Understanding the role of RNA in regulating metabolic enzymes in multi-drug resistant Staphylococcus aureus

耐甲氧西林表皮葡萄金黄色葡萄球菌（MRSA）正在世界范围内引起重大的卫生保健问题，并且越来越难以用现有的抗生素治疗。S.金黄色葡萄球菌是最成功的人类和牲畜病原体之一，并且是可能危及生命的皮肤感染和呼吸道疾病的常见原因。这强调了开发替代治疗方法以对抗感染的重要性。S.金黄色葡萄球菌是这样一种有效的病原体，因为它可以非常迅速地适应环境损害，例如宿主温度的改变和免疫系统的攻击。这使得它不仅能够在恶劣的条件下生存，而且还能在宿主体内存活。为了实现这种快速适应，S。金黄色葡萄球菌在施加压力的几分钟内重塑其转录组。转录因子被认为主要负责指导这一过程，然而，转录后调节因子，如RNA结合蛋白（RBP），现在被认为是通过调节mRNA翻译和/或降解速率来控制适应性反应的关键参与者。虽然在S.金黄色葡萄球菌和被证明是重要的致病性，大多数有未知的功能。这强调了需要彻底表征这些molecules.The项目：虽然RBP被认为是发挥了重要作用，在调节宿主适应性反应过程中的基因表达，目前还不清楚哪些RBP是在这个过程中的关键球员，以及他们如何控制快速基因表达重塑。使用新的蛋白质组学方法（Asencio等人，2018; Urdaneta等人，2018），我们最近发现了大量新的S.金黄色葡萄球菌RBP，包括许多代谢酶（metRBP）。我们目前的工作模型是S。金黄色葡萄球菌metRBP作为新的转录后调节因子发挥作用，其直接改变基因表达以响应营养可用性的变化。为了验证这一假设，学生将有机会学习广泛的技术来功能性地表征与抗生素耐药性和/或宿主生存有关的几种新型代谢RBP。本项目的目标是了解（1）RNA结合如何有助于metRBP的活性。(2)这些metRBP在体内结合什么RNA（货车Nues等，2017年）。(3)这种RNA结合活性对宿主环境的生存有多重要。学生将使用的技术包括（但不限于）：在我们的蛋白质生产设施（EPPF）中生产重组蛋白，使用EMSA和相关生化方法测试与RNA的相互作用，使蛋白质与RNA结合，S.使用CRISPR、蛋白质-RNA相互作用的计算机模拟、使用Python和R编程以及交联和免疫沉淀测定（CRAC;货车Nues et al.，2017年）。因此，该项目为希望在这些领域发展技能的学生提供了一个梦幻般的学习环境。参与转录后调节系统的金黄色葡萄球菌RBP可能揭示改善感染治疗的新途径和/或揭示抗微生物药物开发的新靶点。

Methicillin-resistant S. aureus (MRSA) is causing major health-care problems world-wide and is becoming increasingly difficult to treat with current antibiotics. S. aureus is one of the most successful human and livestock pathogens and a common cause of skin infections and respiratory diseases that can be life threatening. This emphasizes the importance of developing alternative therapeutic approaches to combat infections. S. aureus is such an effective pathogen because it can very rapidly adapt to environmental insults, such as alteration in host temperature and attack by the immune system. This enables it not only to survive in hostile conditions, but also to persist within the host. To achieve such rapid adaptation, S. aureus remodels its transcriptome within minutes of stress imposition. Transcription factors were thought to be mainly responsible for directing this process, however, post-transcriptional regulators, such as RNA-binding proteins (RBPs), are now recognized as key players in controlling adaptive responses by modulating mRNA translation and/or degradation rates. Although several RBPs have been discovered in S. aureus and shown to be important for pathogenicity, the majority have unknown functions. This underscores the need for a thorough characterization of these molecules.The project:Although RBPs are believed to play a fundamental role in regulating gene expression during host adaptive responses, it remains unclear which RBPs are the key players in this process and how they control rapid gene expression remodelling. Using novel proteomic approaches (Asencio et al., 2018; Urdaneta et al., 2018), we have recently uncovered RNA-binding domains for a large number of novel S. aureus RBPs, including many metabolic enzymes (metRBPs). Our current working model is that S. aureus metRBPs function as novel post-transcriptional regulators that directly alter gene expression in response to changes in nutrient availability. To test this hypothesis, the student will be given the opportunity to learn a wide array of techniques to functionally characterize several novel metabolic RBPs that are linked to antibiotic resistance and/or host survival.The goals of this project are to understand (1) how RNA-binding contributes to the activity of the metRBPs.(2) what RNAs these metRBPs bind in vivo (van Nues et al., 2017).(3) how important this RNA-binding activity is for surviving the host environment. Techniques that the student will be using include (but are not limited to): production of recombinant proteins in our Protein Production Facility (EPPF), testing interaction with RNA using EMSA and related biochemical approaches, crystallizing proteins bound to RNA, genetic manipulation of S. aureus using CRISPR, in silico modelling of protein-RNA interactions, programming using Python and R and cross-linking and immunoprecipitation assays (CRAC; van Nues et al., 2017). Thus, the project provides a fantastic learning environment for students that wish to develop skills in these areas.Our vision is that a detailed characterization of S. aureus RBPs involved in post-transcriptional regulatory systems may uncover new avenues for improving treatment of infections and/or reveal new targets for antimicrobial drug development.