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

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.