Toxin neutralization to functionally dissect interbacterial antagonism in the gut microbiome

摘要 几十年的研究表明，人类肠道微生物组的组成直接影响人类的健康。 健康，但我们仍然不了解这种复杂的形成和维持的机制 社区与直觉相反，越来越多的证据表明，竞争性相互作用在 维护社区稳定。虽然基因编码的不同系统的组成部分，涉及细菌间 拮抗作用广泛存在于人类肠道宏基因组中， 仍然难以捉摸介导细菌间拮抗作用的途径之一是Esx分泌系统， 保守于两个重要的肠道门，厚壁菌门和放线菌门。虽然这一途径已被证明是 在体外通过有限数量的革兰氏阳性菌种介导接触依赖性细菌间拮抗作用， 关于它的生理功能还有许多问题。Esx机器分泌的抗菌毒素是 存在于整个人类肠道宏基因组中，这表明Esx介导的拮抗作用可能发生在这种情况下。 社区这些发现促使我提出鉴定Esx介导的细菌间作用的靶点， 拮抗作用在一个模型的自然社区，鼠肠道微生物组。为了实现这一目标，我将 细菌表达同源免疫基因以防止分泌的自身中毒的事实的优点 毒素在目标1中，我将使用生物信息学和体外功能测定来鉴定Esx 毒素和免疫基因编码的模型鼠肠道宏基因组来源于野生捕获的小鼠。我 初步研究结果表明，在这种天然肠道微生物组中编码至少两种抗菌Esx毒素。在 目的2，我描述了一种原位接合策略，将目的1中鉴定的免疫基因传递给所有成员 最大的优势我推测，特异性抗体靶向的细菌中免疫基因的表达 Esx毒素会增加它们在社区中的丰度。因此，通过识别社区的变化， 依赖于免疫基因的组合物，我可以确定Esx介导的拮抗作用的靶点在原位。 这种在生理条件下检查细菌间拮抗作用的独特方法将显著地 提高我们对肠道细菌物种中Esx系统的生理功能的理解。它还将 提供了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 建议开发的技术可以在以后广泛应用于表征不同的拮抗剂， 存在于肠道细菌物种中的途径，这是定义肠道形成机制所需的重要下一步 微生物组的组成和最终的人类健康。通过在约瑟夫·穆戈斯博士的实验室工作， 华盛顿大学和我经验丰富的合作者，我将能够学习各种技术， 实践有效的指导策略，并磨练我的科学沟通技巧。这种高质量的培训 将使我能够成功地作为一个独立的研究人员研究微生物之间的相互作用。

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.