Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF.
Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF.
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DOI：
10.1128/msystems.00062-18
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发表时间：
2018-09
期刊：
影响因子：
6.4
通讯作者：
Dunny GM
中科院分区：
文献类型：
--
作者：
Dale JL;Beckman KB;Willett JLE;Nilson JL;Palani NP;Baller JA;Hauge A;Gohl DM;Erickson R;Manias DA;Sadowsky MJ;Dunny GM
粪肠球菌在宿主外存活并通过口粪传播的强大能力及其高度的内在和获得性抗菌素耐药性都使医院获得性肠球菌感染的治疗复杂化。保守的E.粪肠球菌的核心基因组作为一个重要的遗传支架，在现代卫生保健环境中，这种细菌的进化，也提供了有趣的疫苗和药物的目标。我们使用了一种创新的池化/测序策略来映射E. faecalis OG 1 RF，并产生了覆盖约70%的OG 1 RF基因组的确定突变体的阵列文库。然后，我们使用该文库进行了高通量转座子测序实验，以确定OG 1 RF中胆汁抗性的核心基因组决定因素。该集合是使用传统和高通量方法进行全面的功能性肠球菌基因组学的宝贵资源，并且能够立即恢复感兴趣的突变体。粪肠球菌（Enterococcus faecalis）是动物胃肠道（GI）中常见的肠道细菌，并且是现代卫生保健环境中人类机会性感染的主要原因。E.粪肠球菌OG 1 RF是一种不含质粒的菌株，它含有很少的移动的元件，但保留了大多数E. faecalis基因型为了便于询问核心肠球菌遗传决定因素在胃肠道中的竞争适应性，生物膜形成，内在的抗菌素耐药性和在环境中的生存，我们在OG 1 RF中产生了一组排列的，序列定义的染色体转座子插入。我们使用正交池策略结合Illumina测序来鉴定一组具有独特的、单个基于Himar的转座子插入的突变体。突变体在2,651个注释的开放阅读框中的1,926个（72.6%）中含有插入，并且在大多数假设的蛋白质编码基因和长度大于100 bp的基因间区域中含有插入，其可以编码小RNA。作为该阵列转座子文库的有用性的原理证明，我们创建了包含6，829个突变体的最小输入池，所述突变体被选择用于最大基因组覆盖，并使用我们称为SMarT（序列定义的水手技术）转座子测序（TnSeq）的方法来鉴定大肠杆菌中胆汁抗性的许多遗传决定因素。faecalis OG1RF.这些包括以前与胆汁酸抗性相关的几个基因以及新的基因座。我们的阵列文库允许用相对少量的突变体对大比例的基因组进行功能性筛选，减少了重复检查的潜在影响，并能够在竞争后立即恢复突变体。重要性粪肠球菌在宿主外存活并通过口-粪传播的强大能力及其高度的内在和获得性耐药性都使医院获得性肠球菌感染的治疗复杂化。保守的E.粪肠球菌的核心基因组作为一个重要的遗传支架，在现代卫生保健环境中，这种细菌的进化，也提供了有趣的疫苗和药物的目标。我们使用了一种创新的池化/测序策略来映射E. faecalis OG 1 RF，并产生了覆盖约70%的OG 1 RF基因组的确定突变体的阵列文库。然后，我们使用该文库进行了高通量转座子测序实验，以确定OG 1 RF中胆汁抗性的核心基因组决定因素。该集合是使用传统和高通量方法进行全面的功能性肠球菌基因组学的宝贵资源，并且能够立即恢复感兴趣的突变体。
The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest. Enterococcus faecalis is a common commensal bacterium in animal gastrointestinal (GI) tracts and a leading cause of opportunistic infections of humans in the modern health care setting. E. faecalis OG1RF is a plasmid-free strain that contains few mobile elements yet retains the robust survival characteristics, intrinsic antibiotic resistance, and virulence traits characteristic of most E. faecalis genotypes. To facilitate interrogation of the core enterococcal genetic determinants for competitive fitness in the GI tract, biofilm formation, intrinsic antimicrobial resistance, and survival in the environment, we generated an arrayed, sequence-defined set of chromosomal transposon insertions in OG1RF. We used an orthogonal pooling strategy in conjunction with Illumina sequencing to identify a set of mutants with unique, single Himar-based transposon insertions. The mutants contained insertions in 1,926 of 2,651 (72.6%) annotated open reading frames and in the majority of hypothetical protein-encoding genes and intergenic regions greater than 100 bp in length, which could encode small RNAs. As proof of principle of the usefulness of this arrayed transposon library, we created a minimal input pool containing 6,829 mutants chosen for maximal genomic coverage and used an approach that we term SMarT (sequence-defined mariner technology) transposon sequencing (TnSeq) to identify numerous genetic determinants of bile resistance in E. faecalis OG1RF. These included several genes previously associated with bile acid resistance as well as new loci. Our arrayed library allows functional screening of a large percentage of the genome with a relatively small number of mutants, reducing potential effects of bottlenecking, and enables immediate recovery of mutants following competitions. IMPORTANCE The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.