Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota
Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota
复制标题
DOI：
10.1128/mbio.01762-19
复制
发表时间：
2019-07-01
期刊：
影响因子：
6.4
通讯作者：
Comstock, Laurie E.
中科院分区：
文献类型：
--
作者：
Garcia-Bayona, Leonor;Comstock, Laurie E.
近年来，对肠道微生物群的研究急剧增加，因为这种微生物生态系统对人类健康和疾病的重要性得到了更好的认识。拟杆菌目是健康人体肠道中最丰富的细菌目，具有促进健康和促进疾病的作用。有超过55种肠道类杆菌具有广泛的种内遗传多样性，特别是在参与与其他细菌，宿主和饮食相互作用的分子合成的区域。这种特性需要研究不同的物种和菌株。近年来，研究这些细菌的遗传工具包已经大大扩展，但我们仍然缺乏一个在不同菌株中创建缺失突变体和等位基因替换的简易系统，特别是随着对用于遗传操作的两种抗生素的耐药性迅速增加。在这里，我们提出了一种新的多功能和高效的载体套件，允许创建等位基因缺失和替代多耐药菌株的拟杆菌和副拟杆菌使用功能获得系统的基础上多糖的利用。这些载体还允许使用来自脆弱拟杆菌的VI型分泌系统的毒素进行简单的反选择，而不依赖于产生突变体背景菌株。反向选择过程中的毒素产生是由两种不同分子之一诱导的，从而提供了基于菌株表型的灵活性。该载体家族极大地促进了功能性遗传分析，并扩展了可被遗传修饰的肠道拟杆菌目菌株的范围，以包括目前用现有遗传工具遗传上难以处理的多重抗性菌株。重要性我们已经进入了一个时代，肠道微生物群的研究正从组成和宿主效应的基本问题过渡到理解构成组成变化和宿主效应的基础的微生物分子。介导健康和疾病过程。肠道拟杆菌目对人类健康和疾病的重要性以及它们作为工程活生物治疗药物来源的潜力，使这些细菌成为深入机制研究的特别兴趣。然而，仍然有障碍的遗传分析不同的拟杆菌目菌株，限制了我们的能力，研究重要的主机和社区的表型确定在这些菌株。在这里，我们已经克服了许多这些障碍，通过构建一系列的载体，允许在不同的肠道拟杆菌和副拟杆菌菌株容易的遗传操作。这些构建体满足了一个关键的需求，并允许在不同的肠道类杆菌中进行简化的等位基因替换，包括现代人类肠道中存在的越来越多的耐多药菌株。
Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis. Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools.IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.