Analysis of Maturation Pathway of Heat-Stable Enterotoxin of Escherichia coli

大肠杆菌热稳定性肠毒素成熟途径分析

基本信息

批准号：
09670304
负责人：
OKAMOTO Keinosuke
金额：
$ 2.05万
依托单位：
Tokushima Bunri University
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

It has been reported that enterotoxigenic Escherichia ccli produces tow kinds of heatstable enteotoxin, STI and STII.These STs produced in cytoplasm are efficiently secreted into milieu and these extracellular STs induce diarrhea in host. The STs in cytoplasm are the immature toxins which can not induce diarrhea in host. However, these STs secreted into milieu are mature toxin which can. In this study, we investigated how these inactive STs are converted into their active form and how these active STs are secreted into milieu. We obtained the following results.1. Both STI and STII possess intamoleculatr disulfide bonds which are essential for expression of their activities. The study about the mode of disulfide bond formation revealed that DsbA, a periplasmic enzyme, catalyzes the formation of the bonds in both STI and STII.And the study about the mechanism of secretion of STs revealed that the disulfide bond formation is absolutely required for STII to pass through the outer membrane, but the formation is not necessary for STI to pass through the outer membrane. Furthermore, our study showed that the glutamic acid at position 7 of STI is involved in the reaction of STI with DsbA2. It has been shown that TolC, an outer membrane protein, is involved in the secretion not only of protein but also of small molecules such as antibiotics. To investigate whether TolC is involved in the secretion of STI, we prepared the tolC-mutant strain and examined the secretion of STI from the mutant strain. The result showed that TolC is directly involved in the secretion of STI.And we confirmed that the involvement of ToIC in the secretion of STII using our system.

据报道，产肠毒素大肠埃希菌产生两种耐热肠毒素：STI和STII。这些在细胞质中产生的STs被有效地分泌到环境中，这些细胞外STs引起宿主腹泻。细胞质中的ST是未成熟的毒素，不能引起宿主腹泻。然而，这些分泌到环境中的 ST 是成熟的毒素，可以。在这项研究中，我们研究了这些非活性 ST 如何转化为活性形式，以及这些活性 ST 如何分泌到环境中。我们得到了以下结果： 1． STI和STII都具有分子内二硫键，这对于它们的活性表达至关重要。对二硫键形成方式的研究表明，周质酶DsbA在STI和STII中均催化二硫键的形成。而对ST分泌机制的研究表明，二硫键的形成是STII所必需的。穿过外膜，但 STI 穿过外膜并不需要形成。此外，我们的研究表明，STI 7 位的谷氨酸参与了 STI 与 DsbA2 的反应。研究表明，TolC 是一种外膜蛋白，不仅参与蛋白质的分泌，还参与抗生素等小分子的分泌。为了研究TolC是否参与STI的分泌，我们制备了tolC突变株并检测了突变株对STI的分泌。结果表明，TolC直接参与STI的分泌。我们利用我们的系统证实了ToIC参与STII的分泌。