Development of a method to rapidly quantify persister gene expression

描述（由申请人提供）：据估计，生物膜引起超过80%的细菌感染，并且认为持久性是其复发倾向的原因。持留菌是细胞的亚群，其在很大程度上是非复制的，其能够耐受高浓度的抗生素并在去除抗生素后恢复生长。寄生虫富含生物膜，在这种环境中，它们不仅耐受抗生素治疗，而且还受到免疫系统的保护。迫切需要抗持久性治疗来挽救生命并减轻传染病对医疗保健系统的负担。为了确定开发针对这些危险表型变体的新型治疗剂的潜在靶点，需要更好地理解持续生理学。然而，现代高通量技术，如转录组，不能用于表征持久，因为它们不能被分离到任何合理的纯度。因此，目前研究持续基因表达的最先进方法限于使用启动子-GFP转录融合、荧光激活细胞分选（FACS）和抗生素耐受性测定一次研究一个启动子。这种方法既费力又耗时，但却导致了对持续生理学的重要见解。在这里，我们建议将这种技术并行化，使基因组规模的调查持续基因表达。为了实现这一点，我们将利用启动子报告文库，流式细胞仪，抗生素耐受性测定， DNA条形码、测序和统计分析。该技术将首先用于大肠杆菌，持久性研究的常见模式生物，然后转化为研究铜绿假单胞菌，一种重要的临床病原体，其中持久性已被证明在体内发生。这项研究将带来几项重要的科学进步。首先，将开发第一种在基因组规模上研究持久性生理学的高通量方法，并将其转化为临床病原体。 被证明。第二，将重建第一个持续转录网络，从而更好地理解持续表型的建立和维持背后的机制。第三，将创建铜绿假单胞菌启动子报告文库，供更大的研究团体使用，促进对这种重要临床病原体的研究。总的来说，这项研究的成功完成将为更深入地理解持久性开发工具，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.