Award details

Understanding antibiotic entry into bacteria

Reference	BB/R004048/1
Principal Investigator / Supervisor	Professor Alex O'Neill
Co-Investigators / Co-Supervisors	Professor Colin Fishwick, Dr Stuart Warriner
Institution	University of Leeds
Department	Sch of Molecular & Cellular Biology
Funding type	Research
Value (£)	431,865
Status	Completed
Type	Research Grant
Start date	02/01/2018
End date	31/10/2021
Duration	46 months

Abstract

Most antibiotic classes employed in the chemotherapy of bacterial infection act on intracellular targets, and consequently must traverse the membrane(s) of bacteria as a prelude to exerting their inhibitory effects. The process of antibiotic entry into bacteria is not understood, and the properties of small molecules that enable this to occur have not been defined. Not only does this constitute a considerable gap in our fundamental understanding, it presents a major barrier to productive antibacterial drug discovery, and in turn, to our ability to combat the escalating problem of antibiotic resistance. Building on promising preliminary studies, this project seeks to address this knowledge gap through two complementary investigations. The first of these aims to define the physico-chemical properties of small molecules required for bacterial entry. Using a novel, validated liquid chromatography-mass spectrometry (LC-MS) approach to measuring compound accumulation in bacteria, we will screen a large and diverse library of small molecules to identify those capable of entering bacteria. The goal of this work is to formulate a set of 'rules of entry' to inform the future rational design of antibacterial drugs. The second will explore the hypothesis that entry of antibiotics into bacteria does not primarily occur by passive diffusion through the lipid bilayer(s) as is generally thought, but instead predominantly involves uptake via carrier (transport) proteins. Preliminary evidence to support his idea has been obtained - in the form of bacterial strains exhibiting reduced antibiotic susceptibility as a consequence of lacking particular transporters - and this project will employ a suite of approaches to confirm and expand upon this observation. Confirmation that antibiotic entry into bacteria relies on carriers would underpin a strategy for making new antibiotics by designing compounds that have regions of similarity to natural molecules that bacteria import.

Summary

Antibiotics have made possible the treatment and cure of life-threatening bacterial infections. Unfortunately, the rise of antibiotic-resistant "superbugs" is dramatically undermining the effectiveness of these important drugs, a problem that represents one of the current greatest threats to global public health. A crucial aspect of addressing the problem will involve the discovery and development of novel antibiotics active against bacteria that have become resistant to our existing antibiotics. However, a major scientific challenge stands in the way of achieving this aim. Although it is now feasible to identify or make new chemical compounds that interfere with the correct functioning of the biological machinery of bacteria (a requisite property of any potential antibiotic), these compounds are in the main unable to cross the membrane(s) that surround bacteria to reach that machinery. If we knew how to modify these compounds to effectively deliver them into bacteria, they would have potential as new antibiotics. This proposal seeks to provide the fundamental scientific knowledge to make such an approach possible. By understanding how small molecules like antibiotics enter bacteria - a phenomenon about which we know very little at present - we will gain the necessary strategic intelligence to allow us to rationally generate new antibiotics. The project will take two complementary approaches to achieve this. The first of these will employ a technique known as liquid chromatography-mass spectrometry (LC-MS) to directly measure the ability of a large and diverse collection of small molecules to penetrate into bacteria. From the results of this study, we will be able to determine what physical and chemical properties a small molecule must have to allow it to enter bacteria, allowing us to formulate a set of rules that can be applied to guide the generation of new antibiotics. The second approach will investigate the mechanism or process by which established antibiotics actually cross the membrane(s) of bacteria. The current view is that, since membranes are fluid in nature, most such compounds simply drift or diffuse across. However, we have evidence to suggest that in fact most (perhaps all) antibiotics take advantage of protein 'pumps' in the membrane(s) that bacteria ordinarily use to import things such as nutrients from the environment. By examining what impact artificially altering the number or levels of these pumps in bacteria has on the ability of antibiotics to enter, we aim to more clearly demonstrate the role of these pumps in antibiotic entry. The implications of the idea that antibiotics enter bacteria via such pumps are considerable. For example, it would mean that for antibiotics to get into bacteria they need to have similar physical or chemical features to 'desirable' compounds that bacteria import from the environment; features that allow them to 'hijack' the corresponding pumps. Furthermore, it would offer a strategy to make new antibiotics by designing compounds that have regions of similarity to natural compounds that bacteria import.

Impact Summary

Antibiotic resistance dramatically undermines our ability to effectively treat bacterial infection, and is considered one of the greatest threats currently facing human health. The proposed study will contribute to the necessary re-growth of UK expertise and capacity in what has become an under-represented area of research, yet one that is of undeniable importance - fundamental science capable of directly underpinning antibacterial drug discovery efforts. The appointed PDRA will receive multidisciplinary, cutting-edge training in three established laboratories possessing distinct expertise, which will ultimately deliver a highly skilled researcher with an excellent knowledge of antibiotic entry into bacteria and a breadth of experience that encompasses molecular microbiology and LC-MS analysis, and with strong onward employment prospects in either academia or industry. In addition, the proposed project will further foster an existing collaboration capable of bringing world-leading expertise and facilities to bear on novel approaches to help address the escalating problem of antibiotic resistance. Results generated within the lifetime of the study will both enhance the scientific knowledgebase and make a major contribution to efforts to design and/or optimise novel antibacterial drug candidates. Robust confirmation that antibiotic entry is predominantly carrier-mediated (Objective II) would not only offer the field a fundamental understanding of how antibiotics enter cells, but would also prompt an immediate step-change in the approach (and likely effectiveness) of antibacterial drug discovery by providing the key insight that chemical similarity to cellular metabolites that bacteria ordinarily import is key to delivering inhibitors into cells. This understanding would be substantially augmented via delineation of the properties of small molecules capable of entering bacteria (Objective I), thereby yielding a tangible set of 'rules of entry' that would constitute vital strategic intelligence for current and future antibacterial drug discovery programmes in both academia and industry. Thus, in the long term this project has the potential to deliver impact to the economy (through pharmaceutical companies developing improved antibacterial therapeutics), to healthcare services and professionals (through availability of improved treatment options) and to patients (through improved treatments and outcomes).

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Microbiology, Pharmaceuticals
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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