Toxin neutralization to functionally dissect interbacterial antagonism in the gut microbiome

毒素中和以功能性剖析肠道微生物组中细菌间拮抗作用

• 批准号: 10534604
• 负责人: Beth A Shen
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534604
• 关键词: Actinobacteria class; Affect; Animals; Anti-Bacterial Agents; Bacteria; Bioinformatics; Biological Assay; Catalogs; Cells; Communication; Communities; Complex; Data; ELF3 gene; Environment; Firmicutes; Foundations; Gene Delivery; Genes; Genus Mycobacterium; Gnotobiotic; Goals; Health; Human; Immunity; In Situ; In Vitro; Individual; Intoxication; Learning; Maintenance; Mediating; Mentors; Methods; Microbe; Modeling; Monitor; Mus; Nutrient; Organism; Outcome; Pathway interactions; Phenotype; Physiological; Play; Prevalence; Property; Research; Research Personnel; Role; Shapes; Shotgun Sequencing; Structure; System; Techniques; Testing; Toxin; Training; Universities; Virulence; Washington; Work; antagonist; base; design; experience; experimental study; gut metagenome; gut microbiome; gut microbiota; improved; in vivo; innovation; insight; member; metagenomic sequencing; microbial; microbial community; microbiome; microbiome composition; microbiome sequencing; mouse model; prevent; skills

Abstract Decades of research have revealed that the composition of the human gut microbiome directly impacts human health, yet we still do not understand the mechanisms underlying formation and maintenance of this complex community. Counterintuitively, increasing evidence suggests that competitive interactions play a role in maintaining community stability. While genes encoding components of diverse systems involved in interbacterial antagonism are widespread in human gut metagenomes, the specific antagonistic interactions occuring in situ remain elusive. One of the pathways mediating interbacterial antagonism is the Esx secretion system, which is conserved in two prominent gut phyla, Firmicutes and Actinobacteria. While this pathway has been shown to mediate contact-dependent interbacterial antagonism by a limited number of Gram-positive species in vitro, many questions remain about its physiological function. Antibacterial toxins secreted by the Esx machinery are present throughout human gut metagenomes, suggesting that Esx-mediated antagonism could occur in this community. These findings led me to propose to identify the targets of Esx-mediated interbacterial antagonism in a model natural community, the murine gut microbiome. To accomplish this goal, I will take advantage of the fact that bacteria express cognate immunity genes to prevent self-intoxication by secreted toxins. In Aim 1 of my proposed studies, I will use bioinformatics and in vitro functional assays to identify Esx toxin and immunity genes encoded in a model murine gut metagenome derived from wild-caught mice. My preliminary findings indicate at least two antibacterial Esx toxins are encoded in this natural gut microbiome. In Aim 2, I describe an in situ conjugation strategy to deliver the immunity genes identified in Aim 1 to all members of the murine gut community. I hypothesize that expression of immunity genes in bacteria targeted by specific Esx toxins will increase their abundance in a community. Therefore, by identifying changes to community composition that depend on immunity genes, I can determine the targets of Esx-mediated antagonism in situ. This unique approach to examine interbacterial antagonism under physiological conditions will significantly improve our understanding of the physiological function of the Esx system in gut bacterial species. It will also provide the first direct characterization of antagonism between gut microbiome constituents. The methods I propose to develop may additionally later be applied broadly to the characterization of diverse antagonistic pathways present in gut bacterial species, an essential next step needed to define the mechanisms shaping gut microbiome composition and ultimately human health. By working in the lab of Dr. Joseph Mougous at the University of Washington and with my experienced collaborators, I will be able to learn diverse techniques, practice effective mentoring strategies, and hone my scientific communication skills. This high-quality training will enable me to succeed as an independent researcher studying microbe-microbe interactions.

抽象的 数十年的研究表明，人类肠道微生物组的组成直接影响人类 健康，但是我们仍然不了解这种复合物的形成和维护的机制 社区。违反直觉，越来越多的证据表明竞争互动在 保持社区稳定。而基因编码参与细菌的各种系统的组件 拮抗作用在人类肠道元基因组中广泛，这是在原位发生的特定拮抗相互作用 保持难以捉摸。 ESX分泌系统是介导细菌间拮抗作用的途径之一 在两个著名的肠道门，富有肠细菌和静脉细菌中保守。虽然已显示此途径 通过有限数量的革兰氏阳性物种在体外介导接触依赖性的细菌间拮抗作用， 关于其生理功能仍然存在许多问题。 ESX机械分泌的抗菌毒素是 存在整个人类肠道元基因组，表明ESX介导的拮抗作用可能发生在此中 社区。这些发现使我提议识别ESX介导的细菌间的目标 模型自然社区中的对抗，鼠肠道微生物组。为了实现这一目标，我将采取 细菌表达同源免疫基因以防止分泌自我毒化的事实的优势 毒素。在我提出的研究的目标1中，我将使用生物信息学和体外功能测定法来识别ESX 毒素和免疫基因在模型中编码的鼠类肠道元基因组衍生自野生小鼠。我的 初步发现表明至少两个抗菌ESX毒素在此天然肠道微生物组中编码。在 AIM 2，我描述了一种原位共轭策略，以将AIM 1中确定的免疫基因交付给所有成员 鼠肠道社区。我假设特异性针对的细菌中免疫基因的表达 ESX毒素将增加社区的丰富性。因此，通过确定社区的变化 取决于免疫基因的组成，我可以确定ESX介导的拮抗作用的原位靶标。 这种在生理条件下检查细菌间拮抗作用的独特方法将显着 提高我们对肠道细菌中ESX系统生理功能的理解。它也会 提供肠道微生物组成分之间拮抗作用的第一个直接表征。方法i 提议开发可能会额外扩展到多种拮抗作用的表征 肠道细菌物种中存在的途径，定义构成肠道的机制所需的下一步 微生物组组成，最终是人类健康。通过在约瑟夫·穆格斯博士的实验室工作 华盛顿大学以及我经验丰富的合作者，我将能够学习多种技术， 练习有效的指导策略，并磨练我的科学沟通技巧。这种高质量的培训 将使我能够成为研究微生物微生物相互作用的独立研究人员。