Novel antimicrobial agents for bacterial pathogens of livestock: light-activated CO-releasing molecules

Antimicrobial resistance is a growing problem in the UK and worldwide. We urgently need ways to counteract the effects of the antimicrobial resistance of bacteria in human and animal health. One promising approach is through novel alternatives or additions to antibiotics, so that antibiotics are used less, resistance is countered, and costs are lowered.

Carbon monoxide (CO) is a colourless and odourless gas, and an infamous respiratory poison, notorious as the 'silent killer' originating from domestic gas appliances, motor car exhausts and various industrial processes. However, it is also known to be a vital small signaling molecule (or 'gasotransmitter') in microbes, animals and plants. We still know relatively little about how CO gas exerts its many important effects in inflammation, the cardiovascular system and elsewhere. One reason for this is that it is difficult to deliver and manipulate CO doses in biological systems. When CO is applied by inhalation, as is currently being explored in medicine, the final distribution of the gas is difficult to tune, while application to localised sites, e.g. of microbial infection, is virtually impossible. However, because, in small doses, CO has beneficial and essential roles in biology, researchers have started in the last 10 years to evaluate CO-releasing molecules (CORMs) as easy-to-handle, safer and more selective ways to administer CO. Remarkably, these compounds can kill bacteria and the way they work appears entirely different from currently prescribed antibiotics.

This proposal focuses on the antimicrobial applications of CORMs in which CO release is triggered on demand by light (PhotoCORMs) so that we can deliver the gas selectively to sites of microbial infection and at chosen times. We aim to understand the antibacterial effects of CORMS in general, and PhotoCORMs in particular, in comparison with more established antimicrobial compounds.

In this project we will:
a) Develop and synthesise new PhotoCORMs with better biological properties and potential for use as alternatives to antibiotics, or supplements to antibiotics, against bacterial infections. In collaboration with chemists, we will design and make improved molecules in which CO release can be adjusted for particular needs. We will also improve the specific targeting of cells and tissues, again by molecular design;
b) Test these compounds for their ability to inhibit the growth or kill bacteria that infect poultry (avian pathogenic E. coli, APEC) by entering the cells and damaging important sites and enzymes;
c) Understand how PhotoCORMs get into bacterial cells and whether bacteria take up these compounds more readily than do animal cells;
d) Unravel the complex responses that bacteria make when challenged with these compounds and identify perhaps resistance strategies that the bacteria may mount and that could influence future use of PhotoCORMs as antimicrobial agents;
e) Exploit this basic knowledge to study how and if these compounds can kill bacteria in in the laboratory, in invertebrate models of pathogenesis in chickens;

It is intended that these studies will lead to the future application of CORMs to aid in the battle against antibiotic-resistant infections in animals and man.

Carbon monoxide (CO) is a vital signaling molecule in biology. However, the mode(s) of action of CO are unclear, mainly due to the difficulty of delivering and manipulating CO. Therefore, there is intense interest in CO-releasing molecules (CORMs, mostly transition metal carbonyl complexes) as easy-to-handle molecular storage and carrier systems for CO. Remarkably, these compounds have potent, unexplained, bactericidal and bacteriostatic effects and the mode of action appears entirely distinct from current antibiotics so that exciting opportunities exist for novel therapeutic applications.

This proposal focuses on the antimicrobial applications of CORMs in which CO release is triggered by light (PhotoCORMs). We aim for a molecular understanding of their antibacterial effects in comparison with more established compounds and CO gas. We will:
a) Develop and synthesise new PhotoCORMs with enhanced biological properties and potential for use as pro-drugs against bacterial infection, specifically avian pathogenic E. coli (APEC).
b) In collaboration with chemists, consider how CO release kinetics and the specific targeting of cells and tissues can be tuned by molecular design.
c) Understand whether and how PhotoCORMs enter APEC and mammalian cells and whether the greater toxicity of CORMs to bacteria can be explained by transport to the cell interior.
d) Explain the greater effectiveness of CORMs compared to CO gas in antimicrobial activity, exploiting transcriptomics and statistical modelling to unravel bacterial responses to the released CO and the metal coligand fragment.
e) Describe the toxicity of CORMs to APECs, the relationship with oxidative stress and the transcriptional consequences of CORM treatment.
f) Compare the potential of PhotoCORMs with an established CORM, in which CO release occurs via a ligand exchange mechanism, for mitigating infections of cultured avian cells, the Galleria model, avian macrophages and in the whole animal (chickens).

This research is in the priority area of "Combatting antimicrobial resistance". It will evaluate the antimicrobial potential of a new class of metal carbonyl compounds that release carbon monoxide (CO) in biological systems, specifically light-activated CO-releasing molecules (PhotoCORMs). The outcome will be new knowledge of how CORMs act at the cellular and biochemical levels and how they may be used to act as adjuvants or possibly replacements for antibiotics used in the poultry industry, specifically against avian pathogenic E. coli (APEC). The impact will be a contribution to the tackling of antimicrobial resistance in this industry and beyond. The foreseeable impacts on the UK and internationally include:

1. High quality training of early-career bio-scientists;
2. Establishing generic experimental tools and principles essential for deepening our understanding of the role of CO as novel antimicrobial agents or adjuvants to conventional antibiotics;
3. Identifying targets in the form of CO responsive genes and proteins that could be used to control bacterial infections;
4. Publishing quality science in high impact peer-reviewed scientific journals;
5. "Induced" impacts, in which the employment of an individual or stimulating an area of research subsequently results in either economic and social impact;
6. Providing underpinning knowledge and tools that may be applied to other systems, such as many animal pathogens and clinically relevant enteric bacteria;
7. Encouraging multidisciplinary and collaborative research by building upon our successful track records;
8. Engaging with the public to highlight the importance of fundamental underpinning science in advancing measures to counteract antibiotic resistance.