Uncovering the role of polyamines in bacterial survival and antibiotic resistance

细菌在其细胞外环境中遇到许多化学物质。可以想象，这些化学物质共同影响影响细菌存活和抗生素耐药性的细胞反应。这些影响通常在标准体外测定中未检测到，通常忽略关键的毒力和抗生素抗性表型。这类化学物质的一个突出类别是多胺，它在环境中无处不在，在感染期间由植物和动物细胞过量产生。高浓度的多胺杀死细菌。然而，在达到致命水平之前，多胺使细菌对许多抗生素更具耐药性或更敏感，从而改变这些药物的疗效。我的NSERC研究计划的总体目标是了解多胺如何与细菌相互作用，影响它们在压力下的生存和适应。这项拟议的研究推进了我早期的工作，确定了多胺在抗生素耐药性中的作用，并利用高通量化学基因组学方法在全基因组水平上研究细菌的反应。我假设细菌使用以前未被认识到的机制来承受高聚胺浓度。此外，我假设多胺调节细菌对抗生素的反应，通过与特定的细胞靶点的相互作用，抗生素暴露触发调节级联反应连接多胺生产的抗生素反应。为了解决这些假设，我提出了三个平行和互补的工作包，以实现三个具体目标的发现补助金：1。研究革兰氏阳性菌和革兰氏阴性菌如何耐受高浓度的多胺。2.确定多胺介导对抗生素反应改变的机制。3.描述支持多胺在抗生素反应中作用的调控网络。我的实验室将使用全基因组方法来询问数千个突变体，每个突变体都有单个基因缺失，分析它们对多胺和多胺抗生素组合的反应。我们还将构建一个报告文库，以测量暴露于抗生素时多胺生物合成酶的表达。我们将使用S。aureus和E.大肠杆菌分别作为革兰氏阳性多胺非生产菌和革兰氏阴性多胺生产菌的代表。通过这种系统的方法，我们将制定新的假设，使我们能够进行详细的分析，使用传统的生物化学和分子生物学方法，提供机制的见解细菌对多胺和抗生素耐药性的反应。总之，我的研究计划解决了在理解多胺在细菌存活和抗生素耐药性中的重要性方面的重大知识差距。该计划产生的发现将为未来对抗超级细菌的应用铺平道路，并将为医疗保健系统，农业和生物技术部门带来实实在在的好处。

Bacteria encounter many chemicals in their extracellular milieu. Conceivably, these chemicals collectively affect cellular responses influencing bacterial survival and antibiotic resistance. Such effects are typically undetected in standard in vitro assays, often overlooking critical virulence and antibiotic resistance phenotypes. A prominent class of such chemicals is polyamines being ubiquitous in the environment and overproduced by plant and animal cells during infection. High concentrations of polyamines kill bacteria. Yet, before reaching lethal levels, polyamines make bacteria either more resistant or susceptible to many antibiotics, thereby changing the efficacy of these drugs. The overall goal of my NSERC research program is to understand how polyamines interact with bacteria influencing their survival and adaptation under stress. The proposed research advances my earlier work that identified the role of polyamines in antibiotic resistance and harnesses high-throughput chemical genomics approaches to study bacterial responses at the whole-genome level. I hypothesize that bacteria use previously unrecognized mechanisms to withstand high polyamine concentrations. Further, I hypothesize that polyamines modulate the bacterial responses to antibiotics through interaction with specific cellular targets and that antibiotic exposure triggers regulatory cascades linking polyamine production to the antibiotic response. To address these hypotheses, I propose three parallel and complementary work packages to achieve three specific objectives of this discovery grant: 1. Study how model Gram-positive and Gram-negative bacteria tolerate high concentrations of polyamines. 2. Determine the mechanisms by which polyamines mediate altered responses to antibiotics. 3. Characterize the regulatory networks underpinning polyamine role in response to antibiotics. My lab will use a whole-genomic approach to interrogate thousands of mutants, each harboring single gene deletions, profiling their responses to polyamines and polyamine-antibiotic combos. We will also construct a reporter library to measure the expression of polyamine biosynthetic enzymes when exposed to antibiotics. We will use S. aureus and E. coli as representatives of Gram-positive polyamine non-producer and Gram-negative polyamine-producer bacteria, respectively. Through this systems approach, we will formulate new hypotheses that will allow us to undertake detailed analyses using conventional biochemical and molecular biology methods providing mechanistic insights into the bacterial responses to polyamines and antibiotic resistance. Together, my research program addresses a significant knowledge gap in understanding the importance of polyamines in bacterial survival and antibiotic resistance. The discoveries generated from this program will pave the way for future applications to fight superbugs and will have tangible benefits to the healthcare systems, agriculture, and the biotech sectors.