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Development of Type III secretion system inhibitors

III型分泌系统抑制剂的开发

基本信息

批准号：
20790107
负责人：
IWATSUKI Masato
金额：
$ 2.16万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2009
项目状态：
已结题

项目摘要

As a part of screening by the assay utilizing TTSS-induced hemolysis in enteropathogenic Escherichia coli (EPEC), we have screened 20,250 microbial extracts and found that the extracts of the strain Streptomyces sp. K06-0806 and KK-88 showed potent TTSS inhibitory activity. After the fermentation and purification of the active compound, linear polyketides, aurodox and KK-88 compound, novel peptide, were isolated as TTSS inhibitors from the culture broths of K06-0806 and KK-88, respectively.The SDS-PAGE study showed that aurodox inhibited the expression of TTSS-related proteins, Tir, EspB, Map and EspF without the inhibition of EPEC viability and the expression of the house-keeping protein, GroEL The RT-PCR study of the genus of LEE (locus of enterocyte effacement) showed that aurodox reduced the transcription of many T3SS-related genes such as ler (LEE-encoded regulator), eae (LEE2: Type III apparatus), espA, espD, espB, espF (LEE4) and tir (LEE5) without reducing T3SS-nonrelated genes such as rrsB, one of 16S rRNA gene. Aurodox significantly reduced the transcription of ler, a gene of positive regulator of LEE, even at 0.5 μg/mL. This result suggests the specific inhibition of aurodox against the expression of T3SS-related proteins is caused by the inhibition of transcription of ler.In vivo study was carried out using C3H/HeJ mice infected by Citrobacter rodentium. Orally administrated aurodox caused murine survival with the repression of bacterial colonization in the intestinal mucosa. Therefore, aurodox is suggested to reduce the bacterial pathogenesis by the inhibition of T3SS.

作为通过利用TTSS诱导的肠致病性大肠杆菌（EPEC）溶血的测定法进行筛选的一部分，我们筛选了20，250种微生物提取物，发现链霉菌K 06 -0806和KK-88菌株的提取物显示出有效的TTSS抑制活性。经发酵和纯化后，分别从K 06 -0806和KK-88菌株发酵液中分离得到了TTSS抑制剂线性聚酮化合物Aurodox和新型多肽KK-88化合物。SDS-PAGE研究表明，Aurodox抑制TTSS相关蛋白Tir，EspB，MAP和EspF不抑制EPEC活力和管家蛋白的表达，李属植物的RT-PCR研究（肠细胞消失位点）表明，金露减少了许多T3 SS相关基因（例如ler）的转录（LEE编码的调节子）、eae（LEE 2：III型装置）、espA、espD、espB、espF（LEE 4）和tir（LEE 5），而不减少T3 SS非相关基因，如16 S rRNA基因之一的rrsB。Aurodox在0.5 μg/mL浓度下也能显著抑制LEE的正调控基因ler的转录。这一结果表明，Aurodox对T3 SS相关蛋白表达的特异性抑制是通过抑制Ier的转录而实现的。口服给药的aurodox引起小鼠的生存与抑制细菌定植在肠粘膜。因此，建议Aurodox通过抑制T3 SS来减少细菌致病性。