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

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

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.

Technical Summary

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).

Planned Impact

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.

Publications

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Betts JW (2018) Novel Antibacterials: Alternatives to Traditional Antibiotics. in Advances in microbial physiology

 
Description The work proposed is in line with the original grant objectives. Working closely with Prof. Ulrich Schatzschneider (University of Würzburg), new PhotoCORM derivatives have been synthesised that display enhanced activity against avian pathogenic Escherichia coli (APEC). These include the most promising PhotoCORMs developed yet, namely [Mn(CO)3(tqa-k3N)]Br, [Mn(CO)3(bqpa-k3N)]Br and [Mn(bpen-cholamide)(CO)3]Br. The first two compounds have already been subject to extensive testing against APEC and other pathogenic bacteria at the Universities of Sheffield and Surrey. Our findings show that these compounds target bacterial membranes and work in synergy with existing antibiotics in the Galleria mellonella invertebrate infection model. Additional on-going work is required for publication and for assessment of these compounds as potential prodrugs, including in vitro cyto-toxicity testing against eukaryotic cells (avian macrophages) and further functional studies with bacteria to determine the mechanism of uptake of these compounds by APEC, the mechanism of cellular death and the role of CO in biological activity.
The last compound, [Mn(bpen-cholamide)(CO)3]Br, is a newly synthesised derivative of PhotoCORM designed to have specific membrane-targeting activity and exert dual-action via an antimicrobial cholic-acid moiety. This compound is the most potently antimicrobial PhotoCORM developed yet and displays high activity against a wide-range of pathogenic bacterial species in vitro. We are in the final stages of this work: synergy testing to determine if this compound enhances the effectiveness of existing antibiotics against APEC, the mechanism of compound uptake by APEC, the mechanism of action of this compound against APEC and related bacteria, cytotoxicity against eukaryotic cells and its effectiveness in in vivo infection models.
One full peer-reviewed paper has been published and others will follow.
Exploitation Route We hope that novel metal and CO-releasing molecules can be developed as adjuvants to existing antibiotics.
Sectors Agriculture, Food and Drink,Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description Chair of the Humanimal Trust
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Chair of the Veterinary Pathology SAC for the RCPath
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description NHS AMR Diagnostics committee
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact The committee provides guidance on the most appropriate diagnostics for use in the NHS and for surveillance.
 
Description Participation in FSA ACSMF AMR committee
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description AMR MOOC 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact AMR MOOC for Vets
Year(s) Of Engagement Activity 2020
 
Description AMR Talk WOHC 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact AMR talk at the 2020 WOHC
Year(s) Of Engagement Activity 2020
 
Description AVS - Jan 2019 - AMR and biofilms - RVC -UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact A talk on AMR for the Association of Veterinary Students.
Year(s) Of Engagement Activity 2019
 
Description MEVMAS conference - Rapid diagnostics in animal health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk on rapid diagnostics and AMR at MEVMAS conference.
Year(s) Of Engagement Activity 2021
 
Description OBN alternatives to antimicrobial talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact OBN alternatives to antimicrobial talk
Year(s) Of Engagement Activity 2020
 
Description Organised microbiology Society Emerging Threats and AMR meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organised a Microbiology Society Surrey meeting on Emerging Threats and AMR.
Year(s) Of Engagement Activity 2018
 
Description Presentation at St George's hospital June 2018 - One health, Zoonoses and AMR 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Presentation at St George's hospital June 2018 - One health, Zoonoses and AMR
Year(s) Of Engagement Activity 2018
 
Description Talk for the ODN 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk on the alternatives to antibiotics for use in animals.
Year(s) Of Engagement Activity 2020
 
Description Talk for the RCGP on AMR a veterinary perspective 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact A talk on AMR for the RCGP's
Year(s) Of Engagement Activity 2018
 
Description Talk on the microbiome and AMR - European College of Veterinary Microbiology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk on microbiomes and AMR at the European College of Veterinary Microbiology Annual Conference.
Year(s) Of Engagement Activity 2019
 
Description WOHC - Use of big data and digital transformation in microbiology and pathology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Use of big data and digital transformation in microbiology and pathology.
Year(s) Of Engagement Activity 2020