Understanding the Mechanism of a Gut Microbial Genotoxin Involved in Colorectal Carcinogenesis

项目摘要 结直肠癌（CRC）是美国第三大最常见的癌症形式，也是第二大导致癌症的原因。 癌症死亡过去几十年的研究表明，肠道微生物群影响CRC， 最近的研究表明，肠道细菌基因毒素是癌症发展和进展的关键效应物。 大肠杆菌素是与癌症关系最密切的细菌基因毒素之一，它是人类产生的代谢产物。 肠道细菌，包括某些E.大肠杆菌菌株，拥有一个生物合成基因簇，称为 pks岛。pks+ E. pks+菌株增强大肠杆菌在CRC患者中的表达的能力 在CRC小鼠模型中的肿瘤发生表明大肠杆菌素可能在癌症进展中起因果作用。 然而，实现大肠杆菌素的遗传毒性和CRC的贡献机制的理解， 由于无法分离和结构表征活性遗传毒性代谢物而受阻。期间 在此资助的前一个资助期，我们获得了关于大肠杆菌素的结构和模式的关键信息。 通过研究参与其生物合成的酶来研究其作用。最值得注意的是，我们确定大肠杆菌素是一种 DNA烷化剂，并提出了一个潜在的结构活性遗传毒素。本报告的总体目标 更新申请是为了阐明大肠杆菌素的活性和作用的其他分子机制， CRC致癌作用。基于大肠杆菌素的遗传毒性活性来自于 DNA链间交联（ICL）的形成，我们的三个具体目标是：1）表征特异性 大肠杆菌素-DNA ICLs的结构; 2）开发抑制微生物中大肠杆菌素生物合成的小分子 阐明大肠杆菌介导的DNA损伤在CRC中的生理位置和时间 发展这些进步将通过我们的多学科方法来实现，该方法将知识和 化学生物学、结构生物学、微生物学、毒理学和癌症生物学的技术。总体而言，这 这项工作将填补阐明大肠杆菌素产生肠道作用所需基础知识的关键空白 细菌在CRC致癌作用中的作用，并最终影响癌症的预防、诊断和治疗。此外，本发明还 通过成功地证明研究和操纵与疾病相关的微生物途径 可以提供关键的机械见解，这项工作也将支持和验证未来的努力，以了解如何 其它肠道微生物活性影响CRC的发生和发展。

PROJECT SUMMARY Colorectal cancer (CRC) is the third most prevalent form of cancer in the US and the second leading cause of cancer deaths. Studies over the last several decades have revealed that the gut microbiota influences CRC, and recent work has implicated gut bacterial genotoxins as key effectors in cancer development and progression. One of the bacterial genotoxins most strongly connected to cancer is colibactin, a metabolite produced by human gut commensal bacteria, including certain E. coli strains, that possess a biosynthetic gene cluster termed the pks island. The increased prevalence of pks+ E. coli in CRC patients and the ability of pks+ strains to potentiate tumorigenesis in mouse models of CRC suggest colibactin may play a causal role in cancer progression. However, achieving a mechanistic understanding of colibactin's genotoxicity and contribution to CRC has been impeded by an inability to isolate and structurally characterize the active genotoxic metabolite(s). During the previous funding period of this grant, we gained critical information about colibactin's structure and mode of action by studying the enzymes involved in its biosynthesis. Most notably, we established that colibactin is a DNA alkylating agent and proposed a potential structure for the active genotoxin. The overall objective of this renewal application is to elucidate additional molecular mechanisms underlying colibactin's activity and role in CRC carcinogenesis. Building off of the central hypothesis that the genotoxic activity of colibactin derives from the formation of DNA interstrand cross-links (ICLs), our three specific aims will: 1) characterize the specificity and structure of colibactin-DNA ICLs; 2) develop small molecules that inhibit colibactin biosynthesis in microbial communities; 3) elucidate the physiological location and timing of colibactin-mediated DNA damage in CRC development. These advances will be enabled by our multidisciplinary approach, which merges knowledge and techniques from chemical biology, structural biology, microbiology, toxicology, and cancer biology. Overall, this effort will fill critical gaps in fundamental knowledge needed to elucidate the role of colibactin-producing gut bacteria in CRC carcinogenesis and ultimately impact cancer prevention, diagnosis, and treatment. Additionally, by successfully demonstrating that studying and manipulating individual disease-associated microbial pathways can provide key mechanistic insights, this work will also support and validate future efforts to understand how other gut microbial activities influence CRC initiation and development.