Molecular dissection of a novel protein-protein interaction: structure and mechanism of the staphylococcal fusidic acid-resistance protein FusB

Resistance to antibiotics among bacteria is an ever-growing problem, since it interferes with our ability to successfully treat bacterial infections. In order to preserve the activity of existing antibiotics, and to guide the development of new antibiotics which are less resistance-prone, it will be essential to understand in detail the mechanisms by which bacteria develop such resistance. Fusidic acid is an important antibiotic for the treatment of bacterial infections caused by the 'superbug', Staphylococcus aureus. The drug works by interfering with the function of a protein known as elongation factor-G (EF-G) that helps the bacterium make proteins. However, an increasing number of strains of S. aureus are becoming resistant to this drug because they produce a protein called FusB that binds to EF-G and protects it from the antibiotic. Recent work in our laboratory suggests that this FusB resistance mechanism is highly unusual, and unlike most other antibiotic resistance mechanisms characterized to date. Furthermore, it appears that the FusB protein not only functions to protect the cell from fusidic acid, but also performs an accessory role in the cellular manufacture of proteins. Consequently, it is of significant fundamental scientific interest. The mechanism by which FusB performs its native role and causes resistance to fusidic acid remain poorly understood. To better understand how FusB achieves this, we aim to solve the 3-D structure of the FusB protein, alone and when bound to the EF-G protein, using a technique known as Nuclear Magnetic Resonance (NMR) spectroscopy. In conjunction with experiments to examine in detail how these proteins bind together, these studies will provide significant insights into FusB and the mechanism by which it carries out its biological role(s). This information may allow us to devise approaches for blocking the activity of FusB in bacterial cells, and overcoming resistance to fusidic acid in S. aureus.

Fusidic acid (FA) is an important antibiotic for the treatment of bacterial infections caused by the human pathogen, Staphylococcus aureus, and other staphylococci. This agent blocks protein synthesis by interfering with the function of the ribosomal translocase, elongation factor G (EF-G). Unfortunately, the clinical efficacy of FA is being eroded by the growing prevalence of strains that carry the fusB determinant. Our current state of knowledge regarding FusB indicates that it represents a novel paradigm in antibiotic resistance, involving interaction with, and direct protection of, the drug target (EF-G). Furthermore, several lines of evidence suggest that FusB has not evolved as a FA-resistance protein, but actually represents a previously uncharacterized accessory factor active in prokaryotic protein translation. Consequently, it is of significant fundamental scientific interest. The mechanism by which FusB mediates its native role and resistance to FA remain poorly understood. Structural information will be essential to elucidate the mechanism of action of this resistance protein, but unfortunately neither FusB nor the FusB:EF-G complex have proven amenable to X-ray crystallography. In the current proposal, we intend to employ Nuclear Magnetic Resonance (NMR) spectroscopy to determine solution structures of FusB, and of a fragment of the EF-G protein with which it interacts, alone and in complex. In conjunction with experiments to precisely map key points of interaction between the two proteins, and to investigate the thermodynamics of the interaction, these studies will provide significant insights into FusB and its mechanism of action.

The proposed programme of work will have numerous positive impacts: (i) An expanded knowledge of antibiotic resistance - Our ability to treat bacterial infections is diminishing as bacteria become resistant to existing antibiotics, precipitating a global public-health crisis. This is particularly the case for infections caused by hospital pathogens such as Staphylococcus aureus. A thorough understanding of the molecular basis for resistance to antibiotic classes currently in clinical use will be crucial for preserving or rejuvenating the activity of existing antibacterial agents, and for informing the development of novel antibacterials less prone to resistance. Our results will be important to those in both academia and industry interested in antibiotic resistance mechanisms or the development of novel antibacterials. (ii) A detailed understanding of fusidic acid (FA) resistance in staphylococci - FA is an important antibiotic primarily employed for treating infections caused by S. aureus, and represents one of the few remaining oral agents effective against infections caused by methicillin-resistant S. aureus (MRSA). The utility of this antibiotic is being eroded by the growing prevalence of FA resistance, which is almost exclusively mediated by FusB and its homologues, and this escalating resistance intensifies the need to understand how FusB-type resistance to FA is mediated. This is particularly the case since FusB represents a completely novel paradigm in antibiotic resistance. In addition to its clinical importance and mechanistic novelty, FusB and homologues appear to play a house-keeping role in protein translation in diverse bacterial genera. Consequently, FusB is of considerable scientific interest. FA is now off-patent, and is currently in Phase III clinical trials in the US (where it has not previously been deployed). Consequently, FA will almost certainly enjoy a higher profile in the coming years, and there has already been substantially increased interest in the mechanisms of resistance to FA from US researchers at recent scientific conferences. Since the identification and characterization of FA resistance mechanisms has to date been driven almost entirely by the UK scientific community (predominantly by AO and collaborators), this development should serve to foster collaborations with research scientists in the US who wish to avail themselves of our established expertise in the area. Furthermore, it offers the potential to attract international research income, and AO is currently in the process of forging collaborative links with Cempra (who are bringing FA to market in the US) with a view to doing just that. We envisage that structural information arising from this project will subsequently enable the development of strategies to overcome FA resistance in clinical strains, thereby rejuvenating the activity of this useful antibiotic. Such strategies could include the development of FA analogues capable of circumventing FusB-type resistance, or of small molecule inhibitors to block the FusB:EF-G interaction, and could make an important contribution both to health and economic competitiveness of the UK. (iii) Novel insights into protein-protein interactions (PPI) - FusB-mediated resistance involves a PPI between FusB and EF-G which exhibits unusual thermodynamic properties that contrast with most PPIs characterized to date. Consequently, structural and thermodynamic characterization of the FusB:EF-G complex will almost certainly provide novel insights into PPIs and contribute to the knowledge base. This is important both from a fundamental scientific perspective and since PPIs are receiving considerable attention as promising targets for chemotherapeutic intervention. Very few protein complexes of the size of FusB:EF-G have been successfully studied by NMR, or characterized with respect to their thermodynamics, and the outcome of our work will thus have significant impact on this basis alone.