Bypassing the restriction barrier to improve transformation in S. epidermidis

绕过限制性屏障以改善表皮葡萄球菌的转化

• 批准号: 9386188
• 负责人: Ambrose Lin Yau Cheung
• 金额: $ 20.25万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-27 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9386188
• 关键词: Adenine; Antibiotic Resistance; Bacteremia; Bacteria; Blood Circulation; Blood Vessels; Bypass; Catheters; Clinical; Cloning; Communities; Complex; Cytosine; DNA; DNA Restriction Enzymes; DNA Restriction-Modification Enzymes; Devices; Escherichia coli; Escherichia coli K12; Excision; Genes; Genetic; Genome; Genomics; Genus staphylococcus; Goals; Hip Prosthesis; Hospitals; Human; In Vitro; Individual; Indolent; Infection; Knowledge; Laboratories; Libraries; Mediating; Medical Device; Methylation; Methyltransferase; Microbial Biofilms; Mind; Modification; Morbidity - disease rate; Mucous Membrane; Nosocomial Infections; Organism; Pacemakers; Pathogenesis; Pathogenicity; Phase; Physiology; Pilot Projects; Plasmids; Positioning Attribute; Process; Prokaryotic Cells; Prosthesis; Publishing; Reporting; Research; Research Personnel; Series; Site; Skin; Specific qualifier value; Specificity; Staphylococcus aureus; Staphylococcus epidermidis; Surface; Symbiosis; System; Time; University Hospitals; Virulent; Work; antimicrobial; base; design; improved; in vivo; methylation pattern; novel strategies; pathogen; plasmid DNA; prevent; single molecule; success; uptake; virtual

Abstract Staphylococcus epidermidis (SE) ranks as one of the most common causes of nosocomial infections in US hospitals. Intravascular devices and prostheses are the usual sites of infection followed by seeding of the bloodstream. Although more indolent than S. aureus, SE infections are difficult to treat due to its persistence as biofilm on abiotic surfaces as well as its penchant for antibiotic resistance. There are two major issues in advancing our understanding of SE pathogenesis: 1) prior work has focused on two laboratory isolates, SE 1457 and RP62A while our knowledge on clinical isolates are virtually non-existent; 2) SE clinical isolates are extremely difficult to transform due to its formidable restriction barrier. As a consequence, we lack the ability to manipulate clinical isolates genetically to dissect the relevant pathogenic steps leading to colonization, biofilm formation, persistence and dissemination. With the advance of genomics and SMRT sequencing, we are in a unique position to analyze the methylation pattern of the restriction modification (RM) system based on the specificity unit (HsdS) and methylase (HsdM). There are four RM systems in SE (types I-IV). Type 1 and type IV are the major restriction barriers that prevent uptake of foreign DNA due to restriction by HsdR. We will exploit the RM system by methylating adenine in plasmid DNA with type I cognate HsdMS complex(es) in E. coli DC10B which is defective in cytosine methylation and can bypass the type IV RM system. While type IV RM system is conserved among S. aureus and SE, the type I RM system is more variable and entails at least 7 distinct groups, each with its unique target recognition motif (TRM) mediated by HsdS. These discrepancies help explain the differences in restriction among SE isolates. We hypothesize that type 1 RM system in a particular SE isolate can be completely bypassed to yield efficient transformation by cloning shuttle plasmids in DC10B that express the cognate hsdMS genes. With this goal is mind, we propose the following two specific aims: I) determine the different groups in type I RM system by performing Pacbio single molecule real time sequencing (SMRT) to detect methylated-adenine residues and the target recognition motif of the HsdMS complex in the SE genomes; II) Construction of DC10B-derived E. coli strains to enable efficient transformation into diverse SE strains. As a utility from this system, we will construct two relevant mariner transposon libraries to be available to the research community.

抽象的 葡萄球菌表皮（SE）是美国诊断感染最常见的原因之一 医院。血管内装置和假体是通常的感染部位 血液。尽管比金黄色葡萄球菌更懒惰，但由于其持久性而难以治疗SE感染 作为非生物表面上的生物膜以及对抗生素耐药性的偏爱。有两个主要问题 促进我们对SE发病机理的理解：1）先前的工作集中于两个实验室分离株，SE 1457和RP62A虽然我们对临床分离株的了解实际上是不存在的； 2）SE临床分离株是 由于其强大的限制性障碍，极其难以改变。结果，我们缺乏能力 在遗传上操纵临床分离株，以剖析导致定植，生物膜的相关致病步骤 形成，持久和传播。随着基因组学和SMRT测序的发展，我们在 基于限制修饰（RM）系统的甲基化模式的独特位置 特异性单位（HSD）和甲基化酶（HSDM）。 SE中有四个RM系统（I-IV型）。类型1和类型 IV是由于HSDR限制而导致外国DNA摄取的主要限制障碍。我们将 用E型质粒DNA中的甲基化腺嘌呤与E中的E.相关HSDMS络合物（ES）来利用RM系统。 大肠杆菌DC10B在胞嘧啶甲基化中有缺陷，可以绕过IV型RM系统。同时IV类 RM系统在金黄色葡萄球菌和SE中是保守的，I型RM系统更可变，至少需要 7个不同的组，每个组具有其独特的目标识别基序（TRM），由HSD介导。这些差异 帮助解释SE分离株之间限制的差异。我们假设该类型1 RM系统 可以通过克隆穿梭质粒在 表达同源HSDM基因的DC10B。有了这个目标，我们提出以下两个特定的 目的：i）通过实时执行PACBIO单分子来确定I型RM系统中的不同组 测序（SMRT）以检测甲基化 - 腺苷残基和HSDMS的靶识别基序 SE基因组中的复合物； ii）构建DC10B衍生的大肠杆菌菌株以实现有效的效率 转化为多种SE菌株。作为该系统的实用程序，我们将构建两个相关的水手 转座库可供研究社区使用。