Bacterial lipocalins: Novel role in bacterial protection against antibiotic-induced membrane lipid peroxidation

Research on antibiotic action and resistance has helped elucidate fundamental biochemical and regulatory pathways in bacteria and fungi operating within the microbial cells. However, whether bacteria produce molecules that interfere with antibiotics before they reach cells has been overlooked. We discovered a previously unrecognized mode of bacterial antibiotic resistance operating in the extracellular space that depends on molecules produced and released by bacteria in response to sublethal antibiotic concentrations. This adaptive mechanism protects less resistant bacterial cells of the same or different species from killing by antibiotics. The molecules involved are putrescine, a polyamine, and secreted lipocalin proteins, which are highly conserved in bacteria. We also discovered that lipocalins are part of a previously ignored mechanism to protect cell membranes from lipid peroxidation. Peroxidation occurs as a consequence of environmental stress including bacterial exposure to near-lethal antibiotic concentrations, cold, and inflammation. Lipid peroxidation ends up with the destruction of membrane lipids and their conversion into highly toxic forms which in turn can oxidise and destroy cellular proteins and the genetic material. This proposal is on fundamental microbiology research combining molecular microbiology, biochemistry, and infection models to elucidate the role of a conserved family of bacterial lipocalins in bacterial cell physiology and antibiotic resistance. Our long-term goals are to establish the function of lipocalins by elucidating their mechanism and physiological roles in bacterial cells, and to utilise this information to gain proof of principle that new molecules can be designed to help prevent extracellular bacterial scavenging of antibiotics, and more importantly accelerate peroxidation leading to bacterial death, thus providing a potentially new solution to combat antibiotic resistance.

This research programme will decode the function(s) and role(s) of a conserved family of bacterial lipocalins(LPs). It builds on our seminal discovery that LPs afford nonspecific antibiotic resistance by a previously unrecognised extracellular scavenging mechanism. LPs are ubiquitous in bacteria, but their physiological role is unknown. New preliminary data point out LPs as components of an uncharacterized system to protect membrane lipids from peroxidation, a common bacterial response to antibiotic-mediated stress. This proposal outlines a innovative, interdisciplinary, and ambitious research programme to elucidate the function of LPs, more specifically their role in imparting protection against lipid peroxidation upon antibiotic mediated stress. We will use relevant opportunistic, multidrug resistant bacterial pathogens to establish the generality of LPs function across different bacteria.

The following specific aims will be carried out:

1.To determine that antibiotic binding is a conserved property of LPs from different multidrug resistant pathogens (KAPE pathogens) and establish its mechanism by: 1.1 investigating antibiotic binding of recombinant LPs from KAPE pathogens; 1.2 Examining the contribution of LPs to antibiotic resistance of KAPE pathogens; 1.3 analysing structure-function of LPs and defining critical amino acid residues making contact with antibiotic molecules.


2.To investigate the function of LPs in the bacterial defense against lipid peroxidation by: 2.1 determining the structure-function of BcnA and its secretion state in protection against lipid peroxidation; 2.2. determining additional components to BcnA and BcoA required to protect bacteria from lipid peroxidation upon exposure to antibiotic stress and in vivo infection; 2.3 studying global effects of BcnA and related proteins in bacterial physiology by comparative transcriptomics on mutants vs. the parental strain pairs both exposed to sublethal concentrations of antibiotics.

This fundamental research proposal will result in novel and significant contributions to UK science and UK knowledge economy, which are outlined as follows:
1) The main path to impact therefore will be through publication of the research in peer-reviewed, open access journals. Publications will be complemented by the presentation of data at national and international scientific conferences, as well as a various fora involving dissemination of new strategies to curtail antimicrobial resistance such as for example the NI-AMR consortium, which gathers multidisciplinary scientits, public health, government and business representatives.

2) Our proposal provides fundamental research that has relevance to the pharmaceutical industry (biopharmaceuticals, antibiotic resistance). The common theme in terms of public benefit arising from this research is the potential to lead to life- enhancing applications when antibiotic resistance becomes more widespread. The problem of antibiotic resistance is even more critical in the case of infections by opportunistic pathogens. The PI is engaged with the UK Cystic Fibrosis Trust and will work with the Trust to disseminate research findings widely to cross-disciplinary audiences beyond the academic setting (for example CF patients/parents groups). Also, both PI and CoI are actively involved in AMR-NI. A tink tank organization in nothern ireland briging together diverse academic fields and commercial interests to find solutions to combat antimicrobial resistance. These activities will help us to translate findings from this research into novel therapeutics singly or integrated with other strategies.

3) Our proposal will apply the outcomes from this research to expand the search for lipocalin antibiotic binding inhibitors for the improvement of inhibitory compounds that can act as antibiotic adjuvants. For example, a strong collaboration with the biotechnology sector is already in place with Biomar Microbial Technologies in Leon, Spain. We will also work with our university's Research & Enteprise Office to obtain financial support and IP protection to exploit promising inhibitory compounds (e.g. Vitamin E derivatives) for future commercial development.

4) This project will contribute to the career development of a PDRA and a Research Technician appointed to the project by ensuring additional training and experience. Similar skills training will be made available to other participants in PhD, MSc and undergraduate positions.

5) The relevance of this research to the biopharmaceutical and biotechnology sectors will benefit public engagement to inform and educate the public about opportunities arising from a knowledge-based economy and to educate about science in general. We intend to take full advantage of available opportunities to do so through Queen's outreach activities and the media. Furthermore, we will use available opportunities to disseminate information about published research success to the general media through the Press and Publicity Units at Queen's.

6) The extenisve use of the Galleria larvae infection model, as proposed in this grant contributes to the 3Rs principles on ethical use of animals, by performing experiments that will result in saving more than 2000 mice in studies where previously use of mice was the only possible alternative.