Power naps: ribosome hibernation in bacterial drug targeting and stress tolerance

当细菌受到药物攻击时，它们会激活各种防御机制来维持生命并减轻药物毒性。这些防御策略使病原菌得以逃脱临床治疗，使耐药感染成为世界各地的主要死亡原因。在这个项目中，我们将通过描述一种以前未知的耐药性机制来帮助解决正在出现的抗菌素耐药性问题，这种机制被称为“巴隆诱导的核糖体冬眠”。简而言之，我们的初步研究已经确定了一种新的细菌蛋白质--Balon，当细菌接受药物治疗时，它会导致细胞“昏迷”：在药物暴露期间，Balon与大多数细胞核糖体(细菌生长所需的蛋白质合成机器)结合，并将它们转化为休眠状态，导致细胞生长停止。当药物从介质中移除时，Balon通过迫使细菌细胞的核糖体重新参与蛋白质合成来“唤醒”细菌细胞。此外，我们的初步研究表明，Balon不仅由许多细菌基因组编码，而且在150多株临床和兽医病原菌中也具有多重耐药性的质粒，包括肠沙门氏菌、铜绿假单胞菌、肺炎克雷伯菌和大肠杆菌。在本研究中，成功的学生将探索Balon诱导耐药性的分子机制。目的1|确定Balon与细菌核糖体结合的冷冻-EM结构首先，利用我们在冷冻-EM显微镜方面的专业知识(Advisor 1)，我们将确定与细菌核糖体结合的Balon的结构。这种结构将揭示Balon保护核糖体免受抗微生物药物(特别是来自氨基糖苷类和大环内酯类临床药物)的分子机制。目的2|确定编码Balon的基因在细菌及其质粒中的出现接下来，利用我们在研究基因组和质粒进化方面的成熟专业知识(Advisor 2)，我们将揭示Balon编码基因在所有细菌和已确定基因组序列的细菌质粒中的分布。这一分析将有助于更好地了解这种耐药因素是如何在细菌中传播的，包括人类和家养动物的病原体。目的3|确定在细菌细胞中刺激Balon合成的生理条件最后，利用我们在转录研究方面的专业知识(Advisor 3)，学生将把Balon编码基因的启动子克隆到DNA报告器中。这些记者将允许我们测试恶劣环境(如抗生素和大量压力条件)如何调节Balon的表达。这一分析将帮助我们检验我们的假设，即某些环境使细菌容易产生更高或更低水平的耐药性。

When bacteria are assaulted with drugs, they activate various defence mechanisms to stay alive and mitigate drug toxicity. These defence strategies allow pathogenic bacteria to escape clinical treatment, making drug-resistant infections a leading cause of death around the world. In this project, we will help address the emerging problem of antimicrobial resistance by describing a previously unknown mechanism of resistance known as "Balon-induced ribosome hibernation". In brief, our preliminary studies have identified a new bacterial protein, Balon, that induces a cellular "coma" when bacteria are treated with drugs: during a drug exposure, Balon binds most cellular ribosomes (machines of protein synthesis required for bacterial growth) and converts them into a dormant state, causing arrest of cell growth. When drugs are removed from the media, Balon "awakens" bacterial cells by forcing their ribosomes to reengage in protein synthesis. What's more, our preliminary study shows that Balon is encoded not only by many bacterial genomes but also by plasmids that confer multidrug-resistance in more than 150 clinical and veterinary isolates of pathogenic bacteria, including Salmonella enterica, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli.In this study, the successful student will explore the molecular mechanism of Balon-induced drug resistance. Aim 1 | Determine the cryo-EM structure of Balon bound to bacterial ribosomesFirst, using our established expertise in cryo-EM microscopy (Advisor 1), we will determine the structure of Balon bound to bacterial ribosomes. This structure will reveal the molecular mechanism by which Balon protects ribosomes from antimicrobial drugs (specifically from clinical drugs from aminoglycoside and macrolide families).Aim 2 | Determine the occurrence of Balon-coding genes in bacteria and their plasmidsNext, using our established expertise in studying the evolution of genomes and plasmids (Advisor 2), we will reveal the distribution of Balon-coding genes across all bacteria and bacterial plasmids with determined genome sequences. This analysis will help to better understand how this drug-resistance factor propagates among bacteria, including pathogens of humans and domesticated animals. Aim 3 | Determine the physiological conditions that stimulate Balon synthesis in bacterial cellsFinally, using our established expertise in transcriptional studies (Advisor 3), the student will clone promoters of Balon-coding genes into DNA reporters. These reporters will allow us test how hostile environments (such as antibiotics and numerous stress conditions) regulate the expression of Balon. This analysis will help us to test our hypothesis that certain environments predispose bacteria to higher or lower levels of drug resistance.