The interplay between cell envelope protein homeostasis and antibiotic resistance in Gram-negative bacteria

项目摘要/摘要 革兰氏阴性细菌是唯一能够击败抗生素的细菌。它们的最外层，即细胞包膜，是 一种天然的渗透性屏障，含有一系列能够中和大多数现有的 抗菌剂。因此，它的存在对耐药感染的治疗造成了重大障碍。 以及新抗生素的开发。 细胞膜也是许多保护其蛋白质组完整性的保守途径的家园。 尽管这些系统在维持蛋白质动态平衡方面起着核心作用，但它们与抗性的相互作用 定位在细胞膜中的蛋白质还没有被检测到。我们假设细胞包膜的活性 折叠催化剂可能对抗性决定因素的功能很重要，我们在 一个关键的蛋白质平衡参与者，二硫键形成系统。我们发现氧化蛋白质折叠 这一途径的活性对于某些流行病学和临床上最相关的病毒的功能是必不可少的 挑战抗性蛋白，即β-内酰胺酶、粘菌素抗性酶和外排泵。引导式 根据从模拟实验室菌株和临床分离株获得的强大初步数据，我们提出了一种新的方法。 深入研究细胞膜蛋白平衡系统在抗生素耐药性中的作用。我们将使用 细菌遗传学、微生物学、生物化学、蛋白质组学、实验进化和人类 疾病建模以追求三个特定的目标：1)识别依赖氧化的抵抗组成分 蛋白质折叠，通过评估对数百个临床重要的二硫键形成的要求 抗性蛋白。2)评估氧化蛋白折叠对耐药感染的影响，通过测试我们的 临床分离株和相关的小鼠慢性感染模型中的生化结果。3)探索 其他细胞膜折叠催化剂在抗生素耐药中的作用 病原菌的菌株，并在模拟实验室菌株中验证我们的结果。 我们预计，我们的整体方法，跨越多种阻力决定因素和折叠催化剂，将打破 在我们理解细胞包膜蛋白平衡在抗性中的作用方面有了新的基础。这一知识将是 适用于许多高度优先的革兰氏阴性病原体，从长远来看，可能会激发出新的广泛作用 克服抗生素耐药性的策略。

PROJECT SUMMARY / ABSTRACT Gram-negative bacteria are uniquely equipped to defeat antibiotics. Their outermost layer, the cell envelope, is a natural permeability barrier that contains an array of resistance proteins capable of neutralizing most existing antimicrobials. As a result, its presence creates a major obstacle both for the treatment of resistant infections and the development of new antibiotics. The cell envelope is also home to numerous conserved pathways that safeguard the integrity of its proteome. Despite the central role of these systems in maintaining protein homeostasis, their interaction with resistance proteins localizing in the cell envelope has not been examined. We hypothesized that the activity of cell envelope folding catalysts may be important for the function of resistance determinants, and we tested this hypothesis on a key proteostasis player, the disulfide bond formation system. We discovered that the oxidative-protein-folding activity of this pathway is essential for the function of some of the most epidemiologically relevant and clinically challenging resistance proteins, namely β-lactamases, colistin resistance enzymes, and efflux pumps. Guided by strong preliminary data obtained from model laboratory strains and from clinical isolates, we propose an in- depth investigation of the role of cell envelope proteostasis systems in antibiotic resistance. We will use a combination of bacterial genetics, microbiology, biochemistry, proteomics, experimental evolution, and human disease modeling to pursue three specific aims: 1) Identify the components of the resistome that rely on oxidative protein folding, by assessing the requirement for disulfide bond formation on hundreds of clinically important resistance proteins. 2) Evaluate the impact of oxidative protein folding on resistant infections, by testing our biochemical findings in clinical isolates and in a relevant murine chronic infection model. 3) Explore the role of other cell envelope folding catalysts in antibiotic resistance, by probing their function in multidrug-resistant clinical strains of pathogenic bacteria and validating our results in model laboratory strains. We expect that our holistic approach, spanning multiple resistance determinants and folding catalysts, will break new ground in our understanding of the role of cell envelope proteostasis in resistance. This knowledge will be applicable to many high-priority Gram-negative pathogens and, in the long term, may inspire novel broad-acting strategies for overcoming antibiotic resistance.