Development of Type III secretion system inhibitors

作为利用TTSS诱导的肠致病性大肠杆菌（EPEC）溶血试验筛选的一部分，我们筛选了20,250个微生物提取物，发现菌株Streptomyces sp. K06-0806和KK-88的提取物具有较强的TTSS抑制活性。活性化合物经发酵纯化后，分别从K06-0806和KK-88培养液中分离到线状聚酮、氧化还原和KK-88化合物作为TTSS抑制剂。SDS-PAGE研究表明，氧化还原抑制了ttss相关蛋白Tir、EspB、Map和EspF的表达，但未抑制EPEC活力和保家蛋白GroEL的表达。对肠细胞消失位点LEE属的RT-PCR研究表明，氧化还原降低了许多t3ss相关基因的转录，如ler （LEE编码调节因子）、eae (LEE2：III型仪器),espA, espD, espB, espF （LEE4）和tir (LEE5)，而不减少t3ss非相关基因，如16S rRNA基因之一rrsB。当浓度为0.5 μg/mL时，Aurodox显著降低了LEE阳性调节基因ler的转录。这一结果表明，氧化还原对t3ss相关蛋白表达的特异性抑制是由抑制ler的转录引起的。采用啮齿柠檬酸杆菌感染的C3H/HeJ小鼠进行体内研究。口服氧化还原通过抑制肠道黏膜细菌定植而使小鼠存活。因此，建议氧化还原通过抑制T3SS来减少细菌发病。

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.