Enzymatic and genetic strategies for targeting disease-associated microbial metabolites

针对疾病相关微生物代谢物的酶和遗传策略

• 批准号: 10686498
• 负责人: Jiahe Li
• 金额: $ 135.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686498
• 关键词: Acceleration; Address; Anabolism; Attention; Bacteria; CRISPR interference; CRISPR/Cas technology; Cancer Intervention; Cell Culture Techniques; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Code; Colorectal Cancer; Communities; DNA; DNA Damage; Disease; Enterobacteriaceae; Environment; Enzymes; Escherichia coli; Etiology; Functional disorder; Gene Cluster; Genetic; Germ-Free; Health; Hybrids; In Situ; In Vitro; Individual; Island; Knock-out; Light; Mediating; Metabolic Pathway; Methodology; Microbe; Mus; Pathology; Peptides; Physiology; Plasmids; Process; Production; Role; Surface; System; Tissues; carcinogenesis; efficacy validation; gene conservation; genetic approach; gut microbiota; host-microbe interactions; member; microbial; microbiome research; microbiota; mouse model; overexpression; polyketide synthase; polyketides; pressure; prevent; secondary metabolite; senescence; translational study

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.

抽象的 微生物组研究越来越强调个体微生物群成员对健康和健康的贡献 疾病。越来越多的证据表明，微生物部分地通过以下方式影响宿主的生理和病理： 微生物代谢产物。为了了解不同微生物代谢物在宿主病理生理学中的作用，大多数 研究重点是通过基因敲除或过度控制单个细菌菌株的代谢产物产生 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， 该策略有其自身的局限性，因为某些微生物代谢物源自多种微生物 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， 有 由于其对结直肠癌和肠道微生物群组成的影响而受到越来越多的关注 功能。 Colibactin 是由不同肠杆菌科细菌产生的杂合聚酮化合物-非核糖体肽 携带高度保守的聚酮合酶（pks）基因簇。然而，理解上的进步 大肠杆菌素+细菌在很大程度上仅限于操纵和表征个体敲除细菌 细胞培养物或无菌小鼠中的菌株，同时忽略了一个事实，即同一环境中存在多种不同的肠道细菌 原生环境可以产生大肠杆菌素，通过获得保守的 pks 岛来影响宿主。 此外，鉴于目前尚未制定针对大肠杆菌素+细菌进行癌症干预的策略。 越来越多的证据表明大肠杆菌素会促进宿主 DNA 损伤、衰老和致癌。致地址 理解和靶向大肠杆菌素+细菌（两种互补且高度整合的细菌）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来灭活酶 多种大肠杆菌素+细菌使用的一种抗大肠杆菌素酶，可防止大肠杆菌素对自身 DNA 造成损伤。 将探索抗大肠杆菌素酶的细菌表面展示，以最大限度地催化失活 大肠杆菌素位于细菌-宿主界面。与此同时，第二种策略是基因抑制，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，这两种 CRISPR 系统是由可自传播的 广泛宿主范围的接合质粒。 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）抑制大肠杆菌素生物合成，而无需 诱导细菌死亡和逃避选择压力。酶抑制系统和遗传抑制系统都会 由基因上易于处理的本地大肠杆菌分离株传递，这些分离株已被证明可以有效定殖 在肠道里。将采用体外细菌-宿主细胞共培养、多种微生物群落和小鼠模型 比较和验证酶法和遗传方法的功效。虽然此应用程序侧重于 如果大肠杆菌素成功，它将开创直接操纵细胞中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。

项目成果

Nonpathogenic E. coli engineered to surface display cytokines as a new platform for immunotherapy.

非致病性大肠杆菌经过改造可表面展示细胞因子，作为免疫治疗的新平台。

• DOI: 10.21203/rs.3.rs-4031911/v1
• 发表时间: 2024
• 期刊: Research square
• 作者: Yang,Shaobo;Sheffer,Michal;Kaplan,IsabelE;Wang,Zongqi;Tarannum,Mubin;Dinh,Khanhlinh;Abdulhamid,Yasmin;Shapiro,Roman;Porter,Rebecca;Soiffer,Robert;Ritz,Jerome;Koreth,John;Wei,Yun;Chen,Peiru;Zhang,Ke;Márquez-Pellegrin,Valeria
• 通讯作者: Márquez-Pellegrin,Valeria