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

据报道，产肠毒素大肠杆菌产生两种耐热性内毒素：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诱导宿主腹泻。细胞质中的STs是不成熟的毒素，不会引起宿主腹泻。然而，这些分泌到环境中的STs是成熟的毒素，可以。在本研究中，我们研究了这些无活性的STs是如何转化为活性形式的，以及这些活性STs是如何分泌到环境中的。我们得到了以下结果。STI和STII都具有分子内二硫键，这是表达其活性所必需的。对二硫键形成模式的研究表明，DsbA是一种周质酶，在STI和STII中都催化了二硫键的形成。通过对STs分泌机制的研究发现，二硫键的形成是STI通过外膜的必要条件，而不是STI通过外膜的必要条件。此外，我们的研究表明STI第7位的谷氨酸参与了STI与DsbA2的反应。研究表明，外膜蛋白TolC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，并检测了突变株的STI分泌。结果表明，TolC直接参与STI的分泌。我们用我们的系统证实了ToIC参与了STII的分泌。

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.