Bacterial GTPases - emerging new targets for antimicrobial drug design

Staphylococcus aureus is a major cause of morbidity and mortality worldwide. The burden caused by this opportunistic pathogen to global human health is compounded by the prevalence of antibiotic resistant strains, which the World Health Organisation predict will contribute to greater than 300 million premature deaths worldwide by 2050. There is, therefore, an urgent need to identify novel therapeutic targets. Ribosomes are responsible for one of the key growth processes in both eukaryotic and prokaryotic cells - the synthesis of proteins. As these molecular machines are so complex, the assembly process is quite tightly regulated. As such, correct maturation of the ribosome requires assembly cofactors. One class of assembly cofactors are the GTPases, which have a range of functions associated with ribosomal development. As assembly cofactors are multiple in number, are crucial for ribosome maturation, and are broadly conserved throughout the bacterial kingdom, they represent novel targets for broad-spectrum antibacterial drug discovery.
Our recent work has utilised a genome-wide screen to identify binding targets for a small nucleotide, ppGpp, in S. aureus. This has led to the discovery of five GTPases with predicted functions as ribosomal assembly cofactors. We show that the activities of all five enzymes are inhibited by ppGpp and initial characterisation confirms that at least one of these enzymes plays a role in ribosomal biogenesis, with a deletion of this gene negatively impacting bacterial growth. In order to determine the contribution of all five GTPases to bacterial survival and initiate the development of an antimicrobial that could potentially target all five simultaneously and so Iimit the development of resistant strains, we will be undertaking a multi-disciplinary approach which includes bacterial genetics, antimicrobial susceptibility testing, enzymatic assays, coupled with protein purification and structural determination of the apo and ppGpp-bound forms of the proteins.
Scientific objectives
1. Examination of the contribution of each GTPase to bacterial growth and ribosome assembly:
Gene deletion mutants, as well as GTPase-inactive protein variants will be created to allow the contribution of each enzyme to bacterial growth, cell morphology and ribosomal assembly in vivo to be examined.
2. Determination of the mechanism of ppGpp-mediated enzyme inhibition:
The GTPase enzymes will be purified and the structures analysed by X-ray crystallography in the presence and absence of ppGpp and GTP (supervised by secondary supervisor) in order to determine precisely how this molecule can inhibit GTPase activity. This will form the basis of future rational drug design based on the structure of ppGpp.
3. Screen to identify other GTPase inhibitors:
A library of 2,000 compounds, including FDA-approved drugs and a natural-products library, is readily available from the small molecule screening facility here at the University. This library will be screened in order to identify molecules that, like ppGpp, can inhibit the GTPase activity of these five proteins. Crystallography will also be performed on any positive interactions to determine the mode of inhibition.
Altogether this project will provide important mechanistic data on the importance of these GTPases for bacterial survival and will lay the foundation for future rational drug design.