidine kinase CpxA and the response regulator, CpxR. It serves as a common stimulus response mechanism, thus allows microbes to sense and respond to diverse environments through a series of phosphorylation reactions. Phosphatidylethanolamine (PE), the major type of phospholipid which presents in V. parahaemolyticus maintaining the function and integrity of the bacterial cell membrane. Phosphatidylserine decarboxylase (Psd, EC 4.1.1.65) is an enzyme which catalyze the formation of PE. It plays a central role in the phospholipid metabolism. In this study, we investigated the role of CpxRA two component system in V. parahaemolyticus in response to different environmental stimuli by constructing the ∆cpxA single deletion mutant and ∆cpxRA double deletion mutant from V. parahaemolyticus no. 93. The CpxR was found to play essential roles in mediating tempeterature, pH stress, osmotic pressure and oxidative pressure tolerance. Moreover, we found two nucleotide sequences which similar to the CpxR conserved binding site via bioinformatics. The two nucleotide sequences located at the promoter region of the psd and the end of psd upstream gene. The transcription start site of psd was 48 nucleotides upstream of the translation start site. We have previously reported that ∆cpxR from V. parahaemolyticus no. 93 significantly downregulated the psd. A comparative transcriptomic analysis of VP93 WT, ∆cpxR, ∆cpxA, ∆cpxRA by real-time quantitative PCR and luminescence assay were subsequently employed to understand how Cpx system affects the psd expression. EMSA analysis was used to determine the interaction between cpxRA, cpxP, psd I as well as psd II promoter region and CpxR. The results indicated that the CpxR was able to specificity bind to the cpxRA promoter region. However, no shift band from EMSA results was observed in cpxP, psd I as well as psd II promoter region. These results indicated that psd is indirectly regulated by CpxR. Furthermore, we investigated the role of autophosphorylation of CpxA in regulation of psd by constructing the CpxA H258A point mutation mutant. The results confirmed that CpxA autophosphorylate, and activate the downstream CpxR, resulting in repression of psd gene expression. In this thesis, we firstly analyzed the CpxRA two component system in V. parahaemolyticus and found that CpxR mediating tempeterature, pH stress, osmotic pressure and oxidative pressure tolerance. Furthermore, real-time quantitative PCR and luminescence assay were used and indicated that psd gene expression is downregulated by CpxRA two component system. However, EMSA results indicated that psd is indirectly regulated by CpxR. At last, the autophosphorylatation of CpxA play an important role in CpxRA two component system. It can activate the CpxR, resulting in repression of psd gene expression. Taken together, these findings first characterize the role of the CpxRA two component system in V. parahaemolyticus conferring stress response tolerance and in phospholipid metabolism, which will deepen our understanding in V. parahaemolyticus.