Development of a method to rapidly quantify persister gene expression

描述（由申请人提供）：据估计，超过80%的细菌感染是由生物膜引起的，持久性细菌感染被认为是其复发倾向的原因。持久性细胞是细胞的亚群，它们大部分是非复制的，能够耐受高浓度的抗生素，并在抗生素去除后恢复生长。坚持者富含生物膜，在这种环境中，它们不仅能耐受抗生素治疗，而且还能免受免疫系统的物理保护。为了挽救生命和减轻传染病对卫生保健系统的负担，迫切需要抗持久性治疗。为了确定针对这些危险表型变异的新型治疗药物的潜在靶点，需要对持久性生理学有更深入的了解。然而，现代高通量技术，如转录组，不能用于表征持久性，因为它们不能被分离到任何合理的纯度。因此，目前研究持久性基因表达的最先进方法仅限于使用启动子- gfp转录融合、荧光活化细胞分选（FACS）和抗生素耐量试验一次研究一个启动子。这种方法既费力又费时，但却对持久性生理学有了重要的见解。在这里，我们提出并行这种技术，使基因组规模的调查，以持久基因的表达。为了实现这一目标，我们将利用启动子报告文库、FACS、抗生素耐受性试验、

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.