"腸炎弧菌絲胺酸蛋白酶PrtA受C-di-GMP, OpaR和RpoS調控之研究"

Abstract

腸炎弧菌(Vibrio parahaemolyticus)又稱為副溶血弧菌，是一株嗜鹽性的革蘭氏陰性菌，為人類的病原菌。其胞外鹼性絲胺酸蛋白酶，PrtA (VPA0227)為腸炎弧菌可能的致病因子之一。鹼性絲胺酸蛋白酶於菌體生長至後對數期時大量分泌，PrtA的表現受到細胞間訊息傳遞之定額感應系統 (quorum sensing system) 的正調控，主要的定額感應調控子為OpaR。本研究預測prtA的上游區域有兩個可能為OpaR的結合位置–3~–145及–229~–361，經由電泳遷移實驗(EMSA)的實驗結果確認OpaR可以結合於此二區域。在EMSA實驗過程中添加廣泛調控菌株的第二傳訊者c-di-GMP，可以觀察到c-di-GMP阻擾OpaR和prtA的結合。此外，本研究探討主要掌管菌株生長穩定期的sigma factor RpoS對於PrtA分泌的影響。本研究以同源重組的方式建構突變株ΔopaR、 ΔrpoS 和ΔopaRΔrpoS，發現NO.93和ΔopaR的生長速率並無明顯差異，然而ΔrpoS 和ΔopaRΔrpoS的生長速率則較NO.93低。透過即時定量聚合酶連鎖反應法(QPCR)觀察野生株NO.93中各目標基因的表現情形，RpoS於菌株培養至穩定生長期時達最高表現量; 定額感應的主要調控子OpaR則於菌株存在高細胞密度(OD600 0.1 )後穩定表現。然而，磷脂質依存溶血素LDH及PrtA則分別於初對數期和後對數期時大量表現。由QPCR及西方墨點法證實RpoS和OpaR 對於prtA基因表現量以及胞外PrtA蛋白表現量為正調控，並且影響胞外PrtA的分泌時間。此發現有助於未來其他具有細胞毒性的蛋白酶探討其致病機制與調控方式。

Vibrio paraheamolyticus is a human pathogen, it cause gastroenteritis and diarrhea in Taiwan, Asia and coast area. It is a gram-negative bacterium. Its extracellular alkine serine protease, PrtA (VPA0227) was seen to be a virulence factor of Vibrio paraheamolyticus No. 93. The extracellular alkine serine protease, PrtA get to maximal expression when growing to late log phase. We know the expression of PrtA would upregulated by quorum sensing system, and the major quorum sensing in Vibrio paraheamolyticus is OpaR. In this study, there are two probable OpaR binding sites upstream of prtA, one located on –3~–145 and another on –229~–361. We purify the OpaR protein and do EMSA to confirm that OpaR would regulate prtA by binding on its promoter region directly. And the second messenger molecule c-di-GMP would interfere with the binding between the OpaR and prtA. Besides, we use homologous recombination to make the deletion mutants ΔopaR, ΔrpoS, ΔopaRΔrpoS for the further studies. We found the growth rate of VP93 and ΔopaR are the same, however ΔrpoS and ΔopaRΔrpoS are lower than VP93. The results from Q-PCR show that the prtA was expressed to the most high level when VP93 was cultured at 3 hour; rpoS was 8 hour ; the ldh was at 1.5 hour. And the quorum sensing regulator opaR express stablely from 1.5 to 12 hour. From the result of Q-PCR and Western, the OpaR and RpoS can promote the expression and secretion of extracellular serine protease PrtA of V. parahaemolyticus NO.93.