Isolation and Systems-Level Characterization of Persistent Bacteria

描述(由申请人提供)：细菌持久者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。细菌持续体是细菌个性的一种表现，其中一部分细胞耐受抗生素，尽管在遗传上是敏感的。这项拟议的研究将开发分离持久者的技术，以表征它们的生理状态。为此，将开发一种新的方法来综合分析蛋白质丰度的群体异质性。这种方法将使用大量带有C末端荧光标签的天然、基因组编码的蛋白质文库来识别重要的持久性病原菌：大肠杆菌、金黄色葡萄球菌和铜绿假单胞菌。这种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仪，在增加持久性的条件下，强有力地分析数千种不同蛋白质的蛋白质丰度分布。这些分布将被分析，以促进荧光标记菌株的合理汇集，用于一种新的选择策略，以识别持久性预测蛋白。选择策略使用荧光激活细胞分类(FACS)，对汇集的文库中蛋白质丰度最高的细胞进行顶端部分的分类。分类的细胞将在持久试验中用高浓度的抗生素处理，从而丰富了含有预测持久性的标记蛋白的细胞的频率。在通过选择方案进行多次迭代之后，将执行DNA测序以确定预测性标记蛋白的身份。这些蛋白质标记将通过流式细胞仪和持久分析进行验证，并将用于分离纯持久种群。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，将被应用于持久者的纯培养，以在系统水平上表征他们的生理特征。所开发的技术将提供对细菌持久性和异质性的深刻洞察，这些发现将加速新抗生素疗法的开发。

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.