Narayanan Ajay M, Ramsey Matthew M, Stacy Apollo, Whiteley Marvin
Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, LaMontagne Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, USA.
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island, USA.
Appl Environ Microbiol. 2017 Jun 30;83(14). doi: 10.1128/AEM.00797-17. Print 2017 Jul 15.
Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen One of this bacterium's key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on is hampered by the field's lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of , revealing a core set of 319 genes. We then generated an arrayed mutant library comprising >1,500 unique insertions and used a sequencing-based approach to define each mutant's position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen. Millions suffer from gum disease, which often is caused by , a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen.
牙周炎是一种微生物感染，会破坏支撑牙齿的结构。虽然它通常是一种慢性疾病，但快速进展的侵袭性形式与口腔病原体有关。这种细菌的一个关键毒力特征是其附着于表面并形成强大生物膜的能力，这些生物膜能够抵抗宿主和抗生素的杀灭。尽管自最初发现以来，人们对它已经有了很多了解，但我们对其基础生物学的一个基本方面仍缺乏深入认识，因为我们不知道它生存所需的全套基因（必需基因组）。此外，该领域缺乏突变体文库阻碍了对它的研究。为了填补这些空白，我们使用快速转座子突变体测序（Tn-seq）来确定两株该细菌的必需基因组，揭示了一组由319个基因组成的核心基因集。然后，我们生成了一个包含超过1500个独特插入位点的阵列突变体文库，并使用基于测序的方法来确定每个突变体在文库中的位置（孔和板）。为了证明其效用，我们筛选该文库以寻找对亚抑制浓度红霉素耐药性减弱的突变体，发现多药外排泵AcrAB是一个关键的耐药因子。在筛选过程中，我们发现红霉素会诱导该细菌形成生物膜。因此，我们设计了一种基于Tn-seq的新型筛选方法来鉴定介导这种表型的特定因子，并在后续实验中证实了4个突变体。总之，这些研究为研究人类病原体的基础生物学和疾病机制提供了新的见解和资源。数百万人患有牙龈疾病，这通常是由一种形成抗生素耐药性生物膜的细菌引起的。为了全面了解任何生物体，我们应该能够回答：它生存需要哪些基因？在这里，我们通过确定其基因组中不能被突变的基因来回答这个关于该细菌的问题。至于那些可以被突变的基因，我们将这些突变体存档到一个文库中，我们用这个文库来寻找对抗生素耐药性有贡献的基因，从而发现抗生素会导致该细菌形成生物膜。然后，我们设计了一种方法来寻找介导这个过程的基因，并证实了4个基因。这些结果揭示了一种人类病原体的新的基本特征。
