Cryptic gut bacterial metabolites that regulate colorectal cancer formation

描述（由申请人提供）：在选定的肠道细菌分离株中发现的孤儿生物合成基因簇与人类肠道中的长期持久性、宿主染色体不稳定性和结直肠癌形成直接相关。在不知道编码分子的身份或其如何被递送到宿主细胞的情况下，评估其特定作用、作用模式、治疗潜力和潜在治疗领域的进一步实验受到严重限制。我们建议确定和结构特征的特定细菌编码的分子，控制主机表型反应。我们将采用三种互补的方法：1）生物测定引导的分级分离以富集调节生理学相关表型的细菌分子，包括抗菌、生物膜分解、DNA损伤，和免疫调节测定~ 2）基因座阳性与基因座阴性细胞的差示液相色谱/质谱分析，以鉴定基因簇特异性分子~和3）蛋白质介导的未知毒素配体的小分子捕获。该毒素的生物活性是通过非常不寻常的细菌-人细胞接触依赖性方式赋予的，其与先前描述的毒素递送策略缺乏遗传或表型相似性。我们还建议操纵细菌的生物合成途径工程毒素衍生物与反应性生物正交功能。我们的探针驱动策略不仅可以阐明将分子引导到哺乳动物细胞中的不寻常的细胞接触依赖性递送机制，而且还可以揭示参与调节染色体不稳定性的潜在新癌症靶点。对毒素不寻常的传递机制的基本理解可能会导致一种自然启发的方法，用于以空间控制的方式将工程分子直接导入哺乳动物细胞，并在益生菌疗法中具有潜在的应用。

DESCRIPTION (provided by applicant): An orphan biosynthetic gene cluster found in select gut bacterial isolates has been directly linked to long-term persistence in the human gut, host chromosome instability, and colorectal cancer formation. Without knowing the encoded molecule's identity or how it is delivered into host cells, further experiments to evaluate its specific role(s), mode(s) of action, therapeutic potential, and potential treatment areas are severely limited. We propose to identify and structurally characterize the specific bacterial encoded molecules that control host phenotypic responses. We will employ three complementary approaches: 1) bioassay-guided fractionation to enrich bacterial molecule(s) regulating the physiologically-relevant phenotypes, including antibacterial, biofilm disassembly, DNA damage, and immunomodulatory assays~ 2) differential liquid chromatography/mass spectrometric analysis of locus positive versus locus negative cells to identify gene cluster-specific molecules~ and 3) protein mediated small molecule capture of unknown toxin ligands. Biological activity of the toxin is imparted through a highly unusual bacteria-human cell contact-dependent manner, which lacks genetic or phenotypic similarities to previously described toxin delivery strategies. We also propose to manipulate the bacterial biosynthetic pathway to engineer toxin derivatives with reactive bioorthogonal functionalities. Our probe-driven strategy could not only illuminate the unusual cell contact-dependent delivery mechanism directing molecules into mammalian cells, but also reveal potentially new cancer targets involved in regulating chromosome instability. A fundamental understanding of the toxin's unusual delivery mechanism could lead to a Nature-inspired approach for directing engineered molecules directly into mammalian cells in a spatially controlled manner with potential applications in probiotic therapies.