Isolation and Systems-Level Characterization of Persistent Bacteria

描述（由申请人提供）：细菌持续存在是许多类型慢性细菌感染的基础，是一个主要的公共卫生问题。细菌持久性是细菌个体性的表现，其中一个亚群的细胞耐受抗生素，尽管遗传上是易感的。拟议的研究将开发分离坚持者的技术，以表征其生理状态。为此，将开发一种新的方法，用于全面分析蛋白质丰度的群体异质性。该方法将使用大型的天然基因组编码蛋白质文库，这些蛋白质具有重要的持久性病原体大肠杆菌的C末端荧光标记。coli、S.金黄色葡萄球菌和铜绿假单胞菌。该方法还将使用开发的培养和固定技术以及高度自动化的流式细胞术，在增加持久性的条件下稳健地分析数千种不同蛋白质的蛋白质丰度分布。将分析这些分布，以促进荧光标记菌株的合理合并，用于新的选择策略，以鉴定持久性的预测蛋白。选择策略使用在合并文库中具有最高蛋白丰度的细胞的顶部级分的荧光激活细胞分选（FACS）。分选的细胞将在持久性测定中用高浓度的抗生素处理，从而富集携带预测持久性的标记蛋白的细胞的频率。在通过选择方案的多次迭代后，将进行DNA测序以确定预测性标记蛋白的身份。这些蛋白标志物将通过FACS和持久性试验进行验证，并将用于分离纯持久性群体。强大的系统生物学技术，包括RNA-seq和体内蛋白质占有率显示，将应用于持久性的纯培养物，以在系统水平上表征其生理学。开发的技术将深入了解细菌的持久性和异质性，这些发现将加速新抗生素治疗的开发。

DESCRIPTION (provided by applicant): Bacterial persisters underlie many types of chronic bacterial infections and are a major public health problem. Bacterial persisters are a manifestation of bacterial individuality in which a subgroup of cells tolerates antibiotics, despit being genetically susceptible. The proposed research will develop techniques to isolate persisters in order to characterize their physiological state. To this end, a novel method for comprehensively analyzing population heterogeneity in protein abundances will be developed. This method will use large libraries of native, genomically-encoded proteins with C-terminal fluorescent tags for the important persistent pathogens E. coli, S. aureus, and P. aeruginosa. This method will also use developed culturing and fixing techniques with highly-automated flow cytometry to robustly analyze the protein abundance distributions for thousands of different proteins under conditions that increase persistence. These distributions will be analyzed to facilitate the rational pooling of fluorescently-tagged strains for a novel selection strategy to identify predictive proteins for persistence. The selection strategy uses fluorescently activated cell sorting (FACS) of the top fraction of cells with the highest protein abundances in a pooled library. The sorted cells will be treated with high concentrations of antibiotics in a persister assay, thereby enriching the frequency of cells harboring tagged proteins that predict persistence. After multiple iterations through the selection scheme, DNA- sequencing will be performed to determine the identity of the predictive tagged proteins. These protein markers will be validated by FACS and persister assays, and will be used to isolate pure persister populations. Powerful systems biology techniques, including RNA-seq and in vivo protein occupancy display, will be applied to pure cultures of persisters to characterize their physiology at the systems level. The developed techniques will provide deep insight into bacterial persistence and heterogeneity, and the findings will accelerate development of new antibiotic treatments.