In Vivo Evidence for a Prodrug Activation Mechanism during Colibactin Maturation
In Vivo Evidence for a Prodrug Activation Mechanism during Colibactin Maturation
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DOI：
10.1002/cbic.201300208
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发表时间：
2013-07-08
期刊：
影响因子：
3.2
通讯作者：
Mueller, Rolf
中科院分区：
文献类型：
--
作者：
Bian, Xiaoying;Fu, Jun;Mueller, Rolf
最近，人们发现，许多肠道和肠外致病性大肠杆菌（ExPEC）菌株的系统发育组B2携带一个基因组岛（pks岛），编码多个非核糖体肽合成酶（NRPS）和聚酮酶（PKS）的生物合成的基因毒素大肠杆菌素。[1]该基因簇也存在于其他肠杆菌科中，例如肺炎克雷伯氏菌、产气肠杆菌和科氏柠檬酸杆菌。[2]含有大肠杆菌素基因簇（clb）的细菌在体外和体内都会诱导真核细胞的DNA双链断裂，[1，3]因此具有致癌潜力。[4]此外，致细胞病变活性是接触依赖性的：clb+细菌培养上清液和细菌裂解物不是致细胞病变的;[1]这增加了分离大肠杆菌素以解析其化学结构的难度。因此，大肠杆菌素的结构仍未被发现。大多数研究都集中在clb基因簇的遗传和功能分析，如获得晶体结构和结构-活性关系的ClbP，一个假设的大肠杆菌素成熟酶。[5，6]最近的报道显示了参与大肠杆菌素生物合成起始的两个NRPS模块（ClbN和ClbB）的体外生物化学表征，并且显示肽酶ClbP通过释放N-酰基-D-天冬酰胺片段来切割推定的前大肠杆菌素的模拟物。[7]因此，在大肠杆菌素生物合成的前药样释放机制，提出了基于晶体结构的ClbP和体外生化研究。[6，7]令人惊讶的是，E。大肠杆菌Nissle 1917，一种被广泛用作肠道疾病（如溃疡性结肠炎和克罗恩病）益生菌治疗的大肠杆菌菌株，[8]也含有功能性大肠杆菌素基因簇，[9]因此激励我们努力通过体内生物合成研究了解更多关于这种有趣的毒素。使用基因失活和异源表达结合比较代谢物分析，我们确定了一个新的化合物从clb+菌株分离。结构鉴定表明，从E. coliNissle 1917培养物以及来自clb基因簇的异源表达。与最近关于大肠杆菌素生物合成中前体释放机制的体外证据一致，[7]该化合物代表了前体大肠杆菌素转化为大肠杆菌素过程中裂解的前体支架。coliNissle 1917，与E. coliIHE 3034中，除了7个核碱基，其中一个导致ClbK中的氨基酸变化（支持信息中的表S1）。用E. coli Nissle 1917显示出细胞病变活性（图S1），这表明该单一突变对clb的细胞病变活性没有影响。然后，我们使用这个clb基因簇和E。coli Nissle 1917进行体内生物合成研究。
Recently, it was found that many commensal and extra-intestinal pathogenic Escherichia coli (ExPEC) strains of the phylogenetic group B2 carry a genomic island (the pks island) that encodes multiple nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) for the biosynthesis of the genotoxin colibactin.[1] This gene cluster is also present in other Enterobacteriaceae, such as Klebsiella pneumonia, Enterobacter aerogenes, and Citrobacter koseri.[2] Bacteria containing the colibactin gene cluster (clb) induce DNA double-strand breaks in eukaryotic cells both in vitro and in vivo,[1, 3] and thus have carcinogenic potential.[4] Furthermore, the cytopathic activity is contact-dependent: clb+ bacterial culture supernatant and bacterial lysate are not cytopathic;[1] this increases the difficulty in isolating colibactin to solve its chemical structure. Hence, the structure of colibactin remains undiscovered. Most studies have focused on genetic and functional analyses of the clb gene cluster, such as obtaining the crystal structure and structure–activity relationship of ClbP, a hypothetical colibactin-maturating enzyme.[5, 6] A very recent report showed the in vitro biochemical characterization of two NRPS modules (ClbN and ClbB) involved in the initiation of colibactin biosynthesis, and peptidase ClbP was shown to cleave a mimic of the putative precolibactin by releasing an N-acyl-D-asparagine fragment.[7] Thus, a prodrug-like release mechanism in colibactin biosynthesis was proposed, based on the crystal structure of ClbP and the in vitro biochemical studies.[6, 7] Amazingly, E. coli Nissle 1917, a commensal strain that has been widely used as probiotic treatment for intestinal disorders, such as ulcerative colitis and Crohn’s disease,[8] also contains a functional colibactin gene cluster,[9] thus motivating our efforts to learn more about this intriguing toxin by in vivo biosynthesis studies. Using gene inactivation and heterologous expression in conjunction with comparative metabolite analysis, we identified a novel compound isolated from clb+ strains. Structure elucidation revealed an N-myristoyl-D-asparagine (1) from E. coli Nissle 1917 culture as well as from heterologous expression of the clb gene cluster. In agreement with recent in vitro evidence for the release mechanism of the precursor in colibactin biosynthesis,[7] this compound represents the prodrug scaffold that is cleaved off during conversion of precolibactin to colibactin.We first cloned and sequenced the colibactin gene cluster from E. coli Nissle 1917; it was found to be identical to that in E. coli IHE3034 except for seven nucleobases, one of which results in an amino acid change in ClbK (Table S1 in the Supporting Information). Transient infection of human U2OS osteosarcoma cells with E. coli Nissle 1917 revealed cytopathic activity (Figure S1), which indicated that this single mutation has no effect on the cytopathic activity of clb. We then used this clb gene cluster and E. coli Nissle 1917 for in vivo biosynthesis studies.