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

耐甲氧西林金黄色葡萄球菌(MRSA)正在世界范围内造成重大的卫生保健问题，并且越来越难以用目前的抗生素进行治疗。金黄色葡萄球菌是人类和家畜最成功的病原体之一，也是皮肤感染和呼吸道疾病的常见原因，这些疾病可能危及生命。这强调了开发防治感染的替代治疗方法的重要性。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侮辱，如宿主温度的变化和免疫系统的攻击。这不仅使它能够在恶劣的条件下生存，而且还能在宿主体内生存。为了实现如此快速的适应，金黄色葡萄球菌在施加压力后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(RBPs)，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角色。虽然已经在金黄色葡萄球菌中发现了几种限制性商业惯例，并证明它们对致病性具有重要作用，但大多数限制性商业惯例的功能尚不清楚。尽管限制性商业惯例被认为在调节宿主适应性反应过程中的基因表达方面发挥了重要作用，但目前尚不清楚哪些限制性商业惯例是这一过程中的关键角色，以及它们如何控制快速的基因表达重塑。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金黄色葡萄球菌限制性商业惯例的RNA结合域，包括许多代谢酶(MetRBPs)。我们目前的工作模式是金黄色葡萄球菌metRBPs作为新的转录后调节因子发挥作用，直接改变基因表达，以响应营养可获得性的变化。为了检验这一假设，学生将有机会学习一系列广泛的技术，以从功能上表征几种与抗生素耐药性和/或宿主生存有关的新型代谢限制性商业惯例。本项目的目标是了解(1)RNA结合如何有助于metRBPs的活性。(2)这些metRBPs在体内结合了什么RNA(van Nue等人，2017)。(3)这种RNA结合活性对于宿主环境的生存是多么重要。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)中生产重组蛋白质，使用EMSA和相关的生化方法测试与RNA的相互作用，结晶与RNA结合的蛋白质，使用CRISPR对金黄色葡萄球菌进行遗传操作，在蛋白质-RNA相互作用的计算机模拟中，使用Python和R编程以及交联和免疫沉淀分析(CRAC；van Nues等人，2017年)。因此，该项目为希望发展这些领域技能的学生提供了一个极好的学习环境。我们的愿景是，对参与转录后调控系统的金黄色葡萄球菌限制性商业惯例的详细特征可能会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.