Structural and mechanistic insights into antibiotic resistance in Staphylococcus aureus

Background: Antibiotic resistance (AR) undermines effective antibacterial chemotherapy and constitutes a major threat to global public health. A comprehensive response to this problem includes gaining a detailed understanding of the mechanisms by which AR is mediated. This project seeks to characterize the structural and molecular mechanism of several antibiotic resistance proteins found in the 'superbug', Staphylococcus aureus; resistance systems that are not currently well understood. In the first instance, we will focus on uncharacterized determinants capable of mediating resistance the aminoglycosides or mupirocin.

Objectives: To elucidate, with the aid of structural characterization, the molecular mechanism by which staphylococcal resistance proteins mediate resistance to clinically-important antibiotics acting on the bacterial translation machinery.

Novelty: Despite their adverse impact on the clinical use of antibiotics, no structural or mechanistic detail currently exists for the resistance proteins that will be studied here. Such information will be crucial to inform the development of strategies to overcome or circumvent these resistance mechanisms.

Timeliness: Antibiotic resistance is a major global concern at present, considered by the WHO to be one of the greatest threats to human health, and features prominently in the funding priorities of the research councils, including the BBSRC. The structural aspects of the study will employ cryo-EM, and will therefore make use of the state-of-the art facilities recently established at Leeds.

Experimental approach: For both initial targets of interest we already have access to purified, soluble protein. For the larger of the two, crystallography has already proven unsuccessful, and we will therefore use cryo-EM for structural characterization. For the smaller protein, we have in preliminary studies been able to generate crystals that require optimization - hence, the initial effort in this case will focus on obtaining an X-ray structure. The structural insights gained in these studies will inform downstream mutagenesis and biochemical experiments to interrogate function.