Confined Genetic Transformation and Exchange of Antibiotic Resistance Genes in Femtoliter Microdroplets

飞升微滴中抗生素抗性基因的有限遗传转化和交换

• 批准号: 9369924
• 负责人: David Eddington
• 金额: $ 22.38万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-05 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9369924
• 关键词: Animal Model; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Base Pairing; Biological Assay; Cell Communication; Cells; Cessation of life; Competence; Cytolysis; DNA; Dangerousness; Disease; Drug resistance; Effectiveness; Engineering; Epithelium; Event; Evolution; Failure; Future; Gene Exchanges; Gene Transfer; Genes; Genetic; Genetic Recombination; Genetic Transformation; Genome; Genomics; Goals; Gram-Positive Bacteria; Horizontal Gene Transfer; Human; Image; Immune; Infection; Intervention; Laboratories; Lung; Medical; Meningitis; Microbial Biofilms; Microfluidics; Modeling; Molecular; Names; Nasopharynx; Nucleotides; Participant; Pathogenicity; Patients; Peptides; Plant Roots; Pneumonia; Population; Process; Property; Reaction; Recovery; Resolution; Sepsis; Serotyping; Streptococcus; Streptococcus pneumoniae; System; Techniques; Testing; Time; Vaccination; Vaccines; Virulence Factors; Work; combat; flexibility; killings; middle ear; novel; pathogen; pathogenic bacteria; pressure; programs; resistance gene; resistant strain; response; theories; tool; uptake; vaccine development

Abstract /Summary Streptococcus pneumoniae is a major global bacterial human pathogen, causing ~1 million deaths annually worldwide, due to pneumonia, sepsis, and meningitis. Two strategies are used to combat such infections. Antibiotics can often cure such infections, and vaccines are used to reduce the circulating populations of the most dangerous serotypes. However, both strategies are failing at an increasing rate. Antibiotic resistant strains are continually arising and spreading globally; vaccination effectiveness is also under challenge, as serotypes not targeted by current vaccine formulations are continually arising and rapidly replace the targeted ones. The cause of these failures is transfer of multiple foreign genes into the bacteria, but the mechanisms creating the new infectious and resistant strain types are unclear. Transfer events are of two types, named as micro- and macro-recombination events. The micro events, involving dozens of hundreds of base pairs, are consistent with the known properties of gene transfer by transformation in pneumococcus. However, the more numerous, and more significant, events involve transfer of multiple blocks of tens of thousands of nucleotides, sometimes all from a single donor strain. These macro-recombination events are difficult to reconcile completely with any known mechanism of gene transfer - whether conjugation, transduction, or transformation. This exploratory project would use microfluidics to create numerous small chambers (droplets) within which attacker-target interactions can be studied and characterized for the first time at both the cellular and molecular levels, by both identifying the participant cells and tracing all gene exchange events at full genome resolution. Medical Relevance. Most pathogenic streptococci share the mechanism of gene transfer by natural genetic transformation. Genetic transformation is an important path for genetic flexibility in pneumococcus, where it is documented as key to vaccine escape and creation and spread of drug-resistance genes. Because Streptococcus pneumoniae is a model organism for the study of DNA uptake, this work on the mechanism that transfers unexpectedly large blocks of genes between strain or species will have broad impact on understanding and targeting many similar peptide regulated gene exchange systems among Gram positive bacteria that are often associated with the ability of these bacteria to cause disease.

摘要/概要 肺炎链球菌是一种主要的全球性细菌性人类病原体，每年造成约100万人死亡 在全球范围内，由于肺炎，败血症和脑膜炎。有两种策略用于防治此类感染。 抗生素通常可以治愈这种感染，疫苗用于减少循环人口的感染。 最危险的血清型然而，这两种策略的失败率都在不断增加。抗生素耐药菌株 疫苗接种的有效性也受到挑战，因为血清型 不被当前疫苗制剂靶向的疫苗不断出现并迅速取代靶向疫苗。的 这些失败的原因是多个外源基因转移到细菌中，但产生这些基因的机制是不确定的。 新的感染性和耐药性菌株类型尚不清楚。传输事件有两种类型，称为微传输事件和 宏重组事件微事件，涉及几十个数百个碱基对，是一致的， 肺炎球菌中通过转化进行基因转移的已知特性。然而，人数越多， 更重要的是，事件涉及数万个核苷酸的多个块的转移，有时全部 来自单一供体菌株。这些宏观重组事件很难与任何 已知的基因转移机制-无论是接合、转导还是转化。这项探索性 该项目将使用微流体技术来创建许多小腔室（液滴），其中攻击目标 第一次可以在细胞和分子水平上研究和表征相互作用， 鉴定参与细胞并以全基因组分辨率追踪所有基因交换事件。 医学相关性。大多数致病性链球菌都具有通过天然遗传途径进行基因转移的机制 转型遗传转化是肺炎球菌遗传灵活性的重要途径， 被证明是疫苗逃逸以及耐药基因产生和传播的关键。因为 肺炎链球菌是研究DNA摄取的模式生物，本研究探讨了其机制， 在菌株或物种之间意外地转移大块基因将对理解 并针对革兰氏阳性细菌中许多类似的肽调节基因交换系统， 通常与这些细菌引起疾病的能力有关。