Enzymatic and genetic strategies for targeting disease-associated microbial metabolites

摘要 微生物组研究越来越强调个体微生物群成员对健康和 疾病越来越多的证据表明，微生物影响宿主的生理和病理，部分是通过 微生物代谢产物为了了解不同微生物代谢产物在宿主病理生理学中的作用， 研究集中于通过基因敲除或过度- 表达来询问微生物、微生物代谢物和宿主过程之间的因果关系。然而，在这方面， 该策略具有其自身的局限性，因为某些微生物代谢物来源于多种微生物， 具有保守基因簇的物种。一个例子是大肠杆菌素，一种细菌次级代谢产物， 具有 由于其对结直肠癌和肠道微生物群组成的影响， 功能大肠杆菌素是由不同肠杆菌科细菌产生的一种杂合聚酮-非核糖体肽 携带高度保守的聚酮合酶（PKS）基因簇。然而，在了解 大肠杆菌素+细菌在很大程度上限于操纵和表征单个敲除细菌 菌株在细胞培养或无菌小鼠，而忽略了这样一个事实，即多种不同的肠道细菌在一个 原生环境可产生大肠杆菌素，通过获得保守的PKS岛来影响宿主。 此外，还没有开发出针对大肠杆菌素+细菌用于癌症干预的策略， 积累了大肠杆菌素促进宿主DNA损伤、衰老和致癌的证据。解决 在理解和靶向大肠杆菌素+细菌方面的局限性， 将开发抑制大肠杆菌素的方法。第一种方法是通过劫持酶失活 一种由多种大肠杆菌素+细菌使用的抗大肠杆菌素酶，用于防止大肠杆菌素对自身DNA的损伤。 将探索抗大肠杆菌素酶的细菌表面展示，以最大限度地催化灭活 大肠杆菌素在细菌-宿主界面。与此同时，第二种策略是遗传抑制，其中保守的 编码大肠杆菌素的pks岛将被两种不同的CRISPR系统抑制， 广宿主范围接合质粒。虽然CRISPR-Cas9（CRISPR敲除）消除了大肠杆菌素+细菌， 通过直接DNA切割，CRISPR-dCpf1（CRISPR干扰）抑制大肠杆菌素的生物合成， 诱导细菌死亡和逃避的选择压力。酶和遗传抑制系统将 由遗传上易处理的天然E.大肠杆菌分离株已被证明有效定植 在肚子里。将采用体外细菌-宿主细胞共培养、多微生物群落和小鼠模型 比较和验证酶和基因方法的有效性。虽然这个应用程序的重点是 大肠杆菌素，如果成功的话，它将开创在细胞内直接操纵微生物代谢物的方法， 组织和有机体水平，以加速基础和转化研究。

Abstract Microbiome research has increasingly highlighted contributions of individual microbiota members to health and disease. Accumulating evidence suggests that microbes influence host physiology and pathology in part through microbial metabolites. To understand the roles of diverse microbial metabolites in host pathophysiology, most studies focus on manipulating individual bacterial strains’ metabolite production with genetic knockout or over- expression to interrogate the causality between microbes, microbial metabolites and host processes. However, this strategy has its own limitations in that certain microbial metabolites are derived from multiple microbial species harboring conserved gene clusters. One example is colibactin, a bacterial secondary metabolite that has garnered increasing attention due to its implications in colorectal cancer and gut microbiota composition and function. Colibactin is a hybrid polyketide-nonribosomal peptide produced by different Enterobacteriaceae carrying a highly-conserved polyketide synthase (pks) gene cluster. However, progress in understanding colibactin+ bacteria has been largely limited to manipulating and characterizing individual knockout bacterial strains in cell culture or germ-free mice, while overlooking the fact that multiple different enteric bacteria in a native environment can produce colibactin to impact the host through the acquisition of the conserved pks island. Furthermore, no strategy has been developed to target colibactin+ bacteria for cancer intervention in light of the accumulating evidence that colibactin promotes host DNA damage, senescence and carcinogenesis. To address the limitations in understanding and targeting colibactin+ bacteria, two complementary and highly integrated approaches will be developed to inhibit colibactin. The first approach is enzymatic inactivation through hijacking an anti-colibactin enzyme employed by diverse colibactin+ bacteria to prevent self-DNA damage by colibactin. Bacterial surface display of the anti-colibactin enzyme will be explored to maximize the catalytic inactivation of colibactin at the bacteria-host interface. In parallel, the second strategy is genetic inhibition, where the conserved pks island coding for colibactin will be inhibited by two different CRISPR systems delivered by a self-transmissible broad-host-range conjugative plasmid. While CRISPR-Cas9 (CRISPR knockout) eliminates colibactin+ bacteria via direct DNA cleavage, CRISPR-dCpf1 (CRISPR interference) suppresses colibactin biosynthesis without inducing bacterial death and selection pressure for evasion. Both enzymatic and genetic inhibition systems will be delivered by genetically tractable native E. coli isolates that have been demonstrated for efficient colonization in the gut. In vitro bacteria-host cell coculture, polymicrobial communities, and mouse models will be employed to compare and validate the efficacy of enzymatic and genetic approaches. While this application focuses on colibactin, if successful, it will pioneer methodologies to directly manipulate microbial metabolites at the cellular, tissue and organismal levels to accelerate fundamental and translational studies.