Macrorecombination in isolated cell pairs via natural genetic transformation

摘要/总结： 肺炎链球菌（肺炎球菌）是一种主要的全球细菌性人类病原体，可引起 全球每年约有 100 万人死于肺炎、败血症和脑膜炎。两种策略 用于对抗此类感染。抗生素通常可以治愈此类感染，而疫苗则是 用于减少最危险血清型的循环种群。然而，两者 战略的失败率越来越高。抗生素耐药菌株不断出现 传播到全球；由于血清型没有针对性，疫苗接种的有效性也受到挑战 当前的疫苗配方不断出现并迅速取代目标疫苗配方。 这些失败的原因是多个外源基因转移到细菌中，但是 产生新的感染性和耐药性菌株类型的机制尚不清楚。转会事件 有两种类型，称为微观重组事件和宏观重组事件。微观事件包括 几十到几千个碱基对，与基因的已知特性一致 肺炎球菌中通过转化进行转移。然而，更重要的事件涉及转让 数万个核苷酸的多个块，有时全部来自单个供体菌株。 这些宏观重组事件很难与任何已知的完全一致 基因转移的机制——无论是接合、转导还是转化。这个项目 将使用微流体技术创建许多小室（液滴），攻击者在其中 首次可以在细胞和细胞层面上研究和表征靶标相互作用 分子水平，通过识别参与细胞和追踪所有基因交换事件 全基因组规模和 200 bp 分辨率。 医学相关性。大多数致病性链球菌都具有相同的基因转移机制： 自然遗传转化。遗传转化是遗传灵活性的重要途径 在肺炎球菌中，它被记录为疫苗逃逸、产生和传播的关键 新的耐药基因。因为肺炎链球菌是一种模式生物 DNA 摄取的研究，这项工作研究了意外转移大块 DNA 的机制 菌株或物种之间的基因将对理解和靶向 革兰氏阳性菌之间有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。

Abstract /Summary: Streptococcus pneumoniae (pneumococcus) 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 replacing the targeted ones. The cause of these failures is transfer of multiple foreign genes into the bacteria, but the mechanisms that create 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 to several thousands of base pairs, are consistent with the known properties of gene transfer by transformation in pneumococcus. However, 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 were difficult to reconcile completely with any known mechanism of gene transfer - whether conjugation, transduction, or transformation. This 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, both by identifying the participant cells and by tracing all gene exchange events at full genome scale and 200-bp 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 new 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 strains or species will have broad impacts on understanding and targeting the many similar peptide regulated gene exchange systems among Gram positive bacteria that are often associated with the ability of these bacteria to cause disease.