Uncovering the role of polyamines in bacterial survival and antibiotic resistance

细菌在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生存和抗生素耐药性的细胞反应。这种效应通常在标准的体外测定中检测不到，往往忽略了关键的毒力和抗生素耐药性表型。这类化学物质中最突出的一类是多胺，多胺在环境中无处不在，在感染期间由植物和动物细胞过量产生。高浓度的多胺能杀死细菌。然而，在达到致死水平之前，多胺会使细菌对许多抗生素产生更强的耐药性或更敏感，从而改变这些药物的功效。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。拟议的研究推进了我早期的工作，即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反应。我假设细菌利用以前未被认识到的机制来承受高浓度的多胺。此外，我假设多胺通过与特定细胞靶点的相互作用来调节细菌对抗生素的反应，而抗生素暴露引发了将多胺的产生与抗生素反应联系起来的调节级联反应。为了解决这些假设，我提出了三个平行和互补的工作包，以实现该发现基金的三个具体目标：研究模型革兰氏阳性和革兰氏阴性细菌如何耐受高浓度多胺。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的作用的调控网络。我的实验室将使用全基因组的方法来调查数千个突变体，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。我们还将构建报告文库，检测多胺生物合成酶在抗生素作用下的表达情况。我们将分别使用金黄色葡萄球菌和大肠杆菌作为革兰氏阳性多胺非产生菌和革兰氏阴性多胺产生菌的代表。通过这种系统方法，我们将制定新的假设，这将使我们能够使用传统的生化和分子生物学方法进行详细分析，为细菌对多胺和抗生素耐药性的反应提供机制见解。总之，我的研究计划解决了理解多胺在细菌生存和抗生素耐药性中的重要性方面的重大知识差距。从这个项目中产生的发现将为未来对抗超级细菌的应用铺平道路，并将对医疗保健系统、农业和生物技术部门产生切实的好处。

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.