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

项目概要/摘要 革兰氏阴性菌具有独特的能力来击败抗生素。它们的最外层，细胞被膜， 一种天然的渗透屏障，含有一系列抵抗蛋白，能够中和大多数现有的 抗菌剂因此，它的存在造成了一个主要障碍， 以及新抗生素的开发。 细胞被膜也是许多保护其蛋白质组完整性的保守途径的所在地。 尽管这些系统在维持蛋白质稳态中起着核心作用，但它们与抗性的相互作用 定位于细胞包膜中的蛋白质尚未被研究。我们假设细胞膜的活性 折叠催化剂可能对抗性决定子的功能很重要，我们在 二硫键形成系统是蛋白质稳定的关键。我们发现氧化蛋白折叠 该途径的活性对于一些流行病学上最相关和临床上最重要的功能是必不可少的。 挑战性耐药蛋白，即β-内酰胺酶、粘菌素耐药酶和外排泵。指导 通过从模型实验室菌株和临床分离株中获得的强有力的初步数据，我们提出了一种 深入研究细胞包膜蛋白质稳态系统在抗生素耐药性中的作用。我们将使用一个 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 疾病建模追求三个具体目标：1）确定依赖于氧化的抵抗体的组分 蛋白质折叠，通过评估数百个临床重要的 抗性蛋白2)通过测试我们的研究，评估氧化蛋白折叠对耐药感染的影响。 临床分离株和相关鼠慢性感染模型中的生化发现。3)探索的作用 其他细胞膜折叠催化剂在抗生素耐药中的作用，通过探讨它们在多药耐药临床中的作用， 病原菌菌株并在模型实验室菌株中验证我们的结果。 我们预计，我们的整体方法，跨越多种抗性决定因素和折叠催化剂，将打破 新的地面在我们的理解中的作用，细胞包膜蛋白质稳态的阻力。这些知识将 适用于许多高优先级的革兰氏阴性病原体，从长远来看，可能会激发新的广泛作用 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.