Exploring Persister Antibiotic Responses as a Source of Biomarkers and Elimination Strategies

 描述（由申请方提供）：细菌持留菌耐受抗生素治疗，并构成生物膜感染复发倾向的基础。对持续存在生理学的更好理解将导致开发针对利用生物膜的病原体（如大肠杆菌、铜绿假单胞菌和金黄色葡萄球菌）的更有效的疗法。在我们对坚持者的理解中，一个关键的知识缺口是他们对抗生素的机械反应。我们还不知道坚持者如何管理抗生素压力，或者这些过程的哪些方面是他们耐受所必需的。这种知识的缺乏很大程度上源于与测量持久性生理学相关的困难。持续存在尚未被分离，因此，荧光激活细胞分选（FACS）已成为最好的技术来量化持续存在的生理。我们假设，关于持续抗生素反应的知识以及它们与死亡细胞的比较可以用于了解持续生存和识别持续生物标志物。坚持者和垂死细胞的抗生素反应之间的相似性和差异将提供关于坚持者如何避免杀死的知识，此外，这些差异将构成潜在的生物标志物。为了验证我们的假设，我们将比较持久的转录反应的细胞死亡，并确定有助于持久的耐受性和/或可以作为持久的生物标志物的功能。为了做到这一点，我们将测量从抗生素死亡的大肠杆菌的mRNA的时间过程，并确定与网络成分分析介导的基因表达变化的监管机构。这些“死亡应答”将用于选择启动子以用于在持留者中进行分析，因为持留生理学的FACS定量是时间和劳动密集型的。选择的启动子将进行荧光标记，并与分选，持久性测定和统计方法进行研究，以量化和比较持久性反应的垂死细胞。将确定相似性和差异性，并通过启动子和调控水平上的响应的遗传重新布线来测试它们对持久耐受性的影响。该提案的结果将填补有关持久性抗生素核心特征的基础知识空白，确定持久性富集的改进生物标志物，通过破坏持久性抗生素应激管理阐明治疗干预的新途径，并影响抗生素耐受性，微生物学和传染病领域。

 DESCRIPTION (provided by applicant): Bacterial persisters tolerate antibiotic treatment, and underlie the propensity of biofilm infections to relapse. An improved understanding of persister physiology will lead to the development of more effective therapies against biofilm-utilizing pathogens such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. A critical knowledge gap in our understanding of persisters is their mechanistic response toward antibiotics. We do not yet know how persisters manage antibiotic stress or what aspects of these processes are required for their tolerances. This lack of knowledge largely originates from difficulties associated with measuring persister physiology. Persisters have yet to be isolated, and therefore, fluorescence activated cell sorting (FACS) has become the best technique to quantify persister physiology. We hypothesize that knowledge of persister antibiotic responses and how they compare to those of cells that die can be used to understand persister survival and identify persister biomarkers. Similarities and differences between the antibiotic responses of persisters and dying cells will provide knowledge of how persisters avoid killing, and in addition, the differences will constitute potential biomarkers. To test our hypothesis, we will compare the transcriptional responses of persisters to those of cells that die, and identify features that contribute to persister tolerances and/or can serve as persister biomarkers. To do this, we will measure time-course mRNA from Escherichia coli dying from antibiotics and identify the regulators mediating gene expression changes with Network Component Analysis. These "death responses" will be used to select promoters for analysis in persisters, because FACS quantification of persister physiology is time- and labor-intensive. Selected promoters will be fluorescently-labelled and studied with sorting, persistence assays, and statistical methods to quantify and compare persister responses to those of dying cells. Similarities and differences will be identified, and both tested for their impacts on persister tolerances through genetic rewiring of the responses at both the promoter and regulatory levels. Results from this proposal will fill fundamental knowledge gaps about the core characteristic of persisters, identify improved biomarkers for persister enrichment, illuminate new avenues for therapeutic intervention through disruption of persister antibiotic stress management, and impact the fields of antibiotic tolerance, microbiology, and infectious disease.