Studies on homoserine lactone and its antagonists which regulate the expression of virulence genes in Pseudomonas aeruginosa

高丝氨酸内酯及其拮抗剂调控铜绿假单胞菌毒力基因表达的研究

基本信息

批准号：
09660106
负责人：
MORIHARA Kazuyuki
金额：
$ 1.92万
依托单位：
The University Of East Asia
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1) Pseudomonas aeruginosa autoinducer bioassayIn Pseudomonas aeruginosa the Las R protein is required for activation of several virulence genes. A diffusible signal molecule, the P.aeruginosa autoinducer (PAI), produced by the bacteiial cell and released into the growth medium, is required for activity of lasR.By cloning a lasB : lacZ fusion and lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI.The plasmid used (pCRTlasI/lacZ/GSTlasR, 10.2 Kb) was constructed, as follows. The pCRlasI (4.4 Kb), containing an ampicillin-resistance marker, was provided by insertion of the promoter region of lasI (666 bp) prepared by PCR into EcoR1 site of pCRII The lacZ fragment (3.1 Kb, Sal digest) of pMC 1871 was inserted in the pCRlasI (Sal1 site) to form the pCRlasI/lacZ (7.5 Kb), in which rrnBT (terminator, EcoRl 180 bp fragment) from pKK 232-8 was inserted. Thus obtained pCRTlasI/lacZ (7.7 Kb) was digested with Xbal and blunted, in which lasR (NarI + TthuIII1 2.4 Kb fragment) from pGEX-3X1asR3455 was inserted to form the final plasmid, pCRTlasI/lacZ/GSTlasR (10.2 Kb).2) Synthesis of homoserine lactone analoguesThe following homoserine lactone analogues were synthesized for determination of their agonist or antagonist activities in the PAl assay system mentioned above ; N-Decanoyl-DL-homo-serine lactone, N-Decanoyl-DL-homocystein thiolactone, N-Dodecanoyl-cyclohexylamine, N-(2-ketobutanoyl)-DL-homoserine lactone, N-Dodecanoyl-DL-homoserine lactone, 4-Oxo-5-azaundecanoyl-DL-homoserine lactone, 3-Oxo-4-azadodecanoyl-DL-homoserine lactone.

1) 铜绿假单胞菌自诱导剂生物测定在铜绿假单胞菌中，Las R 蛋白是几个毒力基因激活所必需的。 lasR 的活性需要一种可扩散的信号分子，铜绿假单胞菌自诱导剂 (PAI)，由细菌细胞产生并释放到生长培养基中。大肠杆菌，我们开发了PAI的定量生物测定法。所用质粒（pCRTlasI/lacZ/GSTlasR, 10.2 Kb)的构造如下。 pCRlasI (4.4 Kb) 含有氨苄青霉素抗性标记，通过将 PCR 制备的 lasI (666 bp) 启动子区域插入 pCRII 的 EcoR1 位点而提供。 pMC 1871 的 lacZ 片段 (3.1 Kb，Sal 消化) 为插入 pCRlasI（Sal1 位点）形成 pCRlasI/lacZ (7.5 Kb)，其中 rrnBT插入来自pKK 232-8的(终止子，EcoRI 180bp片段)。由此获得的 pCRTlasI/lacZ (7.7 Kb) 用 XbaI 消化并平端化，其中插入来自 pGEX-3X1asR3455 的 lasR (NarI + TthuIII1 2.4 Kb 片段)，形成最终质粒 pCRTlasI/lacZ/GSTlasR (10.2 Kb)。2 ) 高丝氨酸内酯类似物的合成以下高丝氨酸合成了内酯类似物，用于在上述PA1测定系统中测定其激动剂或拮抗剂活性； N-癸酰基-DL-高丝氨酸内酯、N-癸酰基-DL-高半胱氨酸硫代内酯、N-十二酰基-环己胺、N-(2-酮丁酰基)-DL-高丝氨酸内酯、N-十二酰基-DL-高丝氨酸内酯、4-氧代-5-氮杂十一酰基-DL-高丝氨酸内酯， 3-氧代-4-氮杂十二酰基-DL-高丝氨酸内酯。