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的慢性细菌感染的基础，是一个主要的公共卫生问题。细菌顽固者是细菌个性的一种表现，其中一组细胞耐受抗生素，尽管在遗传上易感。拟议的研究将开发分离持久性的技术，以表征他们的生理状态。为此，将开发一种综合分析蛋白质丰度群体异质性的新方法。该方法将使用大量天然的、带有c端荧光标记的基因组编码蛋白文库，用于重要的持久性病原体大肠杆菌、金黄色葡萄球菌和铜绿假单胞菌。该方法还将使用先进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，对数千种不同蛋白质的蛋白质丰度分布进行稳健分析。这些分布将进行分析，以促进合理的荧光标记菌株池为一种新的选择策略，以确定预测蛋白的持久性。该选择策略使用荧光激活细胞分选（FACS），对池库中蛋白质丰度最高的细胞的顶部部分进行分选。分选后的细胞将在持久性试验中用高浓度抗生素处理，从而增加携带可预测持久性的标记蛋白的细胞的频率。通过选择方案进行多次迭代后，将进行DNA测序以确定预测标记蛋白的身份。这些蛋白标记物将通过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和体内蛋白质占用显示，将应用于持久者的纯培养，以在系统水平上表征其生理特征。开发的技术将深入了解细菌的持久性和异质性，这些发现将加速开发新的抗生素治疗方法。

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.