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 微生物组研究已增强了个别微生物群成员对健康的贡献 疾病。积累的证据表明，微生物部分影响了宿主的生理和病理学 微生物代谢产物。为了了解潜水微生物代谢产物在宿主病理生理学中的作用，大多数 研究的重点是通过基因敲除或过度操纵单个细菌菌株的代谢产物的代谢产物 表达以询问微生物，微生物代谢产物和宿主过程之间的因果关系。然而， 该策略具有其自身的局限性，因为某些微生物代谢产物来自多个微生物 藏有构成基因簇的物种。一个例子是科利列汀，一种细菌二级代谢产物， 有 由于其对结直肠癌和肠道菌群组成的影响以及 功能。结肠癌是由不同的肠杆菌科产生的杂交聚酮化合物 - 非核糖体肽 携带高度保存的聚酮化合物合酶（PKS）基因簇。但是，理解的进展 科比氏素+细菌在很大程度上仅限于操纵和表征单个基因敲除细菌 细胞培养或无菌小鼠的菌株，同时忽略了一个事实，即 本地环境可以通过获得配置的PKS岛产生肠actin以影响宿主。 此外，根据 累积的证据表明肠胃痛会促进宿主DNA损伤，感应和癌变。解决 理解和靶向Colibactin+细菌的局限性，两个完整且高度整合 将开发方法来抑制结肠癌。第一种方法是通过劫持酶灭活 由Divers Colibactin+细菌采用的一种抗哥伦比辛酶，以防止结肠癌损伤。 将探索抗哥伦比亚素酶的细菌表面显示，以最大化催化失活 在细菌宿主界面处的结肠癌。同时，第二种策略是遗传抑制，其中配置 PKS岛编码为Colibactin的编码将被两种不同的CRISPR系统抑制 广泛的宿主范围偶联质粒。而CRISPR-CAS9（CRISPR淘汰）消除了Colibactin+细菌 通过直接DNA裂解，CRISPR-DCPF1（CRISPR干扰）抑制了Colibactin Biosynsiss，而无需 诱导细菌死亡和逃避选择压力。酶和遗传抑制系统都将 通过已证明以有效定殖的一般可牵引的天然大肠杆菌分离株传递 体外细菌 - 宿主细胞培养，多数菌群和小鼠模型将被录用 比较和验证酶和遗传方法的效率。虽然此应用程序重点是 结肠癌，如果成功，它将开创性方法直接在细胞处操纵微生物代谢物， 组织和有机水平加速基本和翻译研究。

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