Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
mBio. 2019 Aug 13;10(4):e01762-19. doi: 10.1128/mBio.01762-19.
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 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 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 and 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 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 strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools. 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 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 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 and strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut , including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.
近年来，随着人们对肠道微生物群落对人类健康和疾病的重要性认识的提高，对肠道微生物群落的研究有了显著的增加。拟杆菌门是健康人类肠道中最丰富的细菌门，可诱导促进健康和促进疾病的双重作用。肠道中有超过 55 种拟杆菌，具有广泛的种内遗传多样性，特别是在涉及与其他细菌、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有了很大的扩展，但我们仍然缺乏在多种菌株中创建缺失突变体和等位基因替换的简便系统，特别是在对抗生素的耐药性迅速增加的情况下，这两种抗生素用于遗传操作。在这里，我们提出了一种新的多功能和高效的载体套件，允许在多耐药菌株中创建等位基因缺失和替换，并使用基于多糖利用的功能获得系统。这些载体还允许在不创建突变背景菌株的情况下，使用来自 VI 型分泌系统毒素进行简单的反选择，在反选择过程中诱导毒素产生依赖于两种不同的分子之一，为基于菌株表型的提供了灵活性。该载体家族极大地促进了功能遗传分析，并扩展了可进行遗传修饰的肠道拟杆菌菌株的范围，包括目前使用现有遗传工具难以遗传修饰的多耐药菌株。我们已经进入了一个时代，肠道微生物组的研究正在从组成和宿主效应的基本问题向理解构成变化和介导健康和疾病过程的微生物分子的转变。肠道拟杆菌对人类健康和疾病的重要性及其作为工程活生物疗法来源的潜力，使这些细菌成为深入机制研究的特别关注点。然而，对不同拟杆菌菌株的遗传分析仍然存在障碍，限制了我们研究在这些菌株中发现的重要宿主和群落表型的能力。在这里，我们通过构建一系列允许在不同肠道拟杆菌和梭菌菌株中进行简便遗传操作的载体克服了许多这些障碍。这些构建体满足了一个关键需求，并允许在多样化的肠道拟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多抗生素耐药菌株。
