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

摘要/摘要 肺炎链球菌是一种主要的全球细菌性人类病原体，每年造成约100万人死亡 世界范围内，由于肺炎、败血症和脑膜炎。有两种策略被用来对抗这种感染。 抗生素通常可以治愈这种感染，疫苗被用来减少流行性出血热的循环人口。 最危险的血清型。然而，这两种策略都在以越来越快的速度失败。耐药菌株 在全球范围内不断出现和传播；疫苗接种的有效性也受到挑战，如血清型 未被当前疫苗配方靶向的疫苗不断涌现，并迅速取代目标疫苗。这个 这些失败的原因是将多个外来基因转移到细菌中，但创造 新的传染性和抗药性菌株类型尚不清楚。传输事件有两种类型，称为微和 宏观重组事件。涉及数十个数百个碱基对的微观事件与 肺炎球菌通过转化进行基因转移的已知特性。然而，数量越多， 更重要的是，事件涉及数以万计的核苷酸的多个区块的转移，有时全部 来自单一的供体菌株。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。这种探索性的 该项目将使用微流控技术创建许多小室(液滴)，攻击者-目标将在其中 相互作用可以第一次在细胞和分子水平上进行研究和表征， 在全基因组分辨率下识别参与细胞并追踪所有基因交换事件。 医学上的相关性。大多数致病链球菌都有通过自然遗传进行基因转移的机制。 转型。基因转化是肺炎球菌遗传灵活性的重要途径 被记录为疫苗逃逸以及耐药基因产生和传播的关键。因为 肺炎链球菌是一种研究DNA摄取的模式生物，本工作对其机制进行了研究 在菌株或物种之间意外地转移大块基因将对理解产生广泛的影响 并针对革兰氏阳性菌之间许多类似的肽调节基因交换系统 通常与这些细菌致病的能力有关。

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.