Development of a method to rapidly quantify persister gene expression

描述(由申请人提供)：据估计，80%以上的细菌感染是由生物膜引起的，而顽固者被认为是他们复发倾向的原因。持久性细胞亚群主要是非复制的，能够耐受高浓度的抗生素，并在去除抗生素后恢复生长。持久者富含生物膜，在这种环境中，他们不仅耐受抗生素治疗，而且还受到身体保护，免受免疫系统的伤害。为了挽救生命和减轻传染病对医疗系统的负担，迫切需要抗持续感染疗法。为了确定针对这些危险表型变异的新型治疗药物开发的潜在靶点，需要对周围生理学有更多的了解。然而，现代高通量技术，如转录组，不能用来表征持久菌，因为它们不能被分离到任何合理的纯度。因此，目前最先进的研究持久基因表达的方法仅限于使用启动子-GFP转录融合、荧光激活细胞分类(FACS)和抗生素耐受性分析一次研究一个启动子。这种方法费时费力，但对持久生理学有了重要的见解。在这里，我们建议将这项技术并行化，以实现对持久基因表达的基因组规模研究。为此，我们将利用启动子报告文库、流式细胞仪、抗生素耐受性分析、 DNA条形码、测序和统计分析。这项技术将首先在大肠杆菌中使用，这是一种常见的持久性研究模式生物，然后被转化为研究铜绿假单胞菌，这是一种重要的临床病原体，已被证明在体内发生持久性。这项拟议的研究将带来几项重要的科学进步。首先，将开发第一个高通量的基因组水平的遗传生理学研究方法，其对临床病原体的可译性将 被证明。其次，将重建第一个持久体转录网络，从而更好地理解持久体表型建立和维持背后的机制。第三，将创建一个铜绿假单胞菌启动子报告文库，供更大的研究社区使用，促进对这种重要临床病原体的研究。总体而言，这项研究的成功完成将为更深入地了解持久性开发工具，为开发新型抗持久性疗法打开大门，为生物被膜感染的治疗带来突破。

DESCRIPTION (provided by applicant): Biofilms are estimated to cause over 80% of bacterial infections, and persisters are thought to be responsible for their propensity to relapse. Persister are subpopulations of cells, which are largely non- replicating, that are able to tolerate high concentrations of antibiotics and resume growth upon removal of the antibiotic. Persisters are enriched in biofilms, and in this environment they not only tolerate antibiotic treatment but are also physically protected from the immune system. Anti-persister therapies are desperately needed to save lives and reduce the burden of infectious disease on the healthcare system. In order to identify potential targets for the development of novel therapeutic agents against these dangerous phenotypic variants, a greater understanding of persister physiology is needed. However, modern high-throughput technology, such as a transcriptome, cannot be used to characterize persisters because they cannot be isolated to any reasonable degree of purity. Therefore, the current state-of-the-art method to study persister gene expression is limited to investigating one promoter at a time using promoter-GFP transcriptional fusions, fluorescent- activated cell sorting (FACS), and antibiotic tolerance assays. This method is laborious and time-consuming, but has led to important insights into persister physiology. Here we propose to parallelize this technique to enable genome-scale investigations into persister gene expression. To accomplish this, we will utilize a promoter reporter library, FACS, antibiotic tolerance assays, DNA barcoding, sequencing, and statistical analysis. This technique will first be used in Escherichia coli, a common model organism for persistence research, then translated to study Pseudomonas aeruginosa, an important clinical pathogen where persistence has been demonstrated to occur in vivo. The proposed study will result in several important scientific advancements. First, the first high-throughput method for the investigation of persister physiology at the genome scale will be developed and its translatability to clinical pathogens will be demonstrated. Second, the first persister transcription networks will be reconstructed, leading to a greater understanding of the mechanisms behind the establishment and maintenance of the persister phenotype. Third, a P. aeruginosa promoter reporter library will be created and available for use by the greater research community, facilitating the study of this important clinical pathogen. Collectively, successful completion of this research will develop tools for a deeper understanding of persistence, opening the door for the development of novel anti- persistence therapies, bringing about breakthroughs for the treatment of biofilm infections.