腸炎弧菌基因體分析及OpaR調控蛋白酶基因之研究

Abstract

腸炎弧菌 (Vibrio parahaemolyticus) 為海洋細菌，當人類食用受腸炎弧菌污染的海鮮會造成腹瀉等腸胃疾病，是台灣重要的食物病原菌。本研究首先結合Mi-seq、PacBio和Sanger三種定序方法解序腸炎弧菌VP93基因體序列。VP93基因體由一大一小的兩個染色體所組成，全長5,095,022 bp，GC含量 45.26%，有4,709個ORF，第一個染色體長3,396,947 bp，GC 含量45.07%，存在3,181個ORF、31個rRNA、101個tRNA; 第二個染色體較小長1,698,075 bp，GC含量45.63%，存在1,528個ORF、3個rRNA、14個tRNA。當VP93生長至後對數期 (late-log phase) 時胞外蛋白酶的活性達到最高峰。蛋白酶參與弧菌屬菌株營養攝取及感染的機制，菌體透過定額感應系統感知群體密度及環境條件，可以調控菌體中基因的表現。染色質免疫沉澱之定序 (ChIP-seq) 資料顯示VP93基因體有838個OpaR結合序列，歸納OpaR保守性結合序列為ATCAATA。即時定量聚合酶連鎖反應 (qRT-PCR) 和報導基因實驗 (luxAB assay) 確認OpaR經由八個蛋白酶基因附近之OpaR結合位抑制LytM (VPA1649)、Mcp02 (VPA0755)、M6 protease (VP0907)、serine protease (VPA0449)、DegS (VP0432)、protease II (VPA1467)、periplasmic protease (VP2032) 基因表現，以及促進PrtA (VPA0227) 基因表現。電泳遷移 (EMSA) 和足跡 (footprinting) 實驗證實蛋白酶基因附近八個OpaR結合位之結合情形及序列。OpaR於蛋白酶基因之專一性結合序列可歸納為ATCGTTG。胞外鹼性絲胺酸蛋白酶 (PrtA) 具有溶血性和細胞毒性，PrtA主導腸炎弧菌VP93胞外蛋白酶的活性。prtA和上游的反向基因ldh共用啟動子之-10序列。轉錄起始點實驗 (ARF-TSS) 證實ldh和prtA的轉錄起始點分別位於轉譯起始點上游－78的A和－198的G。OpaR於prtA上游有兩個結合位，一個在－269至－246處，另一個位於－88至－68處。其中－269至－246的OpaR結合位重疊於prtA的啟動子之－35序列。本論文提出定額感應調控子OpaR在腸炎弧菌蛋白酶基因的調控中扮演著重要的角色。由臺灣分離之腸炎弧菌VP93基因體解序、OpaR直接調控組 (direct regulon) 及蛋白酶基因調控機制各項研究結果，為海洋弧菌之相關研究領域提供嶄新資訊，對於蛋白酶調控機制提出不同的視野及研究手法。

Vibrio paraheamolyticus is marine bacteria. It causes gastroenteritis and diarrhea when humans are infected with V. parahaemolyticus via consumption of raw contaminated seafood. V. parahaemolyticus is an important foodborn pathogen in Taiwan. In this study, we combine three sequencing methods, Mi-seq, PacBio and Sanger to assemble the VP93 genome. VP93 genome has two chromosomes, containing 5,095,022 bp, 4,709 ORFs and GC content is 45.26%. Chr1 was composed with 3,396,947 bp, 3,181 ORF, 31 rRNA, 101 tRNA and GC content is 45.07%. Chr2 has 1,698,075 bp, 1,528 ORF, 3 rRNA, 14 tRNA and GC content is 45.63%. Extracellular protease activity was highest during the late-log growth phase of VP93. Many protease genes have been shown to be required for nutrient intake and the infection mechanism of Vibrios. Quorum sensing (QS) is a bacterial communication system which uses extracellular chemicals as signals. Bacteria changed their behaviour and synchronised gene expression in a microbial community by QS. ChIP-seq data reveals 838 OpaR binding sites in VP93 genome. The conserved OpaR binding motif is ATCAATA. qRT-PCR and luxAB assay confirmed that OpaR repressed the gene expression of LytM, Mcp02, M6 protease, serine protease, DegS, protease II, periplasmic protease and promoteed prtA expression. EMSA and footprinting results defined the OpaR specific binding sequence of the eight protease genes. The protease specific binding motif of OpaR is ATCGTTG. PrtA is an extracellular serine protease of Vibrio parahaemolyticus and has haemolytic and cytotoxic activities. PrtA is the dominant of VP93 extracellular protease activity. prtA and opposite direction gene, ldh shared the －10 box of the promoter region. ARF-TSS proved that the transcription start site of ldh and prtA is on －78A and －198G position from their translation start site. OpaR bound to prtA upstream sequence at positions －269 to －246 and －88 to －68 from the prtA translational start site. －269 to －246 overlapped the －35 box of prtA promoter. In this study, we report that OpaR, a quorum sensing regulator play important roles in the protease regulation mechanism. The results of clarifying the genome sequence VP93 strain isolated from Taiwan, OpaR direct regulon, protease gene regulation provided novel information and research method on protease regulation of Vibrio research field.