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.

摘要 微生物组研究日益强调单个微生物组成员对健康和健康的贡献 疾病。越来越多的证据表明，微生物影响宿主生理和病理的部分原因是 微生物代谢物。为了了解不同微生物代谢物在宿主病理生理学中的作用， 研究的重点是通过基因敲除或过度敲除来操纵单个细菌菌株的代谢物生产。 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， 这种策略有其自身的局限性，因为某些微生物代谢物是从多个微生物中衍生出来的 拥有保守基因簇的物种。一个例子是Colibactin，一种细菌的次生代谢物 有 由于其对结直肠癌和肠道微生物区系组成的影响而引起越来越多的关注 功能。Colibactin是一种由不同肠杆菌科细菌产生的杂合多酮-非核糖体多肽 携带高度保守的聚酮合成酶(PKS)基因簇。然而，在理解方面的进展 Colibactin+细菌在很大程度上仅限于操纵和表征单个敲除细菌。 在细胞培养或无菌小鼠中的菌株，而忽略了这样一个事实，即在一个 原生环境可以通过获取保守的PKS岛来产生Colibactin来影响宿主。 此外，还没有制定针对Colibactin+细菌进行癌症干预的策略 越来越多的证据表明，Colibactin促进宿主DNA损伤、衰老和致癌。致信地址 理解和靶向Colibactin+细菌这两个互补且高度整合的细菌的局限性 将开发抑制结节蛋白的方法。第一种方法是通过劫持来使酶失活。 一种抗粘连蛋白的酶，被不同的粘连蛋白+细菌用来防止粘连蛋白对自身DNA的损伤。 将探索细菌表面展示抗结肠粘连蛋白酶，以最大限度地使其催化失活 细菌-宿主交界处的大肠菌素。同时，第二种策略是基因抑制，在那里保守的 编码大肠杆菌蛋白的PKS岛将被两个不同的CRISPR系统抑制，该系统由一个自我传播的 宿主范围广的接合质粒。而CRISPR-Cas9(CRISPR基因敲除)可消除结肠肌动蛋白+细菌 通过直接DNA切割，CRISPR-dCpf1(CRISPR干扰)在没有 导致细菌死亡和逃避的选择压力。酶和基因抑制系统都会 由遗传上易驯化的天然大肠杆菌分离株传递，已被证明可有效定植 在肠子里。在体外，将采用细菌-宿主细胞共培养、多菌群落和小鼠模型。 比较和验证酶促方法和遗传方法的有效性。虽然此应用程序关注的是 如果Colibactin成功，它将开创在细胞内直接操纵微生物代谢物的方法， 组织和生物体水平，以加快基础和翻译研究。

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